GSM5211927: In_n2_SUZ12D93_D0; Mus musculus; ChIP-Seq
ChIP was performed as described, with minor modifications (Kanhere et al., 2012). Briefly, cells were trypsinised, resuspended in PBS and then crosslinked by adding 1/10 of cross-linking solution (11% formaldehyde, 0.1 M NaCl, 1mM EDTA pH 8, 0.5 mM EGTA pH 8, 50 mM HEPES pH 8) for 15 min in suspension. Formaldehyde was quenched by adding glycine to a final concentration of 1.25 mM. Cells were then washed twice with ice-cold PBS, centrifuging at 290G at 4 C for 10 min. After last wash, cells were flash-frozen and stored at -80 C. To carry out ChIP, cells were thawed in 5 ml per 3x107 lysis buffer 1 (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 10 % glycerol, 0.5 % IGEPAL CA-630, 0.25 % Triton X-100). Then cells were incubated at 4°C for 10 min with rocking. Cells were recovered by centrifuging at 290 C for 10 min at 4 C. Supernatant was discarded, and nuclei were resuspended in the same volume of lysis buffer 2 (10 mM Tris pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, supplemented with 0.1 DTT and complete protease inhibitor) and incubated at 4°C for 10 min with rocking. Nuclei were pelleted again at 290 G for 10 min at 4°C, and resuspended in 100 ml of lysis buffer 3 (10mM Tris pH 8, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauryl sarcosine, 0.2 % SDS, supplemented with 0.1 DTT and complete protease inhibitor) per 5x106 cells. Cells were incubated for 30 min on ice, to allow lysis. Afterwards, lysates were sonicated for 15 cycles of 30s on and 30s off, using a Diagenode Bioruptor Pico. For EB processing, the same protocol was followed, except using the resulting EB formed by 2 plates per timepoint.  DNA was then quantified using Qubit dsDNA HS Assay kit (Thermofisher) and ChIP was performed using 45 mg of mouse chromatin, with 5 mg of human chromatin extracted from human immortalised fibroblasts (hTERT) cells stably expressing FLAG-tagged SUZ12 for factor, or 5 ug of Drosophila chromatin for histone modifications were added as calibration control for downstream analysis (Furzova et al 2019). The resulting mix of mouse and human chromatin was topped up to 900ul per IP. Then, 100 ml of Triton X-100 10% solution was added, and insoluble material was span down at 17,000 G for 20 mins at 4oC. 2% of the lysate was stored at -20oC to be used as input and whole cell extract and the remaining lysate was incubated overnight at 4oC with 50 ml Protein G Dynabeads preincubated for at least 4 hr with 2.5 mg of FLAG antibody (Sigma, A8592), H3K27me3 (Abcam, ) or H4 pan acetyl (Thermofisher, PA5-40083). Beads were washed and bound complexes were incubated in elution buffer () to reverse crosslinks by heating at 65oC for 1 h. IP and input DNA were then treated with 0.5 mg/ml RNAseA and  mg/ml of proteinase K (Ambion, ), and immunoprecipitated DNA was purified using Kapa Pure beads (Roche, KK8000) using manufacturers recommendations. DNA was resuspended in 60 ml of 10 mM Tris pH 8 and quantified using ds DNA HS Qubit kit (Thermofisher, Q32851) . DNA libraries were generated using NEBNext Ultra II DNA library prep (NEB) using 1.2 ng of DNA and 9 cycles of PCR. Quality and distribution of all libraries was assessed using 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent, 5067-4626) followed by qPCR quantification with Kapa Library Quant Kit (Roche, KK4824).  Sequencing was performed as 75 bp single end reads on Illumina NEXTseq 550 platform for FLAG immunoprecipitation and 138 bp single single end reads on  Novaseq plataform for histones modifications immunoprecipitation, using biological duplicates.