GSM3615267: Yy1-ChIP-serum-Rep1; Mus musculus; ChIP-Seq
Cells were fixed with 1% formaldehyde (Sigma) in ES-medium. Fixed cells were sonicated using a Diagenode Bioruptor UCD-300 for 4x1 minutes (30 seconds on; 30 seconds off). 50µl of chromatin (500,000 cells) were used with 0.5-1µg of H3K27ac antibody (Diagenode) and incubated overnight at 4ºC with rotation. For TF-ChiP-seq, 15-million cells were used per ChIP together with 15 μg anti-GFP antibody (ab290, Abcam) and 3 ChIP-samples were pooled to yield chromatin equal to 30-million cells per transcription factor. Antibody-chromatin complexes were incubated with protein-A/G magnetic beads, eluted, 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. De-crosslinked DNA was used for library construction using KAPA Hifi kit according to manufacturer's instruction. 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 42 bp pair end reads.