Small RNA-seq from HeLa cells exposed to RNA polymerase I inhibitor (BMH21) for 60 min
Adherent HeLa CLL-2 cells were maintained at 37C and 5% CO2 in high glucose Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% Penicillin/Streptomycin. Cells were either treated with 1uM BMH-21 in antibiotic free media for 60 min or 12h, where DMSO for 60 min were used as control. Following treatment media was removed, cells were washed with PBS, collected with trypsinization, and stored at -70oC. Frozen cells were homogenized on Tissue Lyser LT (Qiagen) 2 min at 30 oscillations/second with 5 mm Stainless Steel Beads (Qiagen #69989). RNA was isolated using miRNeasy Micro Kit (Qiagen #217084). Library preparation was done with NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs, Ipswich, MA) with 100 ng of input total RNA with minor customizations: adapters were diluted 1:2, amplification was done for 12 cycles, and size selection for 130 to 190 nt fragments were done on a pre-casted 6% polyacrylamide Novex TBE gel (Invitrogen, Waltham, MA). Gel breaker tubes were used for gel extraction (IST Engineering, Milpitas, CA) in the buffer provided in the NEBNext kit. Pooled libraries were sequenced on NextSeq 500 with NextSeq 500/550 High Output Kit version 2.5, 75 cycles (Illumina, San Diego, CA).