GSM6895823: HA200-T2; Homo sapiens; RNA-Seq
Cells were thawed and transferred into RPMI1640 (Gibco Cat# 11835030) medium containing 10% FBS (Gibco Cat# 10082147). Cells were then collected by centrifugation at 1200 RPM for 7 min and washed twice using ice-cold PBS (Gibco Cat# 10010002). Collected pellets were lysed using Lysis/Binding Buffer (Invitrogen Cat# 61012), and frozen at −80°C. Polyadenylated mRNAs were subsequently isolated from thawed lysates using the Dynabeads mRNA Direct Kit (Invitrogen), following the manufacturer's instructions. RNA-seq libraries were prepared using a protocol modified from the NEBNext Ultradirectional (Illumina® - NEB Cat# E7760L) library preparation protocol. RNA was fragmented at 94°C for 5 minutes to obtain 200–1500 bp fragments in size. SPRISelect beads (Beckman Coulter) were used in all purification steps and size selection. Barcodes from PerkinElmer were added by ligation.