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Extracting RNA from polysome profile fractions for qPCR or TrIP-seq sequencing

Updated 8/14/2019 06:53 PM
Step-by-step
by Stephen Floor

Introduction

Transcript Isoforms in Polysomes sequencing (TrIP-seq) measures the translatability of individual transcript isoforms across the genome. The technique is based on polysome profiling and RNA-seq to identify different populations of RNAs associated with different numbers of ribosomes. Ribosomes are stalled during elongation with cycloheximide before and during lysis and purification, different polyribosomal complexes are separated using sucrose density gradient fractionation, and sequencing libraries are then made from extracted RNA. This protocol will also work to perform qRT-PCR or northerns from gradient fractions.

This protocol contains steps from lysis through sequencing library preparation. Lysis is very sample-dependent, and sequencing library prep can use any of the numerous commercial kits (I typically use the Illumina TruSeq Stranded Total RNA kit)

This protocol includes DNase treatment, which may not be necessary but is advisable to ensure no DNA makes its way into the final RNA for sequencing or otherwise.

This protocol is updated from Floor & Doudna, 2016.

Note: if performing ribosome profiling in parallel, see optional steps below. Ensure you add Superase:In to the sucrose gradients.

Critical: maintain RNase-free conditions throughout this protocol. Change gloves frequently, and it is advisable to use filter tips if you use your pipettes to do minipreps. Use RNase-free or autoclaved microfuge tubes, pipet tips, and water. Sterile filter all reagents. Version history (for full version history see the benchling history) - 2019 07 31 - updated gradient maker instructions - 2017 01 11 - added comments re: performing RP in parallel - 2015 12 21 - initial version updated with acid phenol part number and note re: RNA pellets Todo: Change to directzol kits instead of P:C extraction

Materials

  • 10X Sucrose Gradient Buffer
    • 1M KCl
    • 200mM HEPES (free acid) pH 7.6
    • 50mM MgCl2
  • GlycoBlue (Ambion/Thermo AM9516)
    • 3M NaOAc pH 5.2
      • 0.3M NaOAc pH 5.2
        • 100% Ethanol
          • Acid Phenol:Chloroform 5:1 (Sigma P1944)
            • Acid phenol is used to drive DNA to the organic phase
          • Chloroform:isoamyl alcohol (Sigma C0549)
            • Trizol reagent (Ambion/Thermo 15596-026)
              • RQ1 RNase-free DNase (Promega M6101)
                • 50mg/mL cycloheximide (Sigma C7698)
                  • (make this in DMSO from the Sigma powder)
                • The following required if perfoming parallel ribosome profiling:
                • Hypotonic Lysis Buffer (below for mammalian cell culture; if using a different buffer try to keep salt low and use KCl and include some MgCl2)
                  • 10mM HEPES pH 7.9
                  • 1.5mM MgCl2
                  • 10mM KCl
                  • 0.5mM DTT
                  • 0.2mM PMSF (optional; not typically used)
                  • 1% Triton X-100
                  • 100µg/mL cycloheximide (add immediately prior to use)
                • 10% sucrose in SGB (store at 4 deg)
                  • 1X sucrose gradient buffer (from 10X)
                  • 10% sucrose (w/v)
                  • 1mM DTT (add immediately prior to use)
                  • 100µg/mL cycloheximide (add immediately prior to use)
                • 50% sucrose in SGB (store at 4 deg)
                  • 1X sucrose gradient buffer (from 10X)
                  • 50% sucrose (w/v)
                  • 1mM DTT (add immediately prior to use)
                  • 100µg/mL cycloheximide (add immediately prior to use)
                • 2M sucrose solution
                  • Access to SW-41 or other appropriate ultracentrifuge rotor and ultracentrifuge
                    • Beckman Ultra-Clear SW-41 tubes (Beckman 344059)
                      • Access to Brandel gradient fractionator and ISCO UA-6 UV detector w
                        • This is not absolutely required but is desirable. You can also use a Bio-rad Econo UV monitor or any other UV detector.
                        • http://www.brandel.com/fractgradient.html
                      • Access to a Biocomp gradient maker
                        • This is not absolutely required but is desirable
                      • PBS + 100µg/mL cycloheximide

                        Procedure

                        Make sucrose gradients (~1hr or 1 day ahead of harvesting depending on method)

