GSM7734252: dDHX15.dTAG13.totalRNA.7; Homo sapiens; RNA-Seq
Total and chromatin-associated RNA samples (totalRNA and chRNA) were collected in a paired manner. For each replicate, 5 million cells were seeded on a 15cm plate containing one 25 mm plastic coverslip (Thermanox, VWR 100500-878). At 24 hours after seeding, 6 replicates of each condition were treated with DMSO or 100 nM dTAG13 (Tocris Bioscience, Fisher Scientific 66-055) for 2 hours. Post-treatment, cells growing on coverslips were collected by transferring into PBS-containing 6-well plates for on-plate TRI reagent lysis, and Directi-zol (Zymo R2052) column purification of totalRNA. The remaining ~10 million cells on each 15cm plate were used for cell fractionation as described previously, with minor modifications. To facilitate homogenous RNA lysis, the chromatin pellet was lysed with 1ml TRI reagent and immediately mixed thoroughly by passing through a 1 mL syringe with a 23G needle, followed by Direct-zol column purification of chRNA. Six replicates of totalRNA and chRNA were collected as above. RNA-seq library preparation and sequencing were performed by MIT BioMicro Center. Briefly, for chRNA set, poly(A) depletion was achieved by taking the unbound supernatant after chRNA was incubated with the oligo-d(T) beads from the NEBNext Poly(A) mRNA magnetic isolation module (NEB, E7490L). Then, paired-end, rRNA-depleted, dual-indexed libraries of totalRNA and poly(A)-depleted chRNA were prepared using the NEBNext Ultra II Directional RNA kit (E7760L) with ribosomal RNA (rRNA) depletion module (E6310X). Indexed libraries were sequenced on the Illumina NovaSeq S2 200 platform, resulting in ~20 million paired-end 2 x 100 bp reads per replicate.