GSM9140347: CM_0h_r1; Homo sapiens; ncRNA-Seq
Approximately 4 million cells were collected from 10 cm culture dishes by rinsing the plates with cold PBS twice and resuspended in 50 µL of buffer D. Harvested cells were rapidly frozen in liquid nitrogen and stored at -80°C. PRO-seq was performed on whole nuclei or nuclear condensate fractions in the storage buffer (Buffer D) as described previously (Kwat et al, 2013), with the following modifications. Briefly, isolated fractions were incubated in the nuclear run-on reaction condition (5 mM Tris-HCl pH 8.0, 2.5 mM MgCl2, 0.5 mM DTT, 150 mM KCl, 0.5% Sarkosyl, 0.4 units / μl of RNase inhibitor) with biotin-NTPs and rNTPs supplied (18.75 μM rATP, 18.75 μM rGTP, 1.875 μM biotin-11-CTP, 1.875 μM biotin-11-UTP for uPRO; 18.75 μM rATP, 18.75 μM rGTP, 18.75 μM rUTP, 0.75 μM CTP, 7.5 μM biotin-11-CTP for pChRO) for 5 min at 37°C. Run-On RNA was extracted using TRIzol, and fragmented under 0.2 N NaOH for 15 min on ice. Fragmented RNA was neutralized, and buffer exchanged by passing through P-30 columns (Biorad). 3′ RNA adaptor (/5Phos/NNNNNNNNGAUCGUCGGACUGUAGAACUCUGAAC/3InvdT/) is ligated at 5 μM concentration for 1 hours at room temperature using T4 RNA ligase (NEB), followed by 2 consecutive streptavidin bead bindings and extractions. Extracted RNA is converted to cDNA using template switch reverse transcription with 1 μM RP1-short RT primer (GTTCAGAGTTCTACAGTCCGA), 3.75 μM RTP-Template Switch Oligo (GCCTTGGCACCCGAGAATTCCArGrGrG), 1x Template Switch Enzyme and Buffer (NEB) at 42°C for 30 min. After a SPRI bead clean-up, the cDNA is PCR amplified up to 20 cycles using primers compatible with Illumina Small RNA sequencing.