title: GSM8561171: KH105.2_mutCBS-Car2-prom_ESC_CTCF; Mus musculus; ChIP-Seq
library_construction_protocol: For crosslinking, ESCs with CTCF site mutation in the Car2 promoter (KH105.1 and KH105.2) were dissociated using TrypLE, resuspended in culture medium and counted. 15e6  ESCs were pelleted at 100g for 5 minutes at RT. Supernatant was discarded and cell pellets were resuspended in 5 mL of 10% FBS prepared in 1X PBS. To this, 5 mL of freshly prepared 2% formaldehyde solution was added to get final formaldehyde concentration of 1%. 2% formaldehyde was diluted from 16% methanol-free formaldehyde in 1X PBS (ThermoFisher 28908). Resuspended cells were incubated on rocker to allow for mixing for 10 minutes at RT. Quenching was performed using 0.125M Glycine prepared in 1X PBS and incubated on rocker for 5 minutes at RT. Tubes were then stored on ice for 15 minutes followed by centrifuging at 100g for 5 minutes at 4° C. Supernatant was discarded and cell pellets were washed twice with 5 mL of ice-cold 1X PBS. Cell pellets were resuspended in 1 mL of ice-cold PBS and transferred to 1.5 ml tubes, centrifuged at 100g 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 ESC nuclei were transferred to a pre-chilled 1.5 mL tube and 0.25 x 106 crosslinked HEK nuclei were added as spike-in. Nuclei were pelleted at 400g for 5 minutes at 4°C, supernatant discarded and nuclear pellets resuspended in 900 μL of shearing buffer (0.5% SDS, 10 mM Tris HCl pH 7.5, supplemented with fresh protease inhibitor). Chromatin was sheared using Covaris with settings PIP 105, Duty factor 5, Cycles per burst 200, total time 15 minutes. After shearing, samples were collected in pre-chilled 1.5 mL tubes and spun at 13,000 rpm for 5 minutes at 4° C. Supernatant was collected in a fresh tube. 5% sample was removed as Input and stored at -20°C. For immunoprecipitation (IP), 40 μl Dynabeads/ChIP were prepared by washing them once in 1 mL of 1X ChIP incubation buffer (diluted from 5X ChIP incubation buffer in 1X PBS; 5% TritonX-100, 0.75 M NaCl, 5 mM EDTA, 2.5 mM EGTA, 50 mM Tris-Cl pH7.5) and resuspended in the 40 μL of same buffer. For IP, tubes were incubated overnight on end-to-end rocker at 10 rpm, 4°C. Following overnight incubation, beads were collected using magnetic rack and washed sequentially with different wash buffers for 5 minutes each on end-to-end rocker at 10 rpm, 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). Beads were then washed once each 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) and 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]. Finally, beads were washed 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 end-to-end rocker at 10 rpm for 20 minutes, RT. Beads were then separated using magnetic rack and the eluate (supernatant) was transferred to a fresh tube. 5% 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. For reverse crosslinking, 8 μL of 5 M NaCL and 5 μL of 10 mg/ml Proteinase K was added and incubated overnight at 65°C. Following reverse crosslinking, DNA was eluted using MinElute kit from Qiagen (28604). Eluted DNA was quantified using Qubit and libraries were prepared using NEBNext Ultra II DNA library prep kit as per manufacturer's protocol (E7645S). All of ChIP samples and 4 ng of Input samples were used for library prep. Final library amplification was done for 17 cycles, followed by 0.9X SPRI bead purification (Bulldog Bio CNGS005). Eluted in 10 μL of 10 mM Tris-Cl pH7.5. Entire 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.