GSM5830236: BulkChIPseq_H3K27ac_RANKL_rep_3; Mus musculus; ChIP-Seq
GSM5830236
Following harvesting, organoids were crosslinked with 1 % formaldehyde, and glycine was added to quench the reaction. After a washing step with 1x ice-cold PBS, cell pellets were snap frozen and stored at -80°C until being processed for ChIPmentation. A total of 250.000 cells per replicate were lysed in a buffer containing 20 mM HEPES pH 7,6, 1% SDS, 1x Protease Inhibitor Cocktails (Roche). Chromatin was sheared using the medium setting of the Biorupter Pico (Diagenode) with 6 cycles (20 seconds on/30 seconds off) to achieve fragments between 200 and 600 bp. Samples were then spun at 13.000 rpm for 5 minutes at room temperature and supernatant containing fragmented chromatin was used for ChIPmentation as described by (Schmidl et al., 2015) with the modifications published in Lindeboom et al., 2018. Briefly, before starting the immunoprecipitation, we saved 1% of the input material of either control or RANKL-treated organoids and stored it at 4° C until further processing (tagmentation). Then, chromatin of each replicate was incubated with 1 µg of antibody against H3K27ac (C15410196, Diagenode) or 1 µg of antibody against ONECUT2 (Proteintech, 21916-1-AP, AB_2848180) in dilution buffer (1% Triton X-100, 1,2 mM EDTA, 16,7 mM Tris pH 8, 167 mM NaCl, 1x Protease Inhibitor Cocktail (Roche)), rotating overnight at 4°C. Antibody-bound chromatin was purified using protein A and protein G Dynabeads (Invitrogen) followed by two washing steps with increasing concentration of NaCl and one washing step with TE buffer as described before (Lindeboom et al., 2018). Tagmentation of immunoprecipitated and input chromatin was performed with in-house generated Tn5 enzyme for 10 minutes at 37°C while shaking at 550 rpm, followed by washing steps with decreasing amounts of NaCl and soap (Lindeboom et al., 2018). Lastly, samples were de-crosslinked with 20 µg of proteinase K (Sigma) in elution buffer (0,5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris pH 8) for 1 hour at 55°C and 1.000 rpm shaking followed by an overnight incubation at 65°C and 1.000 rpm shaking. The eluted, tagmented chromatin was treated with 10 µg proteinase K (Sigma) for 1 additional hour at 55°C and purified using 2x AMPure XP beads (Beckman Coulter). The generated libraries were amplified using the KAPA HiFi Hotstart Ready Mix and Nextera Index Kit 1 (i7) and 2 (i5) primers (Illumina). Amplified libraries were purified using a 0,65x AMPure XP beads (Beckman Coulter) followed by a 1,8x AMPure XP beads (Beckman Coulter) purification. Library concentration was measured using the dsDNA Fluorescence Quantification Assays (DeNovix), and library size was determined using the BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). DNA libraries were sequenced with an Illumina NextSeq 500, and 38-bp paired-end reads were generated.