Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: Purified cells were fixed with 1% formaldehyde (Sigma) at a concentration of approximately 10 million cells/ml. Fixed cell preparations were sonicated using a Diagenode Bioruptor UCD-300 for 3x10 minutes (30 seconds on; 30 seconds off). 67µl of chromatin (1 million cells) was incubated with 229µl dilution buffer, 3µl protease inhibitor cocktail and 0.5-1µg of RUNX1 antibody (Abcam: ab23980) and incubated overnight at 4ºC with rotation. Protein A/G magnetic beads were washed in dilution buffer with 0.15% SDS and 0.1% BSA, added to the chromatin/antibody mix and rotated for 60 minutes at 4°C. Beads were washed with 400µl buffer for 5 minutes at 4°C with five rounds of washes. After washing chromatin was eluted using elution buffer for 20 minutes. Supernatant was collected, 8µl 5M NaCl, 3µl proteinase K were added and samples were incubated for 4 hours at 65°C.Finally samples were purified using Qiagen; Qiaquick MinElute PCR purification Kit and eluted in 20µl EB. Illumina library preparation was done using the Kapa Hyper Prep Kit. For end repair and A-tailing double stranded DNA was incubated with end repair and A-tailing buffer and enzyme and incubated first for 30 minutes at 20°C and then for 30 minutes at 65°C. Subsequently adapters were ligated by adding 30µl ligation buffer, 10 Kapa l DNA ligase, 5µl diluted adaptor in a total volume of 110µl and incubated for 15 minutes at 15°C. Post-ligation cleanup was performed using Agencourt AMPure XP reagent and products were eluted in 20µl elution buffer. Libraries were amplified by adding 25µl 2x KAPA HiFi Hotstart ReadyMix and 5µl 10x Library Amplification Primer Mix and PCR, 10 cycles. Samples were purified using the QIAquick MinElute PCR purification kit and 300bp fragments selected using E-gel. Correct size selection was confirmed by BioAnalyzer analysis. Sequencing was performed using Illumina NextSeq 500 machines and generated 43bp paired-end reads.