GSM6124507: K562, LentiLong, Replicate 1, TSS-MPRA; Homo sapiens; OTHER
RNA was harvasted using typical TRIzol LS protocols (Invitrogen) For TSS-MPRA, starting with 10-15 µg RNA (⅓ of the total), non-capped RNAs were dephosphorylated with Quick CIP (NEB) and DNA digested. Samples were mixed well and incubated at 37°C for 90 minutes. For more complete dephosphorylation of uncapped transcripts, RNA was denatured a second time at 75°C for 30 seconds in a pre-warmed water bath and then quickly chilled on ice for 2 minutes before incubating the sample at 37°C for another 30 minutes. As Quick CIP is thermolabile, the brief heat treatment leaves the enzyme active. Following Cap-enrichment, RNA was purified and then vortexed hard and subsequently spun for 10 minutes at 12k g at RT. Following phase separation, the upper layer was taken, and RNA precipitated. RNA pellets were then resuspended and 5' caps removed. Following decapping, 5' adapters were ligated by T4RNA Ligase 1 and RNA extracted. RNA pellets were then resuspended and reverse transcribed. Samples were amplified and sequenced paired-end on a NextSeq 500 (additional details available in the manuscript) TSS-MPRA