GSM5065471: WA01-Input, ChiP-Seq; Homo sapiens; ChIP-Seq
Chromatin was harvested, sonicated, and immunoprecipitated (or not immunoprecipitated in the case of input DNA control) according to manufacturer protocol for Diagenode iDeal ChIP-seq kit for Transcription Factors (Cat# C01010055). Briefly, 25 million cells per replicate were fixed with formaldehyde at room temp for 15 mins before stopping fixation with glycine. Cells were then lysed and chromatin was harvestd and sonicated with a Diagenode Biorupter Pico for 5 cycles of 30 seconds on, 30 seconds off. Sheared chromatin size was checked with agarose gel electrophoresis and shown to be ~200 bp. MEIS1 bound chromatin was immunoprecipitated with MEIS1 ChIP-grade antibody (Cat# ab19867) at 4C overnight before being washed, eluted, and quantified with high sensitivity Qubit. IPs were performed in triplicate for LV-MEIS1 condition and in duplicate for HOXB13ko-LV-MEIS1 condition. Effective pulldown of MEIS1 was validated by western blot before library construction. 5ng of ChIP'd DNA (or of input DNA) was used as starting material for library construction using the KAPA LTP Library prep kit (Roche Cat# 07961863001) following manufacturer protocol for chromatin sheared to 175 bp. Briefly, end repair was performed on fragmented DNA, followed by A-tailing and Illumina TruSeq adapter ligation. Each library was then amplified with 13 cycles of PCR. Final library quality was assessed on an Agilent TapeStation 2200. Libraries were quantified using KAPA Universal Library Quantification Kit (Roche Cat# 07960140001) before pooling libraries in equamolar concentrations. Sequencing was performed on Illumina HiSeq 4000 using single end, 50bp reads.