GSM5579713: Iswi_AID_Ctrl_1_PROseq; Drosophila melanogaster; OTHER
After treatment cells were harvested and nuclei were isolated; Spike in cels (5% total human HCT116 cells) were added at the level of nuclei permeabilization. 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.