                        1. CRITICALThese instructions are for SW-41 rotor gradients; other rotor instructions will differ
                        1. Make 40mL of 10% and 50% sucrose in SGB solutions with DTT and cycloheximide and allow to come to room temperature
                        1. Mark tubes with line for short gradients (optional, if you have the gradient guide)
                        1. Fill tubes with 10% sucrose in SGB to line (or add 6mL)
                        1. Underlay 50% sucrose in SGB so that the 50% solution reaches line (or add 6mL)

                        (use a ~6 inch, ~10 gauge stainless steel blunt syringe needle for this e.g. from VWR 20068-690 or hamilton http://www.hamiltoncompany.com/products/syringes-and-needles/needles/luer-lock-needles/10-gauge-Metal-Hub-NDL-custom-length-0375-to-12-in-point-style-2-3-or-4-6PK)

                        1. Cap tubes and aspirate excess solution from inside the caps
                        1. Ensure there are no air bubbles in any of the tubes on the sides or top
                        1. Save remainder of 10% sucrose in SGB for setting the baseline on the gradient fractionator
                        1. If using gradient maker (Biocomp Gradient Master 108):

                        Turn on the power in the back

                        Use the round bullseye level to check that the plate is flat and adjust with DOWN/UP buttons if not

                        Push DONE when level

                        Choose GRAD

                        Choose LIST

                        Choose SW41 (assuming you're making SW41 gradients)

                        Select "Short Sucr 10-50%" for a 10-50% sucrose gradient (use DOWN/UP to find it if not shown)

                        Push USE

                        Place the samples on the plate in the center

                        Push RUN

                        Allow the program to complete (takes a minute or so; the plate stopping and moving around is normal)

                        When the plate is flat slide the tube holder off the plate (don't pull it as the release will jostle the gradients)

                        1. If using horizontal diffusion (I have not tried this, below is from Bartel lab protocol):

                        Seal the top of the tube with parafilm

                        Gently tilt tube to horizontal and leave in cold room for 6.5 hrs

                        After 6.5 hours tilt tube slowly back to vertical

                        Leave in cold room overnight in SW-41 tube jackets

                        1. Ensure tubes are balanced to within ~50mg - gently add 10% sucrose solution to rebalance them if not

                        Note: 1µL ~= 1mg, so if the tubes are off by 50mg add 50µL to the low one

                        Lyse cells/tissue (below is for mammalian cell culture) (~3hr)

                        1. Note: input should be ~107 cells or more ideally. Also, this procedure will lyse cytoplasm but not nuclei, which is preferred if possible.
                        1. CRITICALEnsure cycloheximide is in all solutions from lysis onward.
                        1. CRITICALStart cooling ultracentrifuge at this point or earlier
                        1. Treat cells with 100µg/mL cycloheximide by adding to media for 5 minutes before harvesting
                        1. Aspirate media, transfer to ice, and replace media with ~5mL ice cold PBS + cycloheximide
                        1. Aspirate PBS and scrape cells into a microfuge tube
                        1. Spin at 16k g for 30 seconds at 4 deg
                        1. Aspirate PBS and resuspend in 3 pellet volumes of lysis buffer + cycloheximide (typically this is ~100µL pellet volume and 300µL lysis buffer in my hands).
                        1. Incubate ten minutes on ice to swell cells
                        1. Triturate ten times through a 26G needle
                        1. Spin at 1500g for 5 minutes at 4 deg to pellet nuclei
                        1. Transfer ~50µL of cytoplasm (supernatant) to a fresh tube to save for total RNA-seq
                        1. Layer 100µL of cytoplasm on top of a gradient and repeat until out of cytoplasm
                        1. Save the tube with the pellet (this is the nuclear fraction)
                        1. If not all tubes were used then add 100µL of water to each
                        1. Spin at 36,000 RPM for 2 hours at 4 degrees

                        Extracting cytoplasm and nuclear fractions

                        1. Add 300µL Trizol to the retained cytoplasmic fraction and to the tube with the nuclear pellet.
                        1. Triturate each tube ten times through a P-1000. The nuclear pellet is usually very clumpy and sticky.
                        1. Follow the trizol extraction protocol, after the lysis step (start at step 2).

