GSM7060134: Input_BAZ2Aend_ChIPseq; Mus musculus; ChIP-Seq
1% formaldehyde was added to cultured cells to cross-link proteins to DNA. For H3K27me3 ChIPs, isolated nuclei were then lysed and sonicated using a Bioruptor® Pico ultrasonic cell disruptor (Diagenode) to shear genomic DNA to an average fragment size of 200 bp. 20 μg of chromatin was diluted to a total volume of 500 μl with ChIP buffer (16.7 mM Tris–HCl, pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100) and pre-cleared with 10 μl packed Sepharose beads for 2 h at 4°C. Pre-cleared chromatin was incubated overnight with ChIP-grade antibody raised against H3K27me3. The next day, Dynabeads™ Protein A (Invitrogen, 10002D) were added and incubated for 4 h at 4°C. After washing, bound chromatin was eluted with the elution buffer (1% SDS, 100 mM NaHCO3). Upon Proteinase K (Thermo Scientific) digestion (50°C for 3 h) and reversion of cross-linking (65°C, overnight), DNA was purified with phenol/chloroform, ethanol precipitated and quantified by qPCR. For BAZ2A ChIPs, crosslinked chromatin has been fragmented into mono-nucleosomes through digestion with MNase. Briefly, isolated and crosslinked nuclei were MNase digested in 400µl MNase digestion buffer (0.3M Sucrose, 50mM Tris pH 7.5, 30mM KCl, 7.5mM NaCl, 4mM MgCl2, 1mM CaCl2, 0.125% NP-40, 0.25% NaDeoxycholate, supplemented with cOmplete™ Protease Inhibitor Cocktail (Roche)) with 100U MNase (Roche) at 37°C for 30 minutes. The digestion was then stopped with 5mM EDTA and the digested chromatin was solubilized in 1% SDS and three pulses of 30sec sonication using a Bioruptor® Pico ultrasonic cell disruptor (Diagenode). 200µg of pre-cleared chromatin was immunopurified with incubation of 20µl of Anti-FLAG® M2 Affinity Gel (Sigma, A2220) over night. The samples were subsequently washed, eluted and the DNA was purified as for histone ChIPs. For ChIPseq normalization with D. melanogaster spike-in chromatin, the samples were prepared and processed as above but with the addition of 200 ng of D. melanogaster S2 chromatin and 0.5 μg of H2Av antibody (Active Motif, 61686) for BAZ2A ChIP and 50 ng of D. melanogaster S2 chromatin and 0.2 μg of H2Av antibody for H3K27me3 ChIP. Quantity and quality of the isolated DNA was determined with Qubit® 4 Fluorometer (Life Technologies). The NEBNext® Ultra™ II DNA Library Prep for Illumina (New England Biolabs, E7645S and E7645L) was used following the manufacturer's protocol. Briefly, ChIP and input samples (10 ng) were end-repaired and polyadenylated before the ligation of Illumina compatible adapters. The adapters contain the index for multiplexing. The quality and quantity of the enriched libraries were validated using Qubit® 4 Fluorometer and the 4200 TapeStation System (Agilent). Sequencing was performed on an Illumina NovaSeq6000 machine with single-end 100 bp reads.