GSM6215553
RNA was harvested with MagMAX RNA Isolation kit compatible with PAXgene tubes (Thermo Fisher) Libraries were prepared using the QuantSeq FWD kit (Lexogen, Vienna, Austria) for multiplexed sequencing according to the recommendations of the manufacturer using both the UDI-adapter and UMI Second-Strand Synthesis modules (Lexogen), as well as the Homo sapiens RS-Globin Block module for globin depletion (Lexogen). The fragment size distribution of the libraries was verified via micro-capillary gel electrophoresis on a LabChip GX system (PerkinElmer, Waltham, MA).   The libraries were quantified by fluorometry on a Qubit fluorometer (LifeTechnologies, Carlsbad, CA), and pooled in equimolar ratios. The library pool was Exonuclease VII (NEB, Ipswich, MA) treated, SPRI-bead purified with KapaPure beads (Kapa Biosystems / Roche, Basel, Switzerland) , and quantified via qPCR with a Kapa Library Quant kit (Kapa Biosystems) on a QuantStudio 5 RT-PCR system (Applied Biosystems, Foster City, CA). Up to forty-eight libraries were sequenced per lane on a HiSeq 4000 sequencer (Illumina, San Diego, CA) with single-end 85 bp reads.