title: GSM7791059: C3H10T1-2_Parental_input_rep3; Mus musculus; ChIP-Seq
library_construction_protocol: Approximately 20 million mMSC cells per cell line were used. Crosslinking was performed in 150 mm cell culture plates using 1% formaldehyde (Sigma) at room temperature with gentle rocking for 10 minutes. The crosslinking reaction was quenched using 1.25 M Glycine with gentle rocking at room temperature for 5 minutes. Fixed cell preparations were washed with ice-cold PBS, scraped, and then washed twice more with ice-cold PBS. Crosslinked pellets were resuspended in 500 μl cell lysis buffer (5 mM pH 8.5 PIPES, 85 mM KCl, 1% (v/v) IGEPAL CA-630, 50 mM NaF, 1 mM PMSF, 1 mM phenylarsine oxide, 5 mM sodium orthovanadate, EDTA-free protease inhibitor tablet) and incubated for 30 minutes on ice. Samples were centrifuged and pellets were resuspended in 500 μl of nuclei lysis buffer (50 mM pH 8.0 Tris-HCl, 10 mM EDTA, 1% (w/v) SDS, 50 mM NaF, 1 mM PMSF, 1 mM phenylarsine oxide, 5 mM sodium orthovanadate, EDTA-free protease inhibitor tablet) and incubated for 30 minutes on ice. Sonication of lysed nuclei was performed using the BioRuptor UCS-300 at maximum intensity for 75-90 cycles (10s on, 20s breaks). Sonication efficiency to achieve fragments between 150 - 500 bp was evaluated using gel electrophoresis on a reversed-crosslinked and purified aliquot from each sample. After sonication, chromatin was diluted to reduce SDS levels to 0.1% and concentrated using Nanosep 10K OMEGA (Pall) columns. Prior to the chromatin immunoprecipitation (ChIP) reaction, 2% sonicated Drosophila S2 cell chromatin was spiked into each sample for quantification of total levels of histone marks. The ChIP reactions were performed using the Diagenode SX-8G IP-Star Compact and Diagenode automated iDeal ChIP-seq Kit for Histones. Dynabeads Protein A (Invitrogen) were washed, then incubated with specific antibodies, 1.5 million cells of sonicated cell lysate, and protease inhibitors for 10 hrs, followed by a 20 min wash cycle using the provided wash buffers (Diagenode Immunoprecipitation Buffers, iDeal ChIP-seq Kit for Histones). Reverse cross-linking was then performed using 5M NaCl at 65℃ for 4 hrs. ChIP samples were then treated with 2 μl RNase Cocktail at 65℃ for 30 min followed by 2 μl Proteinase K at 65℃ for 30 min. Samples were then purified with QIAGEN MinElute PCR purification kit (QIAGEN) as per the manufacturer's protocol. In parallel, input samples (chromatin from about 50,000 cells) were reverse crosslinked and DNA was isolated following the same protocol. ChIP-seq library preparation was performed using the Kapa Hyper Prep library preparation reagents following the manufacturer's protocol (Kapa Hyper Prep Kit, Roche 07962363001). ChIP libraries were sequenced using the Illumina HiSeq 4000 at 50 bp single reads or NovaSeq 6000 at 100 bp single reads. ATAC-seq library preparation was performed according to the Omni-ATAC protocol. 50,000 C3H10T1/2 cultured cells were resuspended in 1 ml of cold ATAC-seq resuspension buffer (RSB; 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2 in water). Cells were centrifuged at 500 rcf for 5 min in a pre-chilled (4°C) fixed-angle centrifuge. After centrifugation, supernatant was aspirated and cell pellets were then resuspended in 50 μl of ATAC-seq RSB containing 0.1% IGEPAL, 0.1% Tween-20, and 0.01% digitonin by pipetting up and down three times. This cell lysis reaction was incubated on ice for 3 min. After lysis, 1 ml of ATAC-seq RSB containing 0.1% Tween-20 (without IGEPAL and digitonin) was added, and the tubes were inverted to mix. Nuclei were then centrifuged for 10 min at 500 rcf in a pre-chilled (4°C) fixed-angle centrifuge. Supernatant was removed and nuclei were resuspended in 50 uL transposition mix (2x TD Buffer, 100 nM final transposase, 16.5 uL PBS, 0.5 uL 1% digitonin, 0.5 uL 10% Tween-20, 5 uL H2O ) (Illumina Tagment DNA Enzyme and Buffer Small Kit, 20034197). Transposition reactions were incubated at 37 °C for 30 min in a thermomixer with shaking at 1000 rpm. Reactions were cleaned up with DNA Clean and Concentrator 5 columns (Zymo Research). Illumina Nextera DNA Unique Dual Indexes Set C (Illumina, 20027215) were added and amplified (12 cycles) using NEBNext 2x MasterMix. Sequencing of the ATAC-Seq libraries was performed on the Illumina NovaSeq 6000 system using 100-bp paired-end sequencing.