GSM5918357: Non-transfected RNA-seq rep2; Homo sapiens; RNA-Seq
15 million HEK293xT cells were spun down, supernatant removed, and washed with cold PBS. Cell pellets were flash frozen on dry ice and stored at -80°C. Total RNA was isolated using the miRNeasy Mini Kit (Qiagen). Poly(A) RNA was isolated using Dynabeads Oligo (dT)25 (Thermo Fisher). RNA integrity and purity was measured using the Agilent 4200 TapeStation. To prepare RNA-sequencing libraries, 50 ng of poly(A) selected RNA was heat fragmented in 2X FastAP buffer and then treated with alkaline phosphatase (FastAP, Thermo Fisher) and T4 PNK (NEB). An RNA adapter was then ligated to the 3′-ends of the mRNA fragments (T4 RNA Ligase, NEB), after which RNA was column purified (Zymo) and reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen), then treated with ExoSAP-IT (Affymetrix) to remove excess oligonucleotides. A 5' Illumina DNA adapter (/5Phos/NNNNNNNNNNAGATCGGAAGAGCGTCGTGT/3SpC3) was ligated to the 3′-end of cDNA fragments with T4 RNA Ligase (NEB) and after on-bead cleanup (Dynabeads MyOne Silane, Thermo Fisher), qPCR was performed on an aliquot of each sample to identify the proper number of PCR cycles. The remainder of the sample was PCR amplified with barcoded Illumina compatible primers (Q5, NEB) and size selected using AMPure XP beads (Beckman). Libraries were quantified using Agilent4200 TapeStation and sequenced on the Illumina NovaSeq platform.