GSM6008594: Assay_RNAseq_1; Homo sapiens; OTHER
Briefly, after detachment following Accutase treatment, cells were pelleted and RNA extracted according to manufacturer's instructions including DNase treatment (Cytiva illustra™ RNAspin Mini Isolation Kit, Fisher Scientific 45-001) using an automated QIAcube platform. DNA: UMIs were introduced with first and second strand synthesis (NEB 2X Hot Start HotFlex Phusion). Amplicons were further amplified using sequences upstream and downstream of the UMIs. Enzymes were removed after every reaction (QIAGEN Qiaquick PCR Purification). RNA: RNA was reverse transcribed using specific primers with UMIs following manufacturer's instructions in a 20µL reaction including RNase H treatment (SuperScript™ IV Reverse Transcriptase, ThermoFisher 180900). Second strand synthesis was generated using a single round of amplification (NEB 2X HotStart HotFlex Phusion) using specific primers with UMIs. Amplicons were further amplified using sequences upstream and downstream of the UMIs. Enzymes were removed after every reaction (QIAGEN Qiaquick PCR Purification).