Matt:LabNotes/2015-5-19

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RNA-Seq of BA8 Tissue Section[edit]

  • Do RNA-Seq on same tissue sections we do DARTFISH on to compare RNA-Seq to DARTFISH
    • This time sequence from WHOLE tissue section (approx 4cm^2) to validate protocol
      • Also use this test to decide whether polydT or Random Primer Mix is better for RT
    • In future cut out small tissue section (approx 1mm^2) to match same region we do DARTFISH on (and maybe another small tissue section on opposite end)
  • Hypothesis: DARTFISH counts will correlate better with the small cut out tissue section than the whole section
    • This will support that DARTFISH can show regional differences in gene expression

Isolate RNA from BA8 tissue section[edit]

  • Using ZR RNA MicroPrep kit from Zymo
  1. Wipe all surfaces, forceps, etc with EtOH and RNase Zap
  2. Scrape tissue off glass with a scalpel and put in 1.5ml tube
  3. Add 400ul RNA Lysis Buffer
    • Vortex 10sec and pipette with 1000p ~10x
  4. Centrifuge at 18,000rcf for 1min
  5. Transfer the 400ul to IIIC column and centrifuge at 8,000rcf for 30sec
  6. Add 320ul 100% EtOH (UV'd) to flow-trhough and mix
  7. Transfer 720ul to IC column and centrifuge at 18,000rcf for 1min
  8. Add 400ul RNA Wash Buffer and centrifuge at 18,000rcf for 30sec
  9. Add 30ul DNase I cocktail and incubate in 37C incubator for 15min
    • 1.5ul DNase I (2U/ul) + 3ul 10X Reaction Buffer + 25.5ul RNA Wash Buffer
    • Centrifuge at 18,000rcf for 30sec
  10. Add 400ul RNA Prep Buffer and centrifuge at 18,000rcf for 1min
  11. Add 800ul RNA Wash Buffer and centrifuge at 18,000rcf for 30sec and repeat with 400ul Wash Buffer
  12. Spin in emptied collection tube at 18,000rcf for 2min
  13. Add 16ul H2O, let stand for 1min, and then centrifuge at 10,000rcf for 30sec
  • Nanodrop Measurement: 6.9ng/ul
    • A260/280 = 1.77

cDNA Synthesis[edit]

  • Using NEB ProtoScript M-MuLV First Strand cDNA Synthesis Kit and NEBNext mRNA Second Strand Synthesis Module
  • Divide 15ul into 3 samples
    • 1 with d(T)VN primer (poly dT)
    • 1 with Random Primer Mix (hexamer and d(T)VN primers)
    • 1 Negative Control with Random Primer Mix (No RTase added to check for DNA contamination)
  • Followed protocol in manual including denaturing RNA at 70C for 5min and incubation at 25C for 5min for Random Primer Mix samples prior to cDNA synthesis
  • Stored first strand cDNA product in -20C overnight
  • Followed second strand synthesis protocol
  • Purified ds cDNA with Qiagen PCR Purification Column and eluted 30ul
  • Quantify with Qubit dsDNA HS kit
    • PolydT: Too low, out of range
    • RanPri: 0.225 ng/ul (x30ul) = 6.75ng
    • NegCtrl: Too low, out of range


Nextera Tagmentation[edit]

  • Minimum input is ~1ng
  1. Add 4 ul 5X Tn5 buffer to each PCR tube
  2. Transfer 14ul of each sample to tubes
  3. Add 2 ul Tn5 (1:50 dilution in 1:1 TE:glycerol) to each sample. Incubated 5 minutes at 55C
    • Old Tn5 from Epicentre
  4. Add 1ul Qiagen protease (20mg/ml) to each sample and incubate 15 minutes at 50C, followed by 20 minutes at 70C

Adapter PCR[edit]

  1. Make PCR master mix (per reaction):
    1. 10 ul 3.3x NPM
    2. 0.66 ul S511 Adapter
    3. 0.66 ul SYBR Green
  2. Add 21ul of Nextera sample
  3. Add 0.66 of N7XX barcode adapters to each sample
    1. PolydT:N701
    2. RanPri:N702
    3. NegCtrl:N703
  4. Add 11.3 ul of PCR master mix to each sample
  5. Incubate in thermocycler:
    1. 72C for 3min
    2. 95C for 30sec
    3. 12 cycles of:
      1. 95C for 10sec
      2. 55C for 30sec
      3. 72C for 30sec
    4. 72C for 5min

File:20150520 RNAseqBA8 Nextera.JPG

  • RNA/cDNA mass was too low
  • Try SMART-seq next time