Matt:LabNotes/2015-5-26: Difference between revisions
Jump to navigation
Jump to search
>Mzcai |
>Mzcai mNo edit summary |
||
Line 112: | Line 112: | ||
*Just prior to adding MM add 6.6ul SMARTScribe Reverse Transcriptase to MM and mix without vortexing | *Just prior to adding MM add 6.6ul SMARTScribe Reverse Transcriptase to MM and mix without vortexing | ||
*Add 7.5ul of MM to each tube and mix with pipette and then spin down | *Add 7.5ul 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--> | *Place tubes in thermalcycler: 42C for 90min -> 70C for 10min -> 4C forever | ||
--> | |||
====Nextera XT Tagmentation==== | ====Nextera XT Tagmentation==== | ||
*Minimum input is ~1ng | *Minimum input is ~1ng | ||
Line 152: | Line 131: | ||
#Add 21ul of Nextera sample | #Add 21ul of Nextera sample | ||
#Add 0.66 of N7XX barcode adapters to each sample | #Add 0.66 of N7XX barcode adapters to each sample | ||
## | ##PosCtrl:N701 | ||
## | ##Exp:N702 | ||
##NegCtrl:N703 | ##NegCtrl:N703 | ||
#Add 11.3 ul of PCR master mix to each sample | #Add 11.3 ul of PCR master mix to each sample | ||
Line 165: | Line 144: | ||
##72C for 5min | ##72C for 5min | ||
<!-- | <!-- | ||
[[File: | 650px]] | [[File: | 650px]] |
Revision as of 18:54, 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
Nextera XT Tagmentation
- 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
- 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
- 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