GSM4743291: BRD4_msAuxin_rep1; Homo sapiens; OTHER
After treatment the cells were harvested and nuclei were isolated. Spike-in control (D. melanogaster S2 cell nuclei; 1% of total human HCT116 cells/nuclei) were added at the level of nuclei permeabilization step. Nuclear-run-on was performed for 3min at 37°C with biotin labeled CTPs (Perkin Elmer #NEL542001EA) followed by RNA extraction and base hydrolysis. Biotin-tagged nuclear-run-on RNA was enriched via M280 streptavidin beads (Invitrogen #112.06D) and precipitated via Phenol-Chloroform treatment. 3' RNA adapters were ligated to isolated RNA and second biotin RNA enrichment followed by RNA 5' cap modification via TAP (Biozym #187005) treatment was performed. Furthermore, 5' hydroxyl repair via PNK (NEB #M0201S) treatment and subsequent 5' adapter ligation was carried out. Afterwards, cDNA was generated from enriched RNA via reverse transcription (Super Script III Reverse Transcriptase, Invitrogen #18080-044). 10µl of the cDNA library was amplified via KAPA Amplification reaction (Roche #7959028001) on a qPCR machine (Biorad CFX Connect RealTime System). KAPA reaction: 10µl cDNA, 1µl forward primer 35µM (RP1-RP20), 1µl of reverse primer 35µM (RP1: 5'-AATGATACGGCGACCACCGAGATCTACAGTTCAGAGTTCTACAGTCCGA-3'), 25µl 2x KAPA SYBER master mix, 13µl water. PCR program: 98°C - 45 sec, 98°C - 15 sec, 60°C - 30 sec, 72°C - 30 sec, 72°C - 10 sec. Samples were removed from the qPCR machine after 12-15 cycles and cleaned with Ampure beads (Beckman #A63881) in a 1:1.4 (DNA:beads) ratio. DNA bound to the beads was eluted in 11µl of water and subjected to single-end deep sequencing.