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)
- This time sequence from WHOLE tissue section (approx 4cm^2) to validate protocol
- 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
- Wipe all surfaces, forceps, etc with EtOH and RNase Zap
- Scrape tissue off glass with a scalpel and put in 1.5ml tube
- Add 400ul RNA Lysis Buffer
- Vortex 10sec and pipette with 1000p ~10x
- Centrifuge at 18,000rcf for 1min
- Transfer the 400ul to IIIC column and centrifuge at 8,000rcf for 30sec
- Add 320ul 100% EtOH (UV'd) to flow-trhough and mix
- Transfer 720ul to IC column and centrifuge at 18,000rcf for 1min
- Add 400ul RNA Wash Buffer and centrifuge at 18,000rcf for 30sec
- 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
- Add 400ul RNA Prep Buffer and centrifuge at 18,000rcf for 1min
- Add 800ul RNA Wash Buffer and centrifuge at 18,000rcf for 30sec and repeat with 400ul Wash Buffer
- Spin in emptied collection tube at 18,000rcf for 2min
- 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
- Add 4 ul 5X Tn5 buffer to each PCR tube
- Transfer 14ul of each sample to tubes
- Add 2 ul Tn5 (1:50 dilution in 1:1 TE:glycerol) to each sample. Incubated 5 minutes at 55C
- Old Tn5 from Epicentre
- Add 1ul Qiagen protease (20mg/ml) to each sample and incubate 15 minutes at 50C, followed by 20 minutes at 70C
Adapter PCR[edit]
- Make PCR master mix (per reaction):
- 10 ul 3.3x NPM
- 0.66 ul S511 Adapter
- 0.66 ul SYBR Green
- Add 21ul of Nextera sample
- Add 0.66 of N7XX barcode adapters to each sample
- PolydT:N701
- RanPri:N702
- NegCtrl:N703
- Add 11.3 ul of PCR master mix to each sample
- Incubate in thermocycler:
- 72C for 3min
- 95C for 30sec
- 12 cycles of:
- 95C for 10sec
- 55C for 30sec
- 72C for 30sec
- 72C for 5min
File:20150520 RNAseqBA8 Nextera.JPG
- RNA/cDNA mass was too low
- Try SMART-seq next time