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===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
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