                        Fractionating gradients (~1hr)

                        1. CRITICAL30 minutes prior to spin ending, turn on all frationation equipment
                        1. CRITICALKeep tubes on ice while fractionating
                        1. Flow the leftover 10% sucrose in SGB solution through the fractionator and press "auto-baseline" on the detector to zero the absorbance trace
                        1. Insert a tube into the fractionator
                        1. Puncture the bottom with a ~16G needle connected to a syringe pump or a peristaltic pump and pump at ~2mL/min
                        1. Manually collect fractions corresponding to the monosome, and all polysome peaks as high as can be separated by hand (typically ~8 ribosomes) and then collect the remainder, all in microfuge tubes.
                        1. This is an example of how I would fractionate the polysome profile:
                        1. Ensure no tube has more than 500µL.
                        1. Add 50µL of 3M NaOAc pH 5.2 to each tube
                        1. Add 2.5µL of GlycoBlue to each tube
                        1. Mix well
                        1. Add 1mL 100% EtOH to each tube
                        1. Mix well
                        1. PAUSEStore at -80 for 1 hour to overnight or at -20 overnight

                        DNase treat fractions (~1.5hr)

                        1. Pellet precipitations from above at 16k RPM in a microfuge for 30 minutes. Note carefully the location of the tube hinge and point it to the outside to locate the pellet on that side of the tube.
                        1. Keep tubes on ice when removing from the centrifuge
                        1. The pellets from this will likely be very small. It is recommended to save the supernatant until RNA is quantified after DNase treatment in the event that re-precipitation to obtain more RNA is desired. Typically quantification is reserved until the end to maximize yield. Pellets in most fractions should be barely visible by eye; poly2, poly3 and poly4 fractions tend to be the most difficult to see. Pellets from the RNP fraction, if collected, will be large and very chalky due to the large amount of protein in these fractions.
                        1. Aspirate supernatant carefully without disturbing the pellet with a P-1000 - recommended to not get too close to the pellet.
                        1. Quick-spin the tubes to collect the remainder of the ethanol
                        1. Aspirate the remainder of the ethanol with a P-10, again avoiding the pellet
                        1. Uncap the tubes and rest for 5 minutes at room temperature
                        1. Add 80µL RNase-free water, 10µL 10X RQ1 DNase buffer and 10µL RQ1 DNase
                        1. Incubate at 37 degrees for 30 minutes

                        Phenol-chloroform extraction (~1hr) [or optionally use a Directzol kit (Zymo)]

                        1. Perform these steps at room temperature
                        1. Add 200µL of 0.3M NaOAc pH 5.2 to each tube and mix
                        1. Add 300µL of acid phenol:chloroform and vortex for 5 seconds
                        1. Spin at 16k RPM in cold microfuge for 5 minutes
                        1. Carefully aspirate the aqueous layer with a P-200 and transfer to a new tube
                        1. Add 300µL chloroform:isoamyl alcohol and vortex for 10 seconds
                        1. Spin at 16k RPM in cold microfuge for 5 minutes
                        1. Carefully aspirate the aqueous layer with a P-200 and transfer to a new tube - avoid transferring any of the organic phase
                        1. Add 2.5µL glycoblue to each tube and mix well
                        1. PAUSEAdd 1mL 100% ethanol to each tube and store at -80 for > 1 hour or overnight

                        Resuspend RNA, quantify, and begin library prep

                        1. Pellet precipitations from above at 16k RPM in a microfuge for 30 minutes. Note carefully the location of the tube hinge and point it to the outside to locate the pellet on that side of the tube.
                        1. Keep tubes on ice when removing from the centrifuge
                        1. Aspirate supernatant carefully without disturbing the pellet with a P-1000 - recommended to not get too close to the pellet.
                        1. Quick-spin the tubes to collect the remainder of the ethanol
                        1. Aspirate the remainder of the ethanol with a P-10, again avoiding the pellet
                        1. Uncap the tubes and rest for 5 minutes at room temperature
                        1. Resuspend the pellet in 30µL H2O
                        1. Measure concentration with nanodrop or qubit (recommended)
                        1. Begin library prep or use as-is for qPCR/etc.

                        Comments

                        Sevimli Yaratik
                        4/3/2016 04:25 AM
                        Hi, great write up!

                        I would appreciate if you could answer my questions regarding to your protocol.

                        You are fractionating ~12-13mL of material at a speed of 2mL/min, but your graph ends in 180sec which is quite short to me. Could you please clarify this.

                        You are using Brandel gradient fractionator and ISCO UA-6 UV detector, what sensitivity setting you choose while baselining?

                        Gradient Master has pre-set programs for different gradient options, why did you prefer a custom program over pre-set ones?


                        Have you tried Qiagen's Rnaeasy column based extracting kit instead of precipitation based extraction?

                        Thank you!
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