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Version 1 CHARGED tRNA Library Preparation for RNA004

Updated 4/16/2025 10:19 PM
Step-by-step
by Kezia Dobson

Introduction

This protocol will take you through the Hesselberth lab's tRNA library preparation to capture both acylated and deacylated tRNAs for nanopore sequencing using the RNA004 chemistry. To begin this protocol you must have a pellet of cells from your organism of interest that you intend to extract RNA from. The protocol will take you through our extraction method which is optimized to keep tRNAs acylated as well as subsequent library preparation.

GOOD RNA PRACTICES: You need to be working in an RNase-free environment. Unless stated otherwise, RNA and reagents should be kept on ice at all times. To store overnight, RNA is always frozen at -80ºC.

INSTRUCTIONS ON ACCESS: This protocol will be made available via a PDF and a Benchling link. Anyone can open the shared link and view the protocol even when they are signed out of a Benchling account (or don't have one). This viewer will allow you to edit the tables only in a way that will not impact the master copy. You will not be able to edit the text on this viewer. The viewer also has a step-by-step feature which allows one to check off completed steps and start timers. This is probably the best electronic way to access the protocol if you do not use Benchling. If you do use Benchling and want to add this protocol to your personal protocols, you can also do that. Just open the shared link while signed into your Benchling account. You will see a clock symbol underneath the dark blue share symbol in the top right margin of the protocol. You will see verion history here. Select the most recent version and click clone from version in the bottom right of this popup. You can then select one of your folders to clone the protocol into. Once you've done that, you now have your own copy that you can edit and add to entries as you please.

USEFUL RESOURCE: I like this online calculator for converting between moles and grams for nucleic acids.

Materials

  • MATERIALS FOR SAMPLE PREP
    • Yeast or other cell culture of interest ready for RNA harvest
    • AES (10mM sodium acetate pH 4.5, 1mM ETDA pH 8, 0.5% SDS) Pre-chill but it will precipitate if it gets too cold.
    • 125:24:1 acid phenol: chloroform: amyl alcohol
    • 10mM NaOAc pH 4.5
    • DEPC treated, nuclease free water (Ambion)
    • Zymo RNA Clean and Concentrator-25 kit (Zymo Research, R1018) - This kit enables rapid clean up of the small RNA fraction (17-200 nt) from up to 50µg of total RNA, using the manufacturer-provided “Purify Small and Large RNAs in Separate Fractions” protocol. You can expect ~10% yield of small RNA from total RNA.
  • MATERIALS FOR EDC-ACTIVATION
    • EDC powder - This must be kept at -20ºC under vacuum or at 4ºC in a desicator. It cannot get wet.
    • 100µM Gel-purified charged 3' adapter (see Appendix 2 for details)
    • 1M imidazole pH 7.0
    • 10mM imidazole pH 8.0
    • 99.5+% Acetone saturated with perchlorate powder
    • 99.5+% Acetone
    • 1:1 acetone: diethyl ether
    • 10mM imidazole pH 8
  • MATERIALS FOR CHEMICAL LIGATION
    • 0.5M MES pH 5.5
    • 1M HEI pH 6.5 (Sigma Aldrich 576166)
    • 200mM MgCl2
    • 100µM gel-purified 5' adapter (see Appendix 2 for details)
    • 200µM IMID-3'-adapter - you make this during EDC activation from 100µM Gel-purified charged 3' adapter (see Appendix 2 for details)
  • MATERIALS FOR CHEMICAL LIGATION GEL PURIFICATION
    • 10% TBU minigel
    • Crush and soak buffer: 300mM NaOAc (pH 5.5), 1mM EDTA, 0.1% SDS
    • 2X RNA Dye (NEB B0363)
    • Low range ssRNA ladder (NEB N0364S)
    • 0.45 µm cellulose filters (we use costar Spin-X, from Corning, #8163)
    • 100% and 70% EtOH
    • Glycoblue (Invitrogen AM9515)
    • 10mM NaOAc pH 4.5
  • MATERIALS FOR ENZYMATIC SPLINT ADAPTER LIGATION OF DEACYLATED tRNAs
    • 10µM mix of gel-purified uncharged 3' adapter + gel-purified 5' splint adapter (see Appendix 2 for details)
    • PEG 8000 (NEB B1004S)
    • T4 RNA ligase 2 and its buffer (NEB B0239S)
    • RNase inhibitor (Watchmaker 7K0088-500UL)
  • T4 DNA ligase and its buffer (Watchmaker 7K004 - 500µL or NEB, M0202M, 2,000,000U/mL)
    • BioDynami tRNA beads (40054S)
      • Unlike single stranded RNA oligos of equivalent size, tRNAs do not readily bind SPRI beads. The Hesselberth lab has tested these beads for purifying ligated tRNAs, and confirmed their capacity to purify tRNAs and tRNA + adapter ligation products over a 75 - 177 nt range.
    • Ampure XP beads (Fisher Scientific A63881)
      • RNA004 ONT sequencing kit (contains RT adapter (RTA), though we can also generate in house RTA mixes)
        • RNA004 ONT flow cell priming kit
          • RNA004 ONT flow cell wash kit
            • HS D1000 Tapestation reagents
              • HS DNA Qubit reagents

                Procedure

                Reagent Preparation

                1. Especially if this is your first time through this protocol, there are a lot of stocks and reagents to prepare. Do not try to make these all the same day you begin the protocol. All the reagent recipes are found in Appendix 1.
                1. You must gel-purify your syntheses of the splint adapters. Make sure this is done before you begin the protocol. See Appendix 2.

                1. Sample Preparation

                We have optimized the sample preparation to retain amino acylated tRNAs optimally. The most important things to consider when doing and acylated extraction is to keep you RNA cold, keep the extraction total time short, and keep everything acidic. Do not EtOH precipitate because some deacylation occurs if you do.

                1. Grow your organism in conditions of interest. Collect cells in a pellet. This exact process for this will depened on the organism.
                1. Chill your AES on ice for 5 minutes. DO NOT EXCEED 5 MINUTES OR IT MIGHT PRECIPITATE. Equilibrate Ampure XP beads to RT.

                RNA Extraction

                1. Thaw pellet of cells on ice and resuspend in 400µL cool AES.
                1. MOVE TO THE FUME HOOD.
                1. Add 400µL cold acid phenol chloroform.
                1. Vortex samples for 15 seconds.
                1. Let samples rest on ice for 20 minutes, vortexting every 5 minutes.

