GSM5942524: 24h Enzalutamide treatment rep1; Homo sapiens; OTHER
Cell permeabilization, run-on reactions, and RNA isolation was carried out as previously described (PMID: 27442863) For PRO-cap, approximately 10 to 30 million cells were processed per sample. Library preparations for two biological replicates each consisting of two technical replicates per condition were processed separately. Cells were permeabilized and run-on reactions were carried out as previously described(PMID: 27442863). Following RNA isolation, two adaptor ligations using T4 RNA Ligase 1 (catalog no. M0204; NEB) and reverse transcription using SuperScript III Reverse Transcriptase (catalog no. 18080044; Invitrogen) were performed. Between adaptor ligations, cap state selection reactions were performed by treating the samples with CIP (catalog no. M0290; NEB) to reduce uncapped RNAs to 5′ hydroxyls and make them incapable of ligating to 5′ adaptor and Cap-Clip (catalog no. C-CC15011H; Cambio) to remove the 5′ cap of transcripts that had undergone guanylation and allow them to be incorporated into the library through 5′ adapter ligation. RNA washes, phenol:chloroform extractions and ethanol precipitations were carried out between reactions. All steps were performed under RNase-free conditions and following manufacturer protocols. Libraries were sequenced on an Illumina HiSeq 3000 following PCR amplification and library clean-up. PRO-cap