title: GSM8545089: EA19.1_ESC_dTAG_input_rep3; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were double crosslinked in DSG followed by formaldehyde. First, cells were dissociated using TrypLE (ESCs) or Accutase (NPCs) and resuspended in culture medium, and 15 million cells were pelleted at 100g for 5 minutes at RT. Supernatant was discarded and cell pellets were washed once in PBS. Cells were resuspended in 10 mL PBS, and DSG (dissolved in DMSO) was added to a final concentration of 2 mM. Cells were fixed at room temperature for 45 minutes with rotation. Cells were then pelleted at 200g for 5 minutes and washed three times with PBS (each time pelleting at 200g for 5 minutes). Cells were then resuspended in 5 mL of 10% FBS prepared in PBS. To this, 5 mL of freshly prepared 2% formaldehyde solution was added to reach a final formaldehyde concentration of 1%. 2% formaldehyde was diluted from 16% methanol-free formaldehyde in 1X PBS with 10% FBS. Resuspended cells were fixed for 10 minutes at RT with rotation. To quench, 0.525 mL of 2.5 M glycine solution in PBS was added (final concentration 0.125 M glycine), and cells were incubated for 5 minutes at RT followed by 15 minutes on ice. Cells were pelleted by centrifuging at 200g for 5 minutes at 4° C. Cell pellets were resuspended in 1 mL of ice-cold 0.125M glycine in PBS, transferred to 1.5 ml tubes, and centrifuged at 300g for 5 minutes at 4° C. Supernatant was discarded and pellets were snap frozen on dry ice and stored at -70°C. Fixed cells were thawed on ice, resuspended in 1 mL of ice-cold lysis buffer [10 mM Tris HCl pH 7.5, 10 mM NaCl, 0.2% NP-40 (IGEPAL), 0.2% TritonX-100, 1 mM EDTA, 0.5 mM EGTA, supplemented with fresh protease inhibitor] and incubated on ice for 10 minutes. ~9.75 x 106 mouse nuclei were combined with ~0.25 x 106 double crosslinked HEK nuclei as spike-in. Nuclei were pelleted at 400g for 5 minutes at 4°C, supernatant discarded and nuclear pellets resuspended in 1 mL of shearing buffer (0.5% SDS, 10 mM Tris HCl pH 7.5, supplemented with fresh 1x protease inhibitor). Chromatin was sheared using a Covaris with settings PIP 105, Duty factor 5, Cycles per burst 200, total time 23 minutes. After shearing, samples were spun at 13,000 rpm for 5 minutes at 4° C. 2.5% of the sample was removed to use as input, and the remainder of the supernatant was used for ChIP. The ChIP reaction was assembled as follows: 900 ul chromatin, 442 μL 5X ChIP incubation buffer (5% TritonX-100, 0.75 M NaCl, 5 mM EDTA, 2.5 mM EGTA, 50 mM Tris-Cl pH7.5), 40 uL Dynabeads (pre-washed in ChIP incubation buffer), 10 ug HA antibody, 22.5 uL protease inhibitor, and 835.5 uL water. Tubes were incubated overnight at 4°C on a rocker. Following overnight incubation, beads were collected using a magnetic rack and washed sequentially with different wash buffers for 5 minutes each on a rocker at 4°C. Beads were washed twice with 500 μL of cold wash buffer 1 (0.1% SDS, 0.1% Sodium deoxycholate, 1% TritonX-100, 0.15 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH 8), once with 500 μL of cold wash buffer 2 (0.1% SDS, 0.1% Sodium deoxycholate, 1% TritonX-100, 0.5 M NaCl, 1 mM EDTA, 10 mM Tris pH 8), once with 500ul of cold wash buffer 3 [0.25 M LiCl, 0.5% Sodium deoxycholate, 0.5% NP-40 (IGEPAL), 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH 8], and twice with 500 μL of cold wash buffer 4 (1 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH 8). For elution, beads were resuspended in 200 μL of elution buffer (1% SDS, 0.1M NaHCO3) and incubated on a rocker for 20 minutes at RT. Beads were then separated using a magnetic rack and the eluate (supernatant) was transferred to a fresh tube. Input samples were thawed on ice and elution buffer was added to bring the volume to 200 μL. To each sample, 2 μL of 1 mg/ml RNaseA was added and incubated at 37°C for 30 minutes. To reverse crosslinks, 8 μL of 5 M NaCL and 5 μL of 10 mg/ml Proteinase K was added and samples were incubated overnight at 65°C. DNA was eluted using MinElute kit from Qiagen (28604). Libraries were prepared using NEBNext Ultra II DNA library prep kit according to the manufacturer's protocol (E7645S). The entire ChIP sample and 50 ng of the input samples were used for library prep. Libraries were amplified for 16 cycles, followed by 0.9X SPRI bead purification (Bulldog Bio CNGS005). The eluate was run on a 2% E-gel (Invitrogen G401002) and fragments between 200-600bp were excised and eluted using MinElute gel extraction kit (Qiagen 28604). Quality and quantity of libraries were assessed using Qubit and Bioanalyzer High Sensitivity DNA kit. Libraries were sequenced on NextSeq 2000 using 65 bp single-end reads. Some libraries were sequenced in a second lane for additional reads.