                Tighten the lids before every vortex session.

                1. Spin at 14000+ rpm for 10 minutes at 4ºC. (18000 rcf)
                1. IN THE FUME HOOD: remove the aqueous layer (top) into a new tube. Do NOT disturb the middle layer of cell debris or bottom layer of phenol or you will have to spin again for 10 minutes.

                You can store aqueous phases at -80ºC. They are often not clear but cloudy.

                1. Blank the nanodrop with AES and nanodrop the aqueous phase. Don't worry about getting an exact measurement by diluting, we just need a ballpark.

                Left-handed Ampure Cleanup

                1. Aliquot up to 1mg of RNA to a PCR tube for bead cleanup.

                Yeast aqueous phases are usually 10-20µg/µL and we move on with 100µL.

                1. Add a 0.4X volume of well-vortexed Ampure beads to the aqueous phase.

                The color of the beads + aqueous will be a light brown cloudy mixture.

                1. Rotate for 2 minutes at RT.
                1. Place on a magnet until clear.
                1. Move the supernant to a new tube and discard the beads.

                Ampure beads do not efficiently bind tRNAs, and so this step removes a portion of larger RNAs while leaving the tRNAs in the supernatant.

                1. Blank with AES and nanodrop the LH cleanup. Again we just want a ballpark concentration.

                Small RNA Cleanup

                1. Use the RNA Clean and Concentrator kit 25, and do a small RNA cleanups according to the manufacturer's instructions.

                Ideally you do 2 columns per sampe, cleaning up 100µg of LH cleanup.

                1. Start by aliquoting 50µg of LH cleanup and bringing the volume up to 50µL with 10mM NaOAc pH 4.5. Do this twice so you have 50µg to go into each of the two cleanups you will do per sample.
                1. Make sure to spin for 5+ minutes to dry the columns after dumping off the last wash buffer. Failure to do this can results in EtOH contamination in the elution.
                1. Elute each column with 15µL of 10mM NaOAc. Pool samples to get 30µL total for each sample.
                1. Nanodrop. Store at -80ºC.

                2. Activating charged 3' adapters with imidazole

                YOU MUST MAKE NEW CHARGED ADAPTERS THE SAME DAY AS DOING CHEMI LIGATIONS.

                EDC degrades rapidly in water and a new stock must be made fresh every time. The imidazolated adapter also hydrolyzes relatively quickly and ligation efficiency goes down if it is not prepared the day it is used. Therefore, it is essential that your samples are prepared before beginning this step.

                1. Get out your EDC powder and warm to room temperature. This reduces condensation. Also start thawing the charged 3' adapter on ice.
                1. Make a 0.5M solution of EDC with a minimum volume of 1045µL.

                For 1045µL solution, weigh 100mg EDC and add 1045 water.

                This solution CANNOT BE SAVED. Throw it out after you use what you need.

                1. Set up the following reaction to imidizolate the 5' end of the gel-purified 3' charged adpater.

                Make sure you select the CHARGED 3' adpater with the sequence specific to charged tRNAs. It MUST be 5' phosphorylated and gel-purified.

                Fill in the desired pmol tRNA you plan to ligate per sample in "Chemi Ligation setup" 30-50pmol recommended, but you can go down to 15pmol if yield is low. Then enter the total sample number in the table below ("EDC-activation reaction setup") and it will auto-populate.

                If your gel-purified 3' adapter final concentration is not exactly 100µM, change the table and it will account for your true concentration.

                Note that this recipe calls for 0.1M imidazole not 1M.

                EDC-activation reaction setup
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                1
                		ReagentStock Concentration (µM, M)Final concentration (µM, M)Molar ratio (adapter: other reagent)Total nmolVolume to add to reaction (µL)Total reaction volume (µL)Final desired concentration of EDC-activated adapter (µM or pmol/µL)
                2
                		3' adapter gel purified (5' phosphorylated) (µM)91501
                #VALUE!
                #VALUE!
                #VALUE!
                		100
                3
                		Imidazole pH 7 (M)0.10.025500
                #VALUE!
                #VALUE!
                		nmol of adapter needed for each ligationVolume 10 mM Imidazole (pH 7.0) to dissolve final product in (µL)
                4
                		EDC (M)0.50.025500
                #VALUE!
                #VALUE!
                #VALUE!
                #VALUE!
                5
                		Water
                #VALUE!
                		Total number of samples to ligate
                6
                		18
                1. Let this reaction incubate for 2 hours at 25ºC in the thermocycler.
                1. Precipitate in 30µL cold 99.5%+ acetone saturated with perchlorate and 1mL cold 99.5%+ acetone on dry ice for 20 minutes.
                1. Spin at max speed for 10 minutes at 4ºC. Remove the supernatant into a small beaker in the fume hood. It evaporates so fast it is hard to collect in a waste container.
                1. Wash with 1mL 1:1 acetone diethyl:ether, remove supernatant into beaker, and wash once more, removing the supernatant again. This will also evaporate rapidly.

                If you disturb the pellet, spin it down again for 1-2 minutes at max speed (4ºC).

                1. Finish drying using a speedvac. It usually takes 10-30 minutes to dry.
                1. Dissolve the pellet in the indicated amount in the above table of 10mM imidazole (pH 7.0). Your final concentration of adapter will be 100µM.

                3. Chemical ligation of acylated tRNAs

                1. Samples beginning at this step should have undergone acidic RNA extraction, a left-handed Ampure XP bead cleanup to remove large RNAs, and a small RNA cleanup with the RNA clean and concentrator kit 5. You small RNAs should be in 10mM NaOAc pH 4.5 (you used this to elute from the small RNA cleanup).
                1. Prepare you tRNAs for chemical ligation and set up the following master mis.

                You should already have entered the pmol tRNAs you plan to ligate. This should be 30-50pmol though 15 is enough to scrape by if you are limited by tRNA amount. The mass tRNAs needed will auto-calculate and you will be given an input volume required for each tRNA sample to attain the desired pmol. Enter the volume required of your most dilute sample to be the total volume in "tRNA Dilutions" (F2). You will dilute all the samples to that total volume using 10mM NaOAc. At this point, all your tRNAs set aside for ligation are an equal volume.

