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==SMART-Seq of BA8 Tissue Section== *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/xxclt_ibcGetAttachment.jsp?cItemId=93858 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 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''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) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''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 <!-- *Realized kit was v3 and not v4 so did new protocol ===SMART-Seq v4 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 8.5ul H2O **NegCtrl: Nuclease-free H2O **PosCtrl: UHRR (10ng/ul) **Exp: Isolated total RNA (16.5ng/ul) *Add 2ul 3' SMART-Seq CDS Primer II A to each *Incubate at 72C for 3min followed by ice for 2min *Make Master Mix {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 5X Ultra Low First-Strand buffer||13.2 |- | SMART-Seq v4 Oligo||3.3 |- | Rnase Inhibitor||1.65 |- | Total||18.15 |} *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 *Place tubes in thermalcycler: 42C for 90min -> 70C for 10min -> 4C forever --> ====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 | 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]]
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