title: GSM8208182: sh823_H2az_chipseq_IN2; Mus musculus; ChIP-Seq
library_construction_protocol: The histones of mNSCs were crosslinked to DNA with 1% formaldehyde for 10 minutes at room temperature. The fixation was stopped by 0.25M glycine. Cells were lysed using SDS CHIP Lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then performed sonication for 40 cycles at the Bioruptor sonicator (Diagenode) (30s ON and 30s OFF). After sonication, the samples were diluted by ChIP dilution buffer (0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8, 167 mM NaCl). After pre-clear, 10% of the lysates were used as input. All the left lysates were used for immunoprecipitation with antibodies and protein A/G agarose beads overnight. The samples were immunoprecipitated with V5 Tag (Thermo Fisher Scientific, Cat#A190-120A) or H2AZ antibody (Abcam, Cat# Ab4174). After immunoprecipitation, the bound DNA was washed with following buffers sequentially to remove nonspecific sequences: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20mM Tris-HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20 mM Tris-HCl pH 8, 500mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8) and TE wash buffer (10 mM Tris-HCl pH 8, 1mM EDTA). The pellet was then eluted in the fresh elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). The eluted samples were then reverse crosslinked with NaCl at 65 °C overnight for library preparation Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.