                The default is total volume for each chemical ligation (H7 in "Chemi ligation setup") is 20µL. If you notice the water per reaction required is negative, you must increase the total volume until the water required is about ~0. It is a good rule of thumb to minimize total reaction volume for ease when gel extracting in the next step.

                tRNA Dilutions
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                1
                		SampleStock Concentration (ng/µL)Desired mass (ng)Volume tRNAs to add (µL)Volume 10 mM NaOAc to add (µL)Total Volume (µL) - input the required to acheive desired mass of your MOST DILUTE sample
                2
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                		ADD
                3
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                		ng per pmol tRNAs
                4
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                		30.65
                5
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                6
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                7
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                8
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                9
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                10
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                11
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                12
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                13
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                14
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                15
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!

                If your gel-purified 5' adapter final concentration is not exactly 100µM, change the table and it will account for your true concentration.

                Chemi Ligation Set up
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                1
                		ReagentsStock Concentration (M, µM, mM, ng/µL)Final Concentration (M, µM, mM)Molar ratio to each otherFinal Amount (pmol)Volume to add to each reaction (µL)Volume to add to master mix (µL)Total number of reactions
                2
                		MES pH 5.5 (mM)500100N/AN/A4.00
                #VALUE!
                		ADD
                3
                		MgCl2 (mM)2002.5N/AN/A0.25
                #VALUE!
                4
                		AA-tRNAsN/AN/A1ADDADD
                5
                		5' adapter (µM)69.7N/A3
                #VALUE!
                #VALUE!
                #VALUE!
                6
                		imid-3'-adapter (µM)100N/A3
                #VALUE!
                #VALUE!
                #VALUE!
                		Total Volume per Reaction (µL)
                7
                		HEI pH 6.5 (mM)100050N/AN/A1.00
                #VALUE!
                		20
                8
                		WaterN/AN/AN/AN/A
                #VALUE!
                #VALUE!
                9
                		Master Mix per reaction (µL)
                10
                #VALUE!
                1. Incubate in the thermal cycler for 30 minutes at 25ºC and then hold at 4ºC (can go overnight). This can be frozen at -80ºC.

                4. Removing free splint adapters

                We cannot have free adpater left behind when we move on to T4 RNL2 ligation of splint adapters to deacylated tRNAs. The imidazole can hydrolyze off the 3' charged adapters leaving behind a 5' P. These adpaters can then be enzymatically ligated onto deacylated tRNAs, causing them to have the sequence specific to acylated tRNAs in nanopore readout despite being deacylated. Therefore we must gel purify the tRNAs and leave behind free adapters.

                1. Add an equal volume of 2X RNA dye to your chemical ligation products.
                1. Load product onto a 10% TBU gel. Depending on the thickness and well-depth, the total volume each well can hold will vary. Do not overfill lanes or you may cross contaminate. Your full chemical ligation should be loaded onto the gel.
                1. Dilute low range ssRNA ladder 1 in 10. Add 5µL of the diluted ladder to 5µL RNA dye and load the full 10µL as a ladder.
                1. Run at 250V for ~35 minutes or until bromophenol blue travels 7+ cm down the gel.
                1. Stain with SYBR gold for 5+ minutes and image (unless your imager uses a type of UV light that mutates nucleic acid! If so, do not image and move to the next step.)
                1. Move the gel to a blue light imager and cut out the ligated and unligated tRNAs. You should cut from underneath the light ~70 nt band (the dark two bands smaller than this are the free adapters, hybridized and free) to the top of the dark 150 nt band.
                1. Move those pieces into a 0.5mL tube that you've punctured with a large guage needle at the bottom. Place this 0.5mL tube into a 1.7mL eppendorf tube.

                If your pieces do not all fit in a 0.5mL tube, you will need to split the sample into multiple tubes,

                1. Spin at max speed for 3 minutes. This will force the gel slices through the needle hole and pulverize the gel allowing for better release of RNA.
                1. Estimate the volume of the tube taken up by the crushed gel and add an equal volume of RT crush and soak buffer.

                We find usually ~300µL of crush and soak buffer is appropriate.

                1. Rotate overnight at 4ºC to release RNA.

                If you are in a pinch timewise, you can also do 37ºC for an hour, but your yield will be less.

                1. Move the eluate to a 0.45 µm cellulose filter. You may need to cut the end of a pipet tip to widen it to transfer the goopy eluate.
                1. Place the filter in a new eppendorf tube. Spin at top speed for 2 minutes or until all the liquid has filtered through.
                1. Ethanol precipitate by adding 1mL of 100% EtOH and 2µL of Glycoblue.

                There is no need to add additional NaOAc because the crush and soak buffer has enough salt to make nucleic acids crash out.

                1. Vortex and incubate at -80ºC for at least 20 minutes (up to as long as you want).
                1. Spin for 15 minutes at max speed at 4ºC.
                1. Remove the supernatant and wash with 1mL 70% EtOH.
                1. Spin for 5 minutes at max speed at 4ºC.
                1. Remove the superntant without sucking up the pellet. Spin down again on the benchtop centrifuge and remove as much of the EtOH as possible. Repeat until you can't suck up any more.
                1. Let the residual EtOH evaporate by leaving the tube open on the bench for 20 minutes or until there is no visible EtOH left.
                1. Resuspend in 11.5µL of 10mM Tris pH 7.0.
                1. Nanodrop with 1µL.

                5. Ligate deacylated tRNAs to splint adapters

                We are now going to enzymatically ligate a set of non-imidazolated splint adapters to the deacylated molecules. The 3' adpater differs in sequence to the one you imidazolated and chemi ligated, allowing you to tell acylated from deacylated tRNAs in the sequencing analysis.

                1. Set up the following 20µL reaction. Add enzymes to master mix last.

                You will first aliquot out the desired mass of each chemi-ligated sample and dilute it to 10µL with 10mM NaOAc pH 4.5. If any of your yields failed to reach the desired mass threshold, you can still progress as long as you have at least 300ng (10pmol). In this case though, you will not be able to use the master mix for this sample because it needs a different amount of splint adapter. Scale down the splint adapter amount so that there are 0.46pmol adapter to 1pmol tRNA. The rest of the component volumes will remain the same.

