Matt:LabNotes/2015-5-26
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SMART-Seq of BA8 Tissue Section
- Repeat RNA-Seq of BA8 Tissue Section but use SMART-Seq v3 Ultra Low Input RNA Kit to amplify since cDNA was too little last time
- v4Protocol
- v3Protocol
Purpose
- Do 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
- 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
- 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 10ul H2O, let stand for 1min, and then centrifuge at 10,000rcf for 30sec
- Nanodrop Measurement: 16.5ng/ul
- A260/280 = 1.75
- A260/230 = 1.60
SMART-Seq v3 RT
- Do work prior to PCR in PCR clean hood
- Prepare 10X Reaction Buffer: 19ul 10X Lysis Buffer + 1ul RNase Inhibitor (avoid bubbles when mixing)
- Add 1ul 10X Reaction Buffer to 1ul of sample and 8ul H2O
- NegCtrl: Nuclease-free H2O
- PosCtrl: UHRR (10ng/ul)
- Exp: Isolated total RNA (16.5ng/ul)
- Add 1ul 3' SMART-Seq CDS Primer II A to each
- Incubate at 72C for 3min followed by ice until next step
- Make Master Mix
Component | Volume |
5X First-Strand Buffer | 13.2 |
DTT (100mM) | 1.65 |
dNTP Mix (10mM, protocol says should be 20mM) | 3.3 |
SMARTer IIA Oligo (12uM) | 3.3 |
Rnase Inhibitor (40U/ul) | 1.65 |
SMARTScribe Reverse Transcriptase (100U/ul) | 6.6 |
Total | 29.7 |
- Just prior to adding MM add the 6.6ul SMARTScribe Reverse Transcriptase to MM and mix without vortexing
- Add 9ul of MM to each tube and mix with pipette and then spin down
- Place tubes in thermalcycler: 42C for 90min -> 70C for 10min -> 4C forever
- Keep at in 4C fridge overnight
SMART-Seq v3 PCR
- Make Master Mix
- Add polymerase just before use (and don't vortex)
Component | Volume |
2X SeqAmp PCR Buffer | 82.5 |
PCR Primer II A v3 (12uM) | 3.3 |
SeqAmp DNA Polymerase | 3.3 |
Nuclease-Free H2O | 9.9 |
Total | 99 |
- Add 30ul to each sample
- Take out of PCR clean hood
Thermalcycle: 95C 1min -> (98C 10s -> 65C 30s -> 68C 3min) x 9 -> 72C 10min -> 4C forever
- Ampure XP bead purification with 50ul beads, elute 17ul with H2O
- 1ul for Qubit HS dsDNA:
- PosCtrl: 2.53 ng/ul
- Exp: 0.452 ng/ul
- NegCtrl: out of range
Nextera XT Tagmentation
- Input is ~1ng
- Add 4 ul 5X Tn5 buffer to each PCR tube
- Transfer 14ul of each sample to tubes
- PosCtrl: 0.4ul sample + 13.6ul H2O
- Exp: 2.2ul sample + 11.8ul H2O
- NegCtrl: 14ul sample
- 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
- Make PCR master mix (per reaction):
- 10 ul 3.3x NPM
- 0.66 ul S511 Adapter
- 1.65 ul SYBR Green (10X, final needs to be 0.5X)
- Add 21ul of Nextera sample
- Add 0.66 of N7XX barcode adapters to each sample
- PosCtrl:N701
- Exp: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
- qPCR shows no increase in fluorescence after 12 cycles
Nextera XT
- Repeat with Derek's/Blue's reagents and protocol
- Input ~0.2ng DNA
- PosCtrl: 0.253ng/ul (diluted 1:10)
- Exp: 0.226ng/ul (diluted 1:2)
- NegCtrl: H2O
- GM12878 gDNA: 0.201ng/ul (diluated 0.803 ng/ul 1:4)
- Add 2.5ul Tagment DNA Buffer
- Add 1.25ul Amplification Tagment Mix
- Add 1.25ul Sample
- Vortex and spin down
- Incubate 55C for 10min and then hold at 10C
- Add 1.25ul NT buffer and incubate at RT for >5min
- Add 3.75ul NPM, 1.25ul Index Primer 1 and 1.25ul Index Primer 2 (S517)
- PosCtrl: N701
- Exp: N702
- NegCtrl: N703
- gDNA: N704
- Vortex and spin down
- Incubate in thermocycler:
- 72C for 3min
- 95C for 30sec
- 12 cycles of:
- 95C for 10sec
- 55C for 30sec
- 72C for 60sec
- 72C for 5min
Gel Check
- Run 1 ul of each sample on PAGE gel with 2ul H2O 3 ul 6X loading dye
- 0.5 ul Low mass Ladder
- 25 min @ 250 V
- Lane1:First PosCtrl
- Lane2:First Exp
- Lane3:First NegCtrl
- Lane4:Low Mass Ladder
- Lane5:Second PosCtrl
- Lane6:Second Exp
- Lane7:Second NegCtrl
- Lane8:Second gDNA
File:2015-05-29 BA8 SMARTSeq Nextera GelCheck.jpg
- Looks like Nextera with 1ng input and Andrew's reagents (SYBR Green) did not work
- Following recommended protocol of 0.1-0.3ng input and Derek/Blue's reagents did work!
- Derek bead purified Second Exp sample for sequencing
- Labeled as 2015 05 29 MC E and quantified