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===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
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