GSM7956901: COLO320, HSR, GRO-seq, rep1; Homo sapiens; OTHER
Purified nuclei were incubated with equal volume of nuclear run on master mix (10mM Tris-HCl pH 8.0, 5mM MgCl2, 1mM DTT, 300mM KCl, 0.5mM ATP, 0.5mM GTP, 0.003mM CTP (unlabeled rNTPs from Roche 11277057001), 0.5mM Bromo-UTP (Sigma B7166), 1% Na-laurylsarcosine, 1ul RNase inhibitor/100ul) and incubated for 5 minutes at 30C. COLO320 samples were then treated with DNAse for 30min, then proteinase K for 1hr, followed by RNA extraction. GBM39 cells were mixed thoroughly with Trizol LS, then chloroform was added and RNA was extracted. Purified RNA from all cell lines was hydrolyzed in 0.2N NaOH for 10min then neutralized with 500mM Tris-HCl pH 6.8. Samples were then treated with T4 PNK (90-120min) and then mRNA decapping enzyme (30min). EDTA was then added to a final concentration of 25mM and samples were heated to 75C. Samples were then added to anti-BrdU agarose beads that had been blocked in 0.1% polyvinulpyrrolidone and 1ug/ml ultrapure BSA) and incubated with rotation for one hour a room temperature. Beads were washed in a series of wash buffers and then eluted at 42C. Eluted RNA was then purified by phenochloroform extraction (COLO320) or RNA cleanup kit (GBM39). NEBNext small RNA library prep kit (New England Biolads E7330) NEBNext multiplex oligos; 14 cycles of amplification; after PCR amplification, libraries were purified with Ampure beads XP (1.8x volume of PCR reaction)