                Chemi ligated tRNA dilutions
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                		SampleStock Concentration (ng/µL)Desired mass (ng)Volume tRNAs to add (µL)Volume 10 mM Tris pH 7.0 to add (µL)Total Volume (µL)
                2
                		ADD613
                #VALUE!
                #VALUE!
                		10
                3
                		ADD613
                #VALUE!
                #VALUE!
                		ng per pmol tRNAs
                4
                		ADD613
                #VALUE!
                #VALUE!
                		30.65
                5
                		ADD613
                #VALUE!
                #VALUE!
                6
                		ADD613
                #VALUE!
                #VALUE!
                7
                		ADD613
                #VALUE!
                #VALUE!
                8
                		ADD613
                #VALUE!
                #VALUE!
                9
                		ADD613
                #VALUE!
                #VALUE!
                10
                		ADD613
                #VALUE!
                #VALUE!
                11
                		ADD613
                #VALUE!
                #VALUE!
                12
                		ADD613
                #VALUE!
                #VALUE!
                13
                		ADD613
                #VALUE!
                #VALUE!
                14
                		ADD613
                #VALUE!
                #VALUE!
                15
                		ADD613
                #VALUE!
                #VALUE!
                Deacylated splint adapter ligation setup
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                1
                		ReagentsStock Concentration (X, mM, µM, %)Final Concentration (X, mM, µM, %)Molar ratio to each otherFinal Amount (pmol)Volume to add to each reaction (µL)Volume to add to master mix (µL)Total number of reactions
                2
                		T4 RNA ligase 2 buffer (X)101N/AN/A2.00
                #VALUE!
                		ADD
                3
                		PEG 8000 (%)5010N/AN/A4.00
                #VALUE!
                4
                		RNase InhibitorN/AN/AN/AN/A1.00
                #VALUE!
                5
                		T4 RNL2N/AN/AN/AN/A2.00
                #VALUE!
                6
                		Chemi ligated tRNAsN/AN/A12010.00Total Volume per Reaction (µL)
                7
                		Uncharged splint adapter mix (µM)10N/A0.4590.90
                #VALUE!
                		20
                8
                		Master Mix per reaction (µL)
                9
                		10
                1. Incubate at 25ºC in the thermal cycler for 30 minutes. Hold at 4ºC after. Equlibrate BioDynami tRNA beads to RT.

                6. Cleaning up splint adapter ligated tRNAs

                Below I have copied the BioDynami protocol with slight modifications.

                1. Add 40µL (2X volume) of WELL-VORTEXED beads to the ligation reaction.
                1. Mix by pipetting and incubate for 5 minutes.
                1. Load onto a magnet.
                1. Incubate for 8 more minutes.
                1. Remove the supernatant.
                1. Wash without disturbing the beads with 180µL 80% EtOH.
                1. Incubate for 1 minute and remove as much of the EtOH as possible. Spin down and remove the last few µLs.
                1. Dry with cap open for 2 minutes. No EtOH should be visible but beads should not look cracked.
                1. Remove from magnet.
                1. Resuspend in 13µL 10mM Tris pH 7.0.
                1. Load onto a magnet.
                1. Incubate for 1 minute and remove the elution to a new tube.

                7. Ligating the RT adapters

                1. Ligate the splint-adapter tRNAs to the RT adapter. There is no need to re-quantify at this step since you put 10-20pmol into the deacylated splint adapter ligation.
                1. Set up the following 20µL ligation. Add enzyme last.
                RTA Ligation Set up
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                1
                		ReagentVolume to add (µL)Master mix volume (µL)Total number of reactions
                2
                		5X Watchmaker T4 DNA ligase buffer or 5X Quick ligation buffer4
                #VALUE!
                		ADD
                3
                		RTA1.5
                #VALUE!
                4
                		RNase Inhibitor0.5
                #VALUE!
                5
                		T4 DNA ligase (from Watchmaker or NEB)1.5
                #VALUE!
                6
                		Splint-adapter ligated tRNAs12.5
                1. Incubate for 30 minutes at 25ºC in the thermal cycler. Hold at 4ºC after.

                8. Cleaning up the RTA Reaction

                1. Take out the HS DNA 1000 Tapestation reagents so they can equilibrate to RT. Also, take out Quant-iT Qubit dsDNA HS reagents out to equilibrate to RT.

                Below I have copied the BioDynami protocol with slight modifications. Beads hsould have already been equilibrated to RT.

                1. Add 40µL (2X volume) of WELL-VORTEXED beads to the ligation reaction.
                1. Mix by pipetting and incubate for 5 minutes.
                1. Load onto a magnet.
                1. Incubate for 8 more minutes.
                1. Remove the supernatant.
                1. Wash without disturbing the beads with 180µL 80% EtOH.
                1. Incubate for 1 minute and remove as much of the EtOH as possible. Spin down and remove the last few µLs.
                1. Dry with cap open for 2 minutes. No EtOH should be visible but beads should not look cracked.
                1. Remove from magnet.
                1. Resuspend in 26µL 10mM Tris pH 7.0.
                1. Load onto a magnet.
                1. Incubate for 1 minute and remove the elution to a new tube.

                9. Assess Quantity via Qubit and Quality via Tapestation

                1. Make sure both Qubit and Tapestation reagents have equilibrated to RT. We will Qubit first.
                1. Get out the number of qubit tubes you need (number of samples + 2).
                1. Set up the following master mix.
                Qubit Master Mix
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                1
                		ReagentVolume per assay (µL)Volume to add (µL)Total number of samples (including 2 standards)
                2
                		Qubit dsDNA HS Buffer1991313.46
                3
                		Qubit dsDNA HS Reagent16.6
                1. Add 10µL of each standard to a Qubit tube.
                1. Add 1µL of each sample (anywhere from 0.2-100ng is acceptable, and you should already be in that range without dilution)
                1. Add 190µL of working solution to each standard and 199µL to each sample.
                1. Vortex all tubes and let sit for 2 minutes.
                1. Read the standards and tubes.
                1. Convert from ng/µL to pg/µL by multiplying by 1000 and enter into the table below ("Molarity Calculation").
                1. The Tapestation accepts 10-1000pg/µL samples. If your Qubit indicates that you exceed this, dilute before moving on. Use 2µL of the original sample in making the dilution so that you remaining volume will be 23µL regardless of whether you dilute or use directly from the sample.

                The instructions for a HS D1000 tape are copied below.

                1. Flick the tape and put it in the Tapestation. Always use up the remaining slots on an opened tape before starting a new tape.
                1. Vortex reagents.
                1. Add 2µL High Sensitivity D1000 Sample Buffer to 2µL of your sample (diluted or undiluted).
                1. Vortex at 2000 rpm for 1 minute.
                1. Spin down. REMOVE CAP!!!
                1. Place tube in Tapestation loading tray. Make sure enough tips are in the Tapestation. Run the program.

