Matt:LabNotes/2015-5-26: Difference between revisions
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==SMART-Seq of BA8 Tissue Section== | ==SMART-Seq of BA8 Tissue Section== | ||
*Repeat [[Matt:LabNotes/2015-5-19|RNA-Seq of BA8 Tissue Section]] but use SMART-Seq | *Repeat [[Matt:LabNotes/2015-5-19|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 | ||
*[http://www.clontech.com/US/Products/cDNA_Synthesis_and_Library_Construction/Next_Gen_Sequencing_Kits/Single_cell_RNA_Seq_Kits_for_mRNA_seq/ibcGetAttachment.jsp?cItemId=104008&fileId=6982600&sitex=10020:22372:US v4Protocol] | *[http://www.clontech.com/US/Products/cDNA_Synthesis_and_Library_Construction/Next_Gen_Sequencing_Kits/Single_cell_RNA_Seq_Kits_for_mRNA_seq/ibcGetAttachment.jsp?cItemId=104008&fileId=6982600&sitex=10020:22372:US v4Protocol] | ||
*[http://www.clontech.com/xxclt_ibcGetAttachment.jsp?cItemId=93858 v3Protocol] | *[http://www.clontech.com/xxclt_ibcGetAttachment.jsp?cItemId=93858 v3Protocol] | ||
Line 75: | Line 75: | ||
| 2X SeqAmp PCR Buffer||82.5 | | 2X SeqAmp PCR Buffer||82.5 | ||
|- | |- | ||
| PCR Primer II A||3.3 | | PCR Primer II A v3 (12uM)||3.3 | ||
|- | |- | ||
| 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 | ||
*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 | |||
<!-- | <!-- | ||
*Realized kit was v3 and not v4 so did new protocol | *Realized kit was v3 and not v4 so did new protocol | ||
Line 112: | Line 117: | ||
*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==== | ||
* | *Input is ~1ng | ||
# Add 4 ul 5X Tn5 buffer to each PCR tube | # Add 4 ul 5X Tn5 buffer to each PCR tube | ||
# Transfer 14ul of each sample to tubes | # 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 | # Add 2 ul Tn5 (1:50 dilution in 1:1 TE:glycerol) to each sample. Incubated 5 minutes at 55C | ||
#*Old Tn5 from Epicentre | #*Old Tn5 from Epicentre | ||
Line 149: | Line 136: | ||
##10 ul 3.3x NPM | ##10 ul 3.3x NPM | ||
##0.66 ul S511 Adapter | ##0.66 ul S511 Adapter | ||
## | ##1.65 ul SYBR Green (10X, final needs to be 0.5X) | ||
#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 152: | ||
##72C for 5min | ##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 | #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 | 650px]] | |||
*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 | |||
*I bead purified Second PosCtrl sample for sequencing | |||
**Labed as 2015 06 16 MC POSCTRL and quantified | |||
[[File:2015-06-01_BA8SmartSeq_BeadPurify_GelCheck.jpg | 250px]] [[File:2015-06-16_UHRRPosCtrlSMARTSeq_BeadPurify_GelCheck.jpg | 200px]] |
Latest revision as of 23:46, 16 June 2015
SMART-Seq of BA8 Tissue Section[edit]
- 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[edit]
- 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[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 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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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
- I bead purified Second PosCtrl sample for sequencing
- Labed as 2015 06 16 MC POSCTRL and quantified
File:2015-06-01 BA8SmartSeq BeadPurify GelCheck.jpg File:2015-06-16 UHRRPosCtrlSMARTSeq BeadPurify GelCheck.jpg