title: GSM7308250: ILC2, H3K4me3, rep1; Mus musculus; ChIP-Seq
library_construction_protocol: Lineage negative, ST2+ cells from the peritoneum and mesenteric lymph nodes were isolated and expanded in culture for 6 days in the presence of recombinant IL-7 and IL-33 as previously described (Bruce et al, J Clin Invest, 2017 and Bruce et al, Blood Advances, 2022). 2.5 - 5x106 cells were pelleted prior to fixation with and fixed with a 1% formaldehyde using the ChIP-IT High Sensitivity Kit (Active Motif). After quenching and washing, pellets were frozen at −80°C. Upon thawing, cells were sheared using a chilled dounce homogenizer using with the ChIP-IT High Sensitivity Kit (Active Motif) and lysed cells were sonicated with nanodroplets for 60 seconds with the Covaris Le220 prior to clarified by centrifuging at full speed for 15 min. Input DNA was prepared with RNase A and Proteinase K prior to clean up and concentration with the Zymo Chip DNA Clean and Concentrate kit. 10-30 ug of ILC chromatin were treated with an anti-H3K4me3 antibody (Cell Signaling Technologies, rabbit mAb 9751, clone C42D8) + blocker mix along with a protease inhibitor cocktail prior to overnight end-to-end rotation at 4°C. 30 μL of Protein G Dynabeads were added to each 240 μL immunoprecipitation reaction, and reactions were incubated for 3 h. Samples were passed through a ChIP Filtration Column and then washed 5x with Wash Buffer AM1. After removal of residual wash buffer, samples were eluted in pre-warmed elution buffer AM4. De-crosslinking was carried out for 2.5h with Proteinase K prior to DNA extraction with the ChIP DNA Clean & Concentrator (Zymo Research) protocol. Chromatin immunoprecipitation quantitative real-time PCR (ChIP-qPCR) was performed using the QuantStudio 6K from Applied Biosystems.