                A good quality library will have peaks ~175 nt and ~300 nt. The RTA ligated product is expected to be 177 nts, and we think the 300 nt band is an artifact. The main thing you want to look out for are large quantities <150 nts. These can be splint adapter ligated or unligated tRNAs. If a large portion of your sample is in this region, the Tapestation will estimate the amount of product in each peak. Scale down your Qubit concentration to just include the peaks of interest when calculating fmol for the final ligation.

                Molarity Calculation
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                		SampleConcentration of ligated tRNAs (pg/µL)Concentration sample (fmol/µL)Total fmol remainingVolume RTA product needed (µL)Volume water to add (µL)~ Size of tRNAs (nts)
                2
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                #VALUE!
                		233
                3
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                #VALUE!
                		fmol/pg (1 nt)
                4
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                #VALUE!
                		2.94
                5
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                #VALUE!
                		Volume sample remaining (µL)
                6
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                #VALUE!
                		23
                7
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                #VALUE!
                		Desired total fmol
                8
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                #VALUE!
                		400
                9
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                #VALUE!
                		Desired total volume (µL)
                10
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                #VALUE!
                		23
                11
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                #VALUE!
                12
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                #VALUE!
                13
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                #VALUE!
                14
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                #VALUE!
                15
                		ADD
                #VALUE!
                #VALUE!
                #VALUE!
                #VALUE!
                1. Aliquot 400fmol and bring the volume up to 23µL with water. Don't exceed this fmol amount. You can drop as little as 50fmol if yields are lower.

                Reagent Amounts to Thaw - Day of Sequencing

                You cannot continue unless you are sequencing today. This last ligation must happen day of sequencing.

                1. Get out at least the indicated volumes or each reagent to begin thawing on ice. DIL, S, and FCF are larger volumes so they can be thawed on the bench and then put on ice.

                If you plan to sequence overnight, don't get out DIL or WMX. Still get out S because sometimes you need it to rescue a flow cell with no pores (See Appendix 4).

                Reagents to thaw day of sequencing
                A
                B
                C
                D
                1
                		ReagentAmount per sample to sequnece (µL)Tota Volume needed for all your samplesNumber of samples you are prepping
                2
                		5X Quick Ligation Buffer8
                #VALUE!
                		ADD
                3
                		RLA6
                #VALUE!
                4
                		T4 DNA Ligase3
                #VALUE!
                5
                		Ampure beads72
                #VALUE!
                6
                		WSB300
                #VALUE!
                7
                		REB33
                #VALUE!
                8
                		RFT25
                #VALUE!
                9
                		FCF975
                #VALUE!
                10
                		SB100
                #VALUE!
                11
                		LIS68
                #VALUE!
                12
                		WMX2
                #VALUE!
                13
                		DIL398
                #VALUE!
                14
                		S500
                #VALUE!

                10. Ligating the RLA adaptors

                1. Set up the following ligation of the helicase-loaded adaptor. You need at least 50fmol tRNA input. Recommended amount is 400fmol.

                If you choose to make a master mix, you should NOT include the RLA adapter. This is the most expensive reagent in the kit: every µL counts. Master mixes waste a small amount of their components and you do not want to waste RLA.

                RLA Adaptor Ligation Set up
                A
                B
                C
                D
                1
                		ReagentVolume to add (µL)Master mix volume (µL)Total number of reactions
                2
                		5X Watchmaker T4 DNA ligase buffer or 5X Quick ligation buffer8
                #VALUE!
                		ADD
                3
                		RLA adaptor6
                4
                		tRNAs ligated to splint adaptors and RTA adaptors23
                5
                		T4 DNA ligase (from Watchmaker or NEB)3
                #VALUE!
                6
                		Master mix volume to add (µL)
                7
                		11.00
                1. Incubate for 30 minutes at 25ºC in the thermal cycler. Hold at 4ºC after.
                1. Start equilibrating your flow cells to RT (20 minutes equilibration needed). Check for condensation on underside and gold connector pins and wipe any off.

                11. Cleaning up the RLA reaction

                1. After flow cells reach RT, check their pores. You must do this before priming the flow cell.
                1. Add 72µL (1.8x volume) Ampure XP beads.

                Because we have ligated 3 adapters at this point, Ampure XP beads can now efficienctly bind our libraries.

                1. Rotate for 5 mins at RT.

                We are using a 1.8X volume of beads instead of ONT's recommened 0.4X volume because tRNAs are smaller than most of the RNAs you would sequence. We need this higher bead volume to help us selectively bind tRNAs and not the 90 nt free RLA adaptor.

                1. Wash twice with 150µL WSB (ONT) resuspending beads by flicking the tube and then reseating on the magnet to settle.
                1. Elute sample:

                FOR PROMETHION: Elute sample in 33µL REB elution buffer (also from ONT kit) with a 10 min incubation rotating at room temperature.

                FOR MinION: Elute sample in 13µL REB elution buffer (also from ONT kit) with a 10 min incubation rotating at room temperature.

                12. Sequencing on a Promethion flow cell

                PROMETHION - Preparing library

                If you are running a MinION, see Appendix 3.

                1. Mix 100µL sequencing buffer (SB) with 68µL library solution (LIS) for each sample.
                1. Add 32µL of library to each SB + LIS mix. Mix by pipetting and do not introduce bubbles.
                1. Keep on ice.

                PROMETHION - Priming the flow cell

                Here is a link to ONT's insturctions on Promethion flushing and loading. It has useful images that can help you identify the different parts of the flow cell if you are unfamiliar.

                1. You should already have equilibrated the flow cell to RT for 20 minutes and checked its pores during the RLA cleanup, but if you didn't do that now. You must do this before priming.
                1. Make the following priming mix and vortex. If you plan on running batches of flow cells, only prep the mix for the ones you are about to run. You need 1000µL per flow cell.
                Promethion Priming Mix Recipe
                A
                B
                C
                D
                1
                		ReagentVolume require per flow cell (µL)Volume to add (µL)Total number of flow cells to be run immediately
                2
                		RNA flush tether (RFT)25
                #VALUE!
                		ADD
                3
                		Flow cell flush (FCF)975
                #VALUE!
                1. If you are using a used flow cell, make sure the inlet port is closed and remove all waste from either of the waste ports.Set a p1000 to 200µL.
                1. Open inlet port by rotating the valve / cover clockwise (it’s the thing with the screw).
                1. Insert the pipet tip into the inlet port (the hole marked 1), and slowly turn the pipet wheel to draw back 20-30µL, or until you see a small volume of yellow buffer entering the tip.

