SMARTseq2 .
day 1 Wipe down bench with RNase zap or similar Dilute oligo - dT30VN primer by adding 37µL of 100µM primer to 63µL of nuclease free H20 .
Prepare RT
mixReagentStock ConcAmt / 1Amt / NFinal ConcNuclease Free H20 - - - 0.29 FS Buffer5x2 1xBetaine5M2 1MMgCl21M0.06 6mMTSO100µM0.1 1µMDTT100mM0.5 5mMSuperscript II200U / µL0.5 100URNase OUT40U / µL 10USample - - - - - - - - - - ( 4.3 ) - - - - - - - - - .
Incubate at 72º for 3m , return to ice .
Take 4.3µL of hybridized oligo - dT + RNA and put in strip tubes .
Add 5.7µL RT mix RTNum CycleGroupTempTime1xA4290m10xB502m B422m1xC7015mholdD4hold Dilute ISPCR primers 1 : 100 Prepare PCR preamp mixComponentStockAmt / 1Amt / NFinal Conc ( First Strand Rxn ) - - 10µL - - - - - - - KAPA HiFi Hot Start2x12.5 1xISPCR primers1µM2.5µL 0.1µM - - - - - - - - - - - - - - - - - - - - - - - - - - Final Volume 25µL .
Add 15µL PCR preamp mix to 10µL RT .
Preamp PCR15 cycles is the current recommendation , but there's enough that I'll try 13 Ampure Cleanup .
Use 0.6 volumes of beads , so 15µL of beads for the 25µL reaction .
Resuspend in 15.5µL EB , then measure concentrations using qubit .
