GSM8751019: FACT_dTAG1h; Mus musculus; OTHER
Cells were  resuspended in 1x DPBS w/o Ca2+ and Mg2+ to reach a solution of 1x106 cells/ml. The cell suspensions were then added 1% Formaldehyde and incubated at RT for 10 minutes on a rotator. The reaction was quenched using 0.25 M glycine and incubated at RT for 5 minutes. The samples were centrifuged at 1000g for 5 minutes, washed once with 1x PBS w/o Ca2+ and Mg2+ with 0.1% BSA and resuspended in 1x PBS w/o Ca2+ and Mg2+ with 0.1% BSA, reaching a solution of 4x106 cells/ml. EGS were dissolved in DMSO and 3 mM EGS were added to the cell suspension prior to incubation at RT for 40 minutes on a rotator. The reaction was quenched using 0.4 M glycine and incubated at RT for 5 minutes. The cells were centrifuged 1000g for 5 minutes and resuspended in 1x PBS w/o Ca2+ and Mg2+ reaching a concentration of 5x106 cells/ml. The crosslinked cells were aliquoted into Eppendorf tubes to generate samples either containing 5x106 or 1x106 cells. The aliquoted samples were centrifuged 2000g for 5 minutes and supernatant were carefully removed. Pelleted cells were snap frozen in liquid nitrogen and stored at -80°C until further processing. To prepare the Micro-C libraries, one vial of 5x106 cells were thawed on ice for 10 minutes and resuspended in 1 ml 1x PBS w/o Ca2+ and Mg2+ with 0.1% BSA and incubated on ice for 20 minutes. Cells were centrifuged 10000g for 5 minutes and resuspended in 0.5 ml MB1 buffer. Samples were centrifuged at 10000g, and pelleted cells were resuspended in 1 ml MB1 buffer and distributed equally in 5 new Eppendorf tubes with 200 μl/tube. With a 15-second interval, 1 µl MNase with the estimated concentration from the MNase titration was added each tube and tubes incubated at 37°C for 10 minutes. With the same order and same 15-seconds interval, 1.6 µl EGTA was added to each tube and tubes incubated at 65°C for 10 minutes. Samples were centrifuged at 10000g, and the pelleted nuclei were resuspended in 0.5 ml 1x ligase buffer pooling the fives tubes into one. Samples were centrifuged at 10000g and resuspended in 0.5 ml 1x ligase buffer. 10% of each suspension were transferred to clean Eppendorf tubes, added 150 µl Tris-HCl, 25 µl 10% SDS and 25 µl 20 ng/ml Proteinase K and incubated overnight at 65°C. The remaining samples were centrifuged at 10000g, and the pellets were resuspended in 90 ul/sample End-Chewing master mix (10x ligase buffer, H2O, 10 U/µl T4 PNK) and incubated at 37°C for 15 minutes with mixing. Following incubation, 10µl Klenow Fragment was added to each sample and samples incubated at 37°C for 15 minutes. 50 µl End-labeling master mix was then added to each sample (1 mM Biotin-dATP, 1 mM Biotin-dCTP, 10 mM dTTP+dGTP, 10x ligase buffer, 20 mg/ml BSA and H2O) and incubated at 25°C for 45 minutes with mixing. To the samples was added 9 µl 0.5 M EDTA and samples incubated at 65°C for 20 minutes, followed by centrifugation at 10000g for 5 minutes. Pelleted nuclei were washed once in 0.5 ml cold 1x ligase buffer, and then resuspended in 500 µl Ligation master mix (10x ligase buffer, 20 mg/ml BSA, 400 U/µl T4 DNA ligase and H2O) and incubated at room temperature for 2.5 hours. Nuclei were then pelleted at 10000g for 5 minutes and resuspended in 0.2 ml Exonuclease master mix (10x NEB buffer 1, 100 U/l Exonuclease III and H2O) and incubated at 37°C for 15 minutes. Samples were then added 25 µl 10% SDS and 25 µl 20 mg/ml Proteinase K and incubated overnight at 65°C. All samples were added 0.5 ml Phenol-Chloroform and centrifuged at 19800g for 5 minutes and the aqueous phase was transferred to new Eppendorf tubes. The DNA was purified using the Qiagen PCR purification kit eluting input samples in 15 µl EB and library samples in 30 µl EB. The samples were added loading buffer and run on a 1.5% agarose gel at 120 V until bands were properly separated, comparing input samples and library samples as a quality control for the ligation efficiency. For all the library samples, the 350 bp band was cut out of the gel and purified using the Qiagen gel purification kit eluting the samples in 150 µl EB.