GSM7140990: priB_PROseq_WT_rep1; Mus musculus; OTHER
trizol extraction PRO-seq was performed as described previously with minor modifications. To isolate nuclei, CH12 cells and Drosphila S2 cells were resuspended in cold Buffer IA (160mM sucrose, 10mM Tris-Cl pH 8, 3mM CaCl2, 2mM MgAc2, 0.5% NP-40, 1mM DTT added fresh), incubated on ice for 3 min and centrifuged at 700g for 5 min. The pellet was resuspended in nuclei resuspension buffer NRB (50mM Tris-Cl pH 8, 40% glycerol, 5mM MgCl2, 0.1mM EDTA). For each run-on, 10 million CH12 nuclei were spiked with 10% Drosophila S2 nuclei in a total of 100µL NRB and incubated at 30°C for 3 min with 100 µL 2x NRO buffer including 5µl of each 1mM NTP (biotinylated ATP and GTP, and unlabelled UTP and CTP). The following steps were performed as described62 with the following changes: (1) we used different adapters, namely, 3' RNA adapter 5Phos/NNNNNNNGAUCGUCGGACUGUAGAACUCUGAAC/3InvdT-3' and 5'RNA adapter: 5'-CCUUGGCACCCGAGAAUUCCANNNN-3'. (2) 3' and 5' ligations which were done at 16°C overnight, and (3) CapClip pyrophosphatase (Cellscript) used for 5' decapping. RNA was reverse transcribed by SuperScript III RT (Invitrogen) with RP1 Illumina primer to generate cDNA libraries. Libraries were amplified using barcoding Illumina RPI-x primers and the universal RP1 and KAPA HiFi Real-Time PCR Library Amplification Kit. Amplified libraries were subjected to electrophoresis on 2.5% low melting agarose gel and amplicons from 150-350 bp were extracted from the gel, multiplexed and sequenced on Illumina platform NextSeq 550 SR75 high. Bioinformatics analyses were performed as described62 but additionally the random 8-mer was used to exclude PCR duplicates and only deduplicated reads were aligned.