GSM5940184: priB_PROseq_rest_rep2; Mus musculus; OTHER
To isolate nuclei, murine and Drosphila S2 cells were resuspended in cold Buffer IA (160 mM Sucrose, 10 mM Tris-Cl pH 8, 3 mM CaCl2, 2 mM MgAc2, 0.5% NP-40, 1 mM DTT added fresh), incubated on ice for 3 min and centrifuged at 700 g for 5 min. The pellet was resuspended in nuclei resuspension buffer NRB (50 mM Tris-Cl pH 8, 40% Glycerol, 5 mM MgCl2, 0.1 mM EDTA). For each run-on, 107 nuclei of the sample and 10% Drosophila S2 nuclei were combined in a total of 100 µL NRB and incubated at 30°C for 3 min with 100 µL 2x NRO buffer including 5µl of each 1mM NTPs. Reactions were terminated with TRIzol and RNA extracted as per the manufacturer's protocol. 3' ligation was performed at 16°C overnight with adapter 5'5Phos/NNNNNNNGAUCGUCGGACUGUAGAACUCUGAAC/3InvdT-3'. RNA was reverse transcribed by SuperScript III RT with RP1 Illumina primer to generate cDNA libraries. Libraries were amplified with barcoding Illumina RPI-x primers and the universal forward primer RP1 using KAPA HiFi Real-Time PCR Library Amplification Kit. Amplified libraries were subjected to gel electrophoresis on 2.5% low melting agarose gel and amplicons from 150 to 350 bp were extracted from the gel, multiplexed and sequenced on Illumina platform NextSeq 550 SR75.