GSM5741842: helaASDMSO_chase2h_rep3; Homo sapiens; OTHER
Cells were lysed directly in TRIzol followed by isolation of total RNA. For experiments involving HF1, K562, and HeLa treated with THZ531 (including controls), the isolated total RNA was further treated with DNAse (TURBO DNA-free Kit; Invitrogen) and a spike-in cocktail of Bru-labeled and unlabeled RNA was mixed in with total RNA. Bru-labeled RNA was immunocaptured using anti-BrdU antibodies (BD Biosciences). Strand-specific libraries for the HeLa-AS experiments were prepared from a modified protocol using the TruSeq Kit (Illumina) as previously described (Paulsen et al., 2014; Paulsen et al., 2013). This protocol used single-index barcoded ligation adapters and universal primers for PCR. For the experiments in HF1, K562, and HeLa cells involving THZ531 treatment, a spike-in cocktail of Bru-labeled and unlabeled RNA (described below) was mixed in with total RNA prior to Bru-RNA IP.  Ribosomal RNA was reduced via QiaSeq FastSelect (Qiagen) prior to first strand synthesis. These strand-specific libraries were made using a universal ligation adapter and dual-index, barcoded primers for PCR. Size selection for all libraries was via agarose gel excision. Bru-seq