GSM5819494: HEK293_GFP_ActD_4h_2; Homo sapiens; RNA-Seq
Total RNA was isolated from HEK293T cells using Bio Tri RNA reagent (Bio-lab) and  mRNA was captured using Oligo d(T)25 magnetic Beads (NEB) according to  manufacturer's protocol. Poly(A)-purified RNAs were taken for library preparation using a  derivation of MARS-seq as described (15). Briefly, 10 ng of poly(A)+ RNA was taken for  the first reverse transcription reaction using illumina barcoded RT1 primer. Resultant  barcoded cDNA samples were subsequently pooled according to Ct values of housekeeping gene (GAPDH) (Quality control 1). Pooled cDNA was treated with Exonuclease  I to remove excess primers followed by second strand synthesis using NEB SSS module  enzyme mix. After that, in-vitro transcription was performed using NEB T7 RNA Pol mix  to generate RNA, which was later fragmented and ligated to an adaptor consisting of RD2  using T4 RNA ligase I (NEB) followed by the second reverse transcription reaction. The  library so formed was amplified using Kapa Hifi ready mix (Roche). The amplified RNA  libraries were sequenced using high-throughput 75 bp kit (Illumina FC404-2005) on  NEXTseq 500 sequencer.