Matt:LabNotes/2015-5-26: Difference between revisions
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| 2X SeqAmp PCR Buffer||82.5 | | 2X SeqAmp PCR Buffer||82.5 | ||
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| PCR Primer II A||3.3 | | PCR Primer II A v3 (12uM)||3.3 | ||
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| SeqAmp DNA Polymerase||3.3 | | SeqAmp DNA Polymerase||3.3 | ||
Line 85: | Line 85: | ||
*Add 30ul to each sample | *Add 30ul to each sample | ||
*Take out of PCR clean hood | *Take out of PCR clean hood | ||
Thermalcycle: 95C 1min -> (98C 10s -> 65C 30s -> 68C 3min) x | Thermalcycle: 95C 1min -> (98C 10s -> 65C 30s -> 68C 3min) x 9 -> 72C 10min -> 4C forever | ||
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*Realized kit was v3 and not v4 so did new protocol | *Realized kit was v3 and not v4 so did new protocol |
Revision as of 18:41, 27 May 2015
SMART-Seq of BA8 Tissue Section
- Repeat RNA-Seq of BA8 Tissue Section but use SMART-Seq v4 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
SMART-Seq v4 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 | 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 __ -> 72C 10min -> 4C forever