                The amount you have to turn pipet may vary depending on brand of pipet tip and how well it is seated in the hole.

                DO NOT REMOVE MORE THAN A FEW µLS OR YOU WILL EXPOSE THE PORES TO AIR.

                1. Slowly load 500µL of the priming mix into the inlet port (same hole marked 1) WITHOUT INTRODUCING AIR. CHECK TO MAKE SURE NO AIR IS AT THE TIP OF YOUR PIPET. IT IS BETTER TO USE A FEW µLs LESS THAN 500 THAN TO INTRODUCE AIR.

                Close the rotating valve to avoid air getting into the port. If you forget, back out another small amount of liquid before adding the rest of the priming mix.

                1. Wait 5 mins, then add another 500µL. DO NOT INTRODUCE AIR.

                PROMETHION - Loading the library and sequencing

                1. Mix library by gentle pipetting just before loading. Do not introduce bubbles.
                1. Insert a p200 tip into the inlet port and add slowly add 190µL of library WITHOUT INTRODUCING AIR. CHECK TO MAKE SURE NO AIR IS AT THE TIP OF YOUR PIPET. IT IS BETTER TO USE A FEW µLs LESS THAN 500 THAN TO INTRODUCE AIR.
                1. Rotate the valve to the closed position (below) to seal the inlet port.
                1. Put the light sheild on. Place a sticker on the light sheild and label the flow cell with the library you just input.
                1. Configure MinKNOW for RNA004 direct RNA sequencing with the following settings:

                POD5 should be the selected output, 20 nts the minimum read size (200 nts may be the default), and do not filter reads.

                1. You can now begin the sequencing run.

                13. Washing and storing Promethion flow cells

                Here is ONT's wash protocol for Promethions. Here is their storage protocol for Promethions.

                1. After your tRNA sequencing run has reached the desired number of reads, you can stop sequencing. Make sure you only tell it to stop sequencing NOT to stop sequencing and basecalling.
                1. The flow cell can be removed before basecalling is finished, but a new flow cell can't be inserted into that position until basecalling is finished. If you do insert one, you won't be able to use that position until basecalling is complete.
                1. Make the following wash mix. You need 400µL per flow cell. If you'll be washing more cells later on, only make enough mix for the cells you plan to wash right away.
                Wash Mix Recipe
                A
                B
                C
                D
                1
                		ReagentVolume require per flow cell (µL)Volume to add (µL)Total number of flow cells to be run immediately
                2
                		Wash Mix (WMX)2
                #VALUE!
                		ADD
                3
                		Wash Diluent (DIL)398
                #VALUE!
                1. Close the inlet port. Remove all liquid from the waste port (labelled 2 or 3).
                1. Open the inlet port. Pull out a SMALL amount of liquid to remove the air gap. DO NOT PULL OUT MORE THAN A FEW µLS OR YOU WILL EXPOSE THE PORES TO AIR.
                1. Add 200µL of the mix mix to the inlet port and close it.
                1. Wait 5 minutes and add the remaining 200µL. If you forget to close the port, reextract a small amount of liquid from the inlet port to remove any air before adding the remaining 200µL wash mix.
                1. Close the port and wait 1 hour for the DNase in the wash mix to work.
                1. With the port closed, extract all the waste.
                1. Open the port and remove a small amount of liquid from the inlet port.
                1. Add 500µL storage buffer to the flow cell. Close the port.
                1. Go check how many pores are still active on the flow call. Write the number down on a sticker on the light sheild.

                We consider any Promethion with less than 200 pores to be dead. Collect dead flow cells somewhere in your lab because ONT offers a free flow cell recycling program for dead cells.

                1. Store at 4ºC until you want to use it again.

                APPENDIX 1: Reagent Preparations

                Suggested total volumes are given in yellow but can be modified and the math in the table will alter to accomodate.

