title: GSM7158584: SLAMseq_K562_REP2_AZ5576; Homo sapiens; OTHER
library_construction_protocol: K562 cells were washed in ice-cold PBS and resuspended in 300μL TRIzol. To extract RNA, one-fifth volume of chloroform was added to TRIzol lysates, followed by shaking, incubation at room temperature for 2 minutes and centrifugation (16000g at 4°C for 15 minutes). The aqueous phase was isolated and RNA was precipitated using DTT (10mM), 100% isopropanol (1 volume) and GlycolBlue co-precipitant, incubated at room temperature for 10 minutes and centrifuged (16000g at 4°C for 20 minutes). Supernatant was removed, RNA pellets were washed in 75% (v/v) ethanol and DTT (100μM) and centrifuged (7500g at room temperature for 5 minutes). Supernatant was removed and RNA pellets dried for 10 minutes prior to reconstitution in Ultrapure DNAse/RNAse-free distilled water supplemented with DTT (1mM) and incubation at 55°C for 10 minutes. Thiol-containing bases were reduced by incubating RNA (10μg) with IAA (10mM, 50 mM NaPO4 pH 8.05 and 50% (v/v) DMSO) in a final volume of 50μL for 15 minutes at 55°C. Reaction was stopped by quenching IAA with DTT (20μM). RNA was precipitated using 3M NaOAc pH 5.2 (5μL), 100% ethanol (125μL) and GlycolBlue co-precipitant, incubated at -80°C for 30 minutes and centrifuged (16000g at 4°C for 30 minutes). Supernatant was removed and RNA pellet was washed in 75% (v/v) ethanol and centrifuged (16000g at 4°C for 10 minutes) twice, dried at room temperature for 10 minutes and reconstituted in Ultrapure DNAse/RNAse-free distilled water. RNA clean-up was performed by incubating RNA solution in 2 volumes of AMPure XP Beads, for 2 minutes at room temperature. Beads were washed in 80% (v/v) ethanol twice, dried at room temperature for 3 minutes and resuspended in Ultrapure DNAse/RNAse-free distilled water. Eluate was collected and S2 spike-in material (5%) was added to RNA. QuantSeq 3′mRNA-seq Library Prep Kit FWD for Illumina (Lexogen, Vienna, Austria) 3'mRNA-seq