GSM7061501: K562 cells, SF3B1 K700E, 4 days, replicate 2 [GROseq_K562_SF3B1K700E_Replicate2]; Homo sapiens; OTHER
Nuclear run-on RNA (NRO-RNA) was extracted with TRIzol LS reagent following the manufacturer's instructions and ethanol precipitated. NRO-RNA was resuspended in water and concentration was determined. RNA was then chemically fragmented by mixing with RNA fragmentation buffer. Fragmented RNA was incubated in Biotinylation Solution. After ethanol precipitation, the biotinylated RNA was resuspended in water and biotinylated-RNA was separated with M280 Streptavidin Dynabeads. 100 ul/sample of beads were washed twice with 2 volumes of freshly prepared wash buffer and resuspended in 1 volume of wash buffer and added to the biotinylated-RNA. After 15 min in rotation at 4°C, beads were washed three times with wash buffer pre-warmed at 65°C and three times with a room temperature wash buffer. 4-S-UTP containing RNA was eluted and purified with RNA Clean and Purification kit with in-column DNaseq reaction to eliminate traces of genomic DNA. The pulled down material and input DNA were then sonicated, size-selected, and ligated to Illumina barcoded adaptors, using TruSeq ChIP Sample Preparation Kit or ThruPLEX® DNA-seq Kit