title: GSM4661962: ESC WT ATRXKD batch2; Mus musculus; ATAC-seq
library_construction_protocol: ATAC-seq was performed as described with minor modification (Corces et al. 2017).  Tn5 transposase was produced in-house as described (Picelli et al. 2014) . Briefly, 100'000 cells were centrifuged at 600g for 5 min at 4˚C. The pellet were resuspended in 200ul lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% NP40, 0.1% Tween-20, 0.01% digitonin) and incubated on ice for 3min. After incubation, cells were centrifuged at 600g for 3min in the pre-chilled swinging-bucket rotor. The nuclei pellets were washed with wash buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.01% digitonin). After washing, the nuclei were pelleted at 600g for 3min, resuspended in 50ul transposase mixture with final concentration of 100nM in-house Tn5 and incubated at 37C for 30min with shaking. After transposition, the mixtures were digested with proteinase K at 63˚C for 1h, followed by phenol-chloroform purification. Illumina sequencing libraries were prepared based as described (Corces et al. 2017).  ChIP-Seq was performed as described in the associated publication. Briefly, cells were cross-linked with 1% methanol-free paraformaldehyde (PFA) in medium for 10 min at room temperature, quenched by Tris-HCl (7.8) and washed twice with ice-cold PBS. 2x10e6 cell aliquots were resuspended in 50 uL Sonication Buffer 1 and chromatin fragmented by using a Bioruptor Plus sonication device (Diagenode) for 80 minutes (30 seconds on/30 seconds off, high) at 4°C to approximately 150-300 bp. Samples were diluted further to a final volume 300 uL with Lysis Buffer. 25μl SureBeads Protein A per IP were incubated with 4 ug a-H3.3 (Millipore, 09-838) or 2.5 ug a-H3K9me3 (Abcam, ab8898). 300-450 uL (2-3 Mio cells) sonicated and cleared chromatin were added to the pre-coupled magnetic beads overnight at 4°C with rotation. Next, the beads were washed each time twice, 5 min each with 1 mL RIPA, 1 mL RIPA high, 1 mL LiCl Buffer and a quick wash with 1 mL TE buffer, then resuspended in 100 uL ChIP elution buffer containing 0.25 mg/mL Proteinase K  and eluted overnight at 63°C. The DNA was isolated using 1x SPRI beads. ChIP libraries were prepared using NEBNext Ultra II DNA library preparation kit (New England Biolabs, E7645) and amplified using NEBNext® Multiplex Oligos for Illumina (New England Biolabs, E7335) according to manufacturer's protocol. Quality  and concentration estimation of the purified library DNA was assessed using Qubit 3 (Life Technologies) and BioAnalyzer 2100 (Agilent). Each library was diluted to 4 nM and combined into a single pool before sequencing on the Illumina NextSeq500 platform (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM4661962