GSM9745842: T-47D PRO-cap biological replicate 1; Homo sapiens; OTHER
Ten million cells were processed per biological replicate. Cells were permeabilized and nuclei were isolated as described previously (PMID: 27442863), prior to downstream PRO-cap library preparation. PRO-cap libraries were prepared using a previously described protocol (PMID: 27442863) with modifications (PMID: 41040139). Briefly, permeabilized cells/isolated nuclei were subjected to nuclear run-on reactions to capture nascent RNA. Total RNA was isolated and split into two rounds of adaptor ligation (i.e. VRA3 and VRA5) using T4 RNA Ligase 1 (NEB; M0204), followed by reverse transcription using SuperScript III (Invitrogen; 18080044) with custom adapters. Between adaptor ligation steps, cap-stable selection was performed using CIP (NEB; M0290) to dephosphorylate uncapped RNAs, thereby preventing ligation to the 5′ adaptor, and Cap-Clip (CellScript; C-CC15011H) to remove 5′ caps from guanylated transcripts, enabling incorporation into the library through 5′ adaptor ligation. RNA washes, phenol-chloroform extraction, and ethanol precipitation were performed between reactions under RNase-free conditions following manufacturer protocols. Following PCR amplification using RP1 and RPI-n primers and library cleanup, sequencing was performed on an Illumina HiSeq 3000 platform.