GSM9589043: mES, dTAG_DGCR8, dTAG3h, PROseq, rep2; Mus musculus; OTHER
RNA was isolated with TRIzol. Permeabilized cells were subjected to nuclear run-on reactions in the presence of biotin-labeled nucleotides. Biotinylated nascent RNA was extracted, fragmented by base hydrolysis, and enriched using streptavidin magnetic beads. Enriched RNA was ligated with a 3' RNA adaptor using T4 RNA ligase I, followed by another round of streptavidin bead enrichment. The 5' ends were then decapped with RNA 5' Pyrophosphohydrolase (RppH) and repaired with Polynucleotide Kinase (PNK) before ligation to a 5' RNA adaptor with T4 RNA ligase I. Adaptor-ligated RNA was enriched again with streptavidin beads. Reverse transcription (RT) was performed using Superscript IV (Thermo Fisher Scientific) with a custom RT primer. The reverse transcription products were proceeded to library amplification using Phusion polymerase (NEB) for 12-14 cycles. Libraries were sequenced on the Illumina NovaSeq platform in paired-end 60/50 bp cycle runs following the manufacturer's protocols.