title: GSM8585602: PROseq_dTAG_rep3; Homo sapiens; OTHER
library_construction_protocol: DMSO or dTAG-treated cells were permeabilized and counted. For each sample, around 1 million permeabilized cells (nuclei) were processed for Run-On reaction containing two biotin-tagged NTPs (PerkinElmer, NEL542001EA, NEL545001EA), which was carried out at 37°C for 6 minutes. The reaction was terminated by adding TRIzol LS, followed by total RNA extraction. Total RNA was denatured by adding NaOH to a final concentration of 200 mM and purified using Bio-Rad RNase-free P-30 columns according to the manufacturer's instructions. The 3′ RNA adaptor was ligated, and the biotin-labeled RNA was enriched using Streptavidin beads. On-bead 5′ hydroxyl repair and de-capping reactions were performed, followed by 5′ RNA adaptor ligation. The biotin-labeled RNA was released from the Streptavidin beads using TRIzol/chloroform extraction and ethanol precipitation. Reverse transcription was performed off-bead to generate cDNA. The resulting libraries were amplified, cleaned up using SPRI beads, and sequenced with single-end 50 bp reads on the Illumina NextSeq 2000, generating approximately 50 million reads per sample.
identifiers.namespace: GEO
identifiers.id: GSM8585602_r1