title: GSM9282520: 1,3 Platin Input Rep 1; Homo sapiens; OTHER
library_construction_protocol: Total RNA was extracted with TRIzol (Thermo Fisher, MAN0001271) according to the manufacturer's protocol. RNA were fragmented by sonication and pre-cleared buy incubation with streptaidin Dynabeads T1. RNA was re-extracted using Trizol LS(Thermo Fisher, MAN0000806). Ten percent lysate was saved as input. Pre-cleared RNA was reacted with 100 µM DBCO-PEG4-Biotin in aqueous solution for 16 h at 37 °C. Following labeling, samples were treated with DNase  at 37 °C for 30 min. RNA was purified from unreacted DBCO-PEG4-Biotin with TRIzol LS. Labeled RNA was incubated with Dynabeads T1 for 30 min at room temperature with rotation. Bead–RNA complexes were washed 12 times: 6 times with 1 ml bead wash buffer II (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl) pre-warmed to 65 °C, followed by 6 times at room temperature. After the final wash, RNA was eluted with TRIzol. Pulldown and input RNA samples were depleted of rRNA using the RiboErase kit (Roche, KK8560) following the manufacturer's protocol. The rRNA-depleted RNAs were processed for stranded cDNA library construction using the KAPA RNA HyperPrep Kit (Roche, KR1351 v4.21).
identifiers.namespace: GEO
identifiers.id: GSM9282520_r1