GSM5857655: MYOD1 mix tdTomato BC SRT Rep2; Homo sapiens; OTHER
Total RNA is harvested using Direct-zol RNA MiniPrep kit (Zymo Research). RNA concentration and integrity was assessed using Nanodrop. Calling card libraries were prepared as described. We performed first-strand reverse transcription reactions in 20 μL total volume using 2 μg of RNA from each in vitro sample. RNA mixed with water and dNTPs was hybridized to oligo-dT primers (1 μL of 50 μM SMART_dTVN) by incubation at 65 ˚C for 5 minutes and immediately transferred onto ice. 0.5 μL of Maxima H Minus Reverse Transcriptase, RNasin RNAse inhibitor, and 5X RT buffer were added and samples are incubated at 50 °C for 60 min for reverse transcription. cDNA is then amplified with primers specific to the SRT before tagmentation.