title: GSM8989246: BI4300.376.Recovered; Homo sapiens; RNA-Seq
library_construction_protocol: RNA extraction was performed using RNeasy Plus Mini Kit (Qiagen) following the manufacturer's protocol. Isolated RNA sample quality was assessed by High Sensitivity RNA Tapestation (Agilent Technologies Inc., California, USA) and quantified by AccuBlue® Broad Range RNA Quantitation assay (Biotium, California, USA). Paramagnetic beads coupled with oligo d(T)25 are combined with total RNA to isolate poly(A)+ transcripts based on NEBNext® Poly(A) mRNA Magnetic Isolation Module manual (New England BioLabs Inc., Massachusetts, USA). Prior to first strand synthesis, samples are randomly primed (5´ d(N6) 3´ [N=A,C,G,T]) and fragmented based on manufacturer's recommendations. The first strand is synthesized with the Protoscript II Reverse Transcriptase with a longer extension period, approximately 30 minutes at 42⁰C. All remaining steps for library construction were used according to the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England BioLabs Inc., Massachusetts, USA). Final libraries quantity was assessed by Qubit 2.0 (ThermoFisher, Massachusetts, USA) and quality was assessed by TapeStation HSD1000 ScreenTape (Agilent Technologies Inc., California, USA). Average final library size was about 450bp with an insert size of about 300bp. Illumina® 8-nt dual-indices were used. Equimolar pooling of libraries was performed based on QC values and sequenced on an Illumina® NovaSeq X Plus 10B (Illumina, California, USA) with a read length configuration of 150 PE for 40M PE reads per sample (20M in each direction).