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    Feb. 2018 Oyster QuantSeq Libraries
    • Notes
    • Metadata
    • Relevant Items
    • QuantSeq 3' mRNA Kit
    • RNA Extraction - Trizol and PureLink Kit
    Editing disabled on read-only entry.

     
    Feb. 2018 Oyster QuantSeq Libraries

    Thursday, 2/22/2018Sample listFriday, 2/23/2018Saturday, 2/24/2018Sunday, 2/25/2018Monday, 2/26/2018Wednesday, 2/28/2018Thursday, 3/1/2018Screenshot from 2018-02-28 17-12-08.pngFriday, 3/2/2018Redo sample listKS_Lib_3_Samples_3-2-18.xlsxMonday, 3/5/2018180305_Pfister-Silliman_SS_KS-Lib3_orig.pdf
    Thursday, 2/22/2018
    Making QuantSeq mRNA libraries for 76 tissue samples from the low pH Olympia oyster experiment in Fall 2016.
    Friday, 2/23/2018
    Extract RNA from 15 samples
    ●
    OR3A_124C, CA3A_118M, OR3A_54C, OR4B_88C, OR3A_122C, CA2A_111C, BC3A_126 M (out of tissue), OR3A_56C, BC3A_57M, BC3A_57C, CA3A_51C, CA1A_105C, M, CA3A_52C, OR1A_99C (out of tissue)
    ●
    eluted in 50uL
    Saturday, 2/24/2018
    Extract RNA from 16 samples. Sorted tissue sample numerically and better labelled boxes.
    ●
    BC3B_C62, BC2A_C35, CA2B_C153, OR3B_C164, BC2B_C41, CA3B_166M, OR2B_37C, BC3B_171C, OR3B_163M, CA2B_45C, BC3B_170C, CA3B_166C, OR2A_28C, CA1B_13M, CA3B_168C, BC3B_170M
    ●
    eluted in 75uL
    Sunday, 2/25/2018
    Extract rest of oly samples
    eluted in 50u
    Monday, 2/26/2018
    Concentrate dilute samples using Zymo Clean & Concentrator -5.
    Left box of new samples on bench overnight (19 hours).... will check RNA quality later.
    Wednesday, 2/28/2018
    Did QuantSeq protocol through Step 11, left libraries on thermocycler at 0degC overnight (15 hours).
    Could not find BC102M, so did not make library. Removed from sample list.
    Thursday, 3/1/2018
    Ran libraries through PCR step #28.
    ●
    bad/dried beads at end of Step 24 purification: 8A, E2, D4
    ●
    Did a qPCR using Lexogen Add-on PCR kit for all samples with less than 400ng starting RNA and then for 9 other samples to estimate the number of PCR cycles for the 2 tissue types:
    ○
    16 PCR cycles for ctenidia (unless qPCR shows otherwise)
    ○
    16 PCR cycles for mantle (unless qPCR shows otherwise)
    ○
    Sample OR_163M did not work in qPCR, so did not continue with that library (74 samples total now)
    ●
    Accidently added 2 idexes to BC_142C, so will NOT pool that sample (73 samples total)
    ●
    Lexogen has a nice tool to estimate color balance based on their indexes
    ●
    Left PCR 19 (10degC) and PCR 18( 0degC) on thermocycler over night after PCR
    Screenshot from 2018-02-28 17-12-08.png
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    Enough reagents for 64(?) more samples
    Friday, 3/2/2018
    Finished library prep- 15 samples had really low library yeilds so will try and remake those.
    Finished library prep of redo samples. Pooled all samples together (70 total) and submitted to sequencing core!
    Sample list with barcodes:
    KS_Lib_3_Samples_3-2-18.xlsx
    Monday, 3/5/2018
    Library QC from Sequencing Core:
    180305_Pfister-Silliman_SS_KS-Lib3_orig.pdf

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