                3M NaOAc pH 4.5
                A
                B
                C
                D
                E
                F
                1
                		ReagentMolar mass (g/mol)Final molesMass/volume to add (g, mL)Total volume (mL)Desired concentration (M)
                2
                		NaOAc powder (g)82.03436492.2120003
                3
                		Water (mL)N/AN/A1500.00
                4
                		Add glacial acetic acid IN THE FUME HOOD until you reach pH 4.5 (it takes a while FYI)
                5
                		Add water up to desired volume
                6
                		Store at RT
                AES
                A
                B
                C
                D
                E
                1
                		ReagentStock concentration (mM, %)Final concentration (mM, %)Volume to add (mL)Total volume (mL)
                2
                		NaOAc pH 4.5 (mM)3000100.1750
                3
                		EDTA pH 8.0 (mM)50010.10
                4
                		SDS (%)200.51.25
                5
                		WaterN/AN/A48.48
                6
                		Store at RT
                1M Imidazole pH 7.0
                A
                B
                C
                D
                E
                F
                1
                		ReagentMolar mass (g/mol)Final molesMass/volume to add (g, mL)Total volume (mL)Desired concentration (M)
                2
                		Imidazole powder (g)68.0770.16.811001
                3
                		Water (mL)N/AN/A60.00
                4
                		Add concentrated HCl IN THE FUME HOOD until you reach pH 7.0 (it takes a while FYI)
                5
                		Add water up to desired volume
                6
                		Store at 4ºC
                Acetone saturated with perchlorate
                A
                B
                C
                1
                		ReagentVolume (mL)Mass
                2
                		99.5+% Acetone10N/A
                3
                		Sodium perchlorateN/AAdd until no longer soluble
                4
                		Don't worry about filtering any bits of sodium perchlorate if there isn't much. Store in a screw cap storage container at 4ºC.
                1:1 aectone: diethyl ether
                A
                B
                C
                1
                		Before beginning, test diethyl ether stock for oxidizer buildup. In the fume hood, suck up and squirt a small amount of the stock diethyl ether onto a KI starch paper using the syringe. If it turns blue, it could be explosive and you should immediately call EHS. If it does not turn a dark color, you are fine.
                2
                		Make this solution in an amber bottle in the fume hood.
                3
                		ReagentVolume to add (mL)Total volume (mL)
                4
                		99.5+% Acetone2550
                5
                		Diethyl ether25
                6
                		Store with flammables at RT.
                0.5M MES pH 5.5
                A
                B
                C
                D
                E
                F
                1
                		ReagentMolar mass (g/mol)Final molesMass/volume to add (g, mL)Total volume (mL)Desired concentration (M)
                2
                		MES free acid powder195.240.0254.88500.5
                3
                		Water (mL)N/AN/A30.00
                4
                		Add concentrated NaOH IN THE FUME HOOD until you reach pH 5.5
                5
                		Add water up to desired volume
                6
                		Store at RT
                1M HEI pH 6.5
                A
                B
                C
                D
                E
                F
                G
                H
                I
                1
                		ReagentConcentration (w/v)Concentration (g/mL)Molar mass (g/mol)Final molesFinal mass (g)Volume to add mL)Total volume (mL)Desired concentration (M)
                2
                		1-(2-Hydroxyethyl)imidazole970.97112.130.022.24262.31201
                3
                		WaterN/AN/AN/AN/AN/A17.69
                4
                		HEI comes as a visous liquid that may preciptate when cold. It will go into solution when warmed. Store at 1M HEI at RT.
                200 mM MgCl2
                A
                B
                C
                D
                E
                F
                1
                		ReagentMolar mass (g/mol)Final molesMass/volume to add (g, mL)Total volume (mL)Desired concentration (M)
                2
                		MgCl2 powder95.2110.010.95500.2
                3
                		Water (mL)N/AN/A50.00
                4
                		Store at RT
                10% TBU gel stock
                A
                B
                C
                D
                E
                1
                		ReagentStock concentration (X, %, g/mol)Final concentration or amount (X, %, mol)Volume or mass to add (mL, g)Total volume (mL)
                2
                		TBE (X)10195.00950
                3
                		19:1 Acyrlamide: Bisacrylamide (%)4010237.50
                4
                		Urea powder (g/mol)60.066.175370.87
                5
                		WaterN/AN/A665.00
                6
                		Wait for urea to dissolve (30 minutes)
                7
                		Add water up to total volume
                8
                		Filter to prevent precipitation. Store at 4ºC.
                Crush and soak buffer
                A
                B
                C
                D
                E
                1
                		ReagentStock concentration (mM, %)Final concentration (mM, %)Volume to add (mL)Total volume (mL)
                2
                		NaOAc pH 5.5 (mM)300030025.00250
                3
                		EDTA pH 8.0 (mM)50010.50
                4
                		SDS (%)200.11.25
                5
                		WaterN/AN/A223.25
                6
                		Store at RT. DO NOT COOL OR IT WILL CRASH OUT. You can warm at 37ºC if it crashes out and it will go back in.

                Note: the pH of NaOAc is 5.5 not 4.5 for crush and soak buffer!

                APPENDIX 2: Gel Purification of splint adapters

                In the table below are specifications for the splint adapters. Both the charged and uncharged 3' adpaters can pair with the 5' splint adapter. Charged and uncharged refer to whether that 3' adapter will be chemically ligated to acylated tRNAs (charged) or enzymatically ligated to deacylated tRNAs (uncharged). You get the best quality libraries when you gel purify these adapters after receiving a synthesis because the synthesis is crude.These adapters contain RNA and need to be stored at -80ºC.

                1. Order a sythesis of the following oligos.
                Splint adapter specifications
                A
                B
                C
                D
                E
                1
                		OligoLigated to?SequenceIDT sequence (DNA/RNA specifications)Adapter length
                2
                		Charged 3' adapterchemically ligated to the amino acid of acyalted tRNAsGGCUUCUUCUUGCUCUUCCAACCUUGCCUUAAAAAAAAAA/5Phos/rGrGrCrUrUrCrUrUrCrUrUrGrCrUrCrUrUrCrCrArArCrCrUrUrGrCrCrUrUAAAAAAAAAA40
                3
                		Uncharged 3' adapterenzymatically ligated to the 3' end deacyalted tRNAsGGCUUCUUCUUGCUCUUAUGGAAGGUAGGCAAAAAAAAAA/5Phos/rGrGrCrUrUrCrUrUrCrUrUrGrCrUrCrUrUrArUrGrGrArArGrGrUrArGrGrCAAAAAAAAAA40
                4
                		5' splint adapter (universal)pairs with either 3' adapter, enzymatically ligated to the 5' end of all tRNAsCCTAAGAGCAAGAAGAAGCCUGGNCCTAAGAGCAAGAAGAAGCrCrUrGrGrN24
                1. Resuspend these oligos to 2mM (2nmol/µL) in water.
                1. Cast a 1.5 mm 6% V16 gel with 10 wells. Load up to 10nmol per lane. Run for 1-1.5 hours at 250V.

                DO NOT STAIN! If you stain you will see a smear that takes up the whole lane.

                1. UV shadow and excise the dark spots and put them into a 50mL falcon tube.
                1. Add ~20-30mL of crush and soak buffer. Use the end of a clean 15mL falcon tube to crush the gel bits into the bottom of the falcon tube.
                1. Parafilm the lid and rotate end over end overnight at 4ºC.
                1. Spin in a fixed angle rotor for 5 minutes at 11,000g at 4ºC to force gel bits to the bottom. Remove the supernatant to a new tube.

                If you want to maximize yield, resuspend the gel bits in 10-15mL of crush and soak buffer. Parafilm the lid and rotate end over end overnight at 4ºC.

                1. Filter the supernatant with a 0.45 µm filter to remove any remaining gel bits.
                1. Split the filtrate into two 50mL falcon tubes. Add a 2.5X volume of cold 100% EtOH to each tube. Do not add a co-precipitant.
                1. Incubate at -80ºC for at least 30 minutes.
                1. Spin for 15 minutes at 11,000g at 4ºC.
                1. Carefully pipet off the sueprnatant, not disturbing the pellet.
                1. Add 5-10mL of cold 70% EtOH to wash.
                1. Spin for 5 minutes at 11,000g at 4ºC.
                1. Remove as much EtOH as possible. Air dry at RT with the lids open for at least 30 minutes or until no EtOH is left.
                1. Input the total nmol you input at the beginning and resuspend in the volume of water indicated by the table. Move to a microcentrifuge tube. You can expect a 30-50% yield on the first elution and a 5-10% yield on the second elution.
                1. Nanodrop the elution. If the OD of the nanodrop exceeds 25, dilute and then use that dilution to calculate what the original concentration in the tube was. Input this into the table and it will tell you the current µM concentration of the adpater. It should be greater than or equal to the desried concentration which is 100µM.
                1. The table will then tell you how much additional water to add to bring the concentration down to the desired amount.
                1. Nanodrop once more. The table will tell you what your final concentration really is. It is ok if it is not exactly the desried concentration, just update in your version the "EDC-activation reaction setup" table with the real concentration of your gel-purified adapters.
                1. Make a 10µM dilution of the 5' adpater and the 3' UNCHARGED adapter (each at 10µM).
                1. Aliquot your adapters and store them at -80ºC.
                Dilution calculations for first elution
                A
                B
                C
                D
                E
                F
                G
                H
                I
                1
                		Total nmol loaded onto gel Conservative estimate of round 1 yield (decimal)Desired final concentration (µM of pmol/µL)Volume water to resuspend in (µL)Starting concentration by nanodrop (ng/µL)Starting concentration by nanodrop converted to (µM) pmol/µLVolume of water to dilute to desired concentration (µL)FINAL concentration by nanodrop (ng/µL)FINAL concentration by nanodrop converted to pmol/µL
                2
                		2000.2510050020001472351400102.9
                3
                		Conservative estimate of round 1 yield (nmol)pmol/ng per ntnt length of adapterTotal water you've resuspended in (µL)
                4
                		502.9440735
                1. If you elected to do a second elution, follow steps 146-160 and use the table below to finish extracting this extra adapter
                Dilution calculations for second elution
                A
                B
                C
                D
                E
                F
                G
                H
                I
                1
                		Total nmol loaded onto gel Conservative estimate of round 2 yield (decimal)Desired final concentration (µM of pmol/µL)Volume water to resuspend in (µL)Starting concentration by nanodrop (ng/µL)Starting concentration by nanodrop converted to (µM) pmol/µLVolume of water to dilute to desired concentration (µL)FINAL concentration by nanodrop (ng/µL)FINAL concentration by nanodrop converted to pmol/µL
                2
                		2000.051001002000147471400102.9
                3
                		Conservative estimate of round 2 yield (nmol)pmol/ng per ntnt length of adapterTotal water you've resuspended in (µL)
                4
                		102.9440147

                APPENDIX 3: Instructions for running a MinION - Preparing the library

                1. Make the following priming mix and vortex. If you plan on running batches of flow cells, only prep the mix for the ones you are about to run. You need 1000µL per flow cell.
                MinION Priming Mix Recipe
                A
                B
                C
                D
                1
                		ReagentVolume require per flow cell (µL)Volume to add (µL)Total number of flow cells to be run immediately
                2
                		RNA flush tether (RFT)25
                #VALUE!
                		ADD
                3
                		Flow cell flush (FCF)975
                #VALUE!
                1. Mix 37.5µL sequencing buffer (SB) with 25.5µL library solution (LIS) for each sample.
                1. Add 12µL of library to each SB + LIS mix. Mix by pipetting and do not introduce bubbles.
                1. Keep on ice.

                MinION - Priming the flow cell

                1. You should already have equilibrated the flow cell to RT for 20 minutes and checked its pores during the RMX cleanup, but if you didn't do that now.
                1. Set p1000 tip to 200µL.
                1. Insert the pipet tip into the priming port (beneath the rotation valve) and slowly turn the pipet wheel to draw back 20-30µL, or until you see a small volume of yellow buffer entering the tip.

                The amount you have to turn pipet may vary depending on brand of pipette tip and how well it is seated in the hole.

                DO NOT REMOVE MORE THAN A FEW µLS OR YOU WILL EXPOSE THE PORES TO AIR.

                1. Pipet 800µL priming mix into the priming port WITHOUT INTRODUCING AIR. CHECK TO MAKE SURE NO AIR IS AT THE TIP OF YOUR PIPET. IT IS BETTER TO USE A FEW µLs LESS THAN INTRODUCE AIR.
                1. Wait 5 minutes.
                1. Pipet the remaining 200µL priming mix into the priming port WITHOUT INTRODUCING AIR.

                MinION - Loading the library and sequencing

                1. Load 80µL library and DO NOT INTRODUCE AIR. IT IS BETTER TO LEAVE BEHIND A FEW µLs THAN INTRODUCE AIR.
                1. Put the light shield on. Label light shield with a sticker.
                1. Configure MinKNOW for RNA004 direct RNA sequencing with the following settings:

                POD5 should be the selected output, 20 nts the minimum read size (200 nts may be the default), and do not filter reads.

                1. You can now begin the sequencing run.

                MinION - Washing flow cells

                Here is ONT's wash protocol for MinIONs. Here is their storage protocol for MinIONs.

                1. After your tRNA sequencing run has reached the desired number of reads, you can stop sequencing. Make sure you only tell it to stop sequencing NOT to stop sequencing and basecalling.
                1. The flow cell can be removed before basecalling is finished, but a new flow cell can't be inserted into that position until basecalling is finished. If you do insert one, you won't be able to use that position until basecalling is complete.
                1. Make the following wash mix. You need 400µL per flow cell. If you'll be washing more cells later on, only make enough mix for the cells you plan to wash right away.
                Wash Mix Recipe - MinION
                A
                B
                C
                D
                1
                		ReagentVolume require per flow cell (µL)Volume to add (µL)Total number of flow cells to be run immediately
                2
                		Mash Mix (WMX)2
                #VALUE!
                		ADD
                3
                		Wash Diluent (DIL)398
                #VALUE!
                1. Close the SpotON port and the priming port. Remove all liquid from the waste port (labelled 2 or 3).
                1. Open the priming port. Pull out a SMALL amount of liquid to remove the air gap. DO NOT PULL OUT MORE THAN A FEW µLS OR YOU WILL EXPOSE THE PORES TO AIR.
                1. Add 200µL of the mix mix to the priming port and close it.
                1. Wait 5 minutes and add the remaining 200µL. If you forget to close the port, reextract a small amount of liquid from the inlet port to remove any air before adding the remaining 200µL wash mix.
                1. Close the port and wait 1 hour for the DNase in the wash mix to work.
                1. With the port closed, extract all the waste.
                1. Open the priming port and remove a small amount of liquid.
                1. Add 500µL storage buffer to priming port and close it.
                1. Go check how many pores are still active on the flow call. Write the number down on a sticker on the light sheild.
                1. Store at 4ºC until you want to use it again.

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