GSM8966098: MCC in SEM cells with MYB endogenously tagged with FKBP12(F36V), replicate 1; Homo sapiens; OTHER
Cells were fixed with 2% formaldehyde for 10 min, then quenched with glycine and PBS-washed. After permeabilized with 0.005% digitonin for 15 min, cells were snap frozen. Thawed cells were pelleted and resuspended in reduced-calcium MNase buffer (10 mM Tris-HCl pH7.5, 1 mM CaCl2), then divided into three aliquots. Cells were titrated with different micrococcal nuclease (NEB) concentrations (typically 10-30 Kunitz U in 800 µl) for 1h at 37 ˚C with shaking at 550 rpm. The reaction was stopped by addition of EGTA to 5 mM, with 200 µl removed to assess the digestion. Remaining cells were pelleted and washed with PBS/EGTA, then cell pellets were resuspended in DNA ligase buffer, supplemented with 400 µM dNTPs and 5 mM EDTA, before addition of DNA Polymerase I large (Klenow) fragment (NEB) to 100 U/µl, T4 polynucleotide kinase (NEB) to 200 U/µl and T4 DNA ligase (Thermo Scientific) to 300 U/µl. The reaction was incubated for 2h at 37 ˚C, then for 8h at 20 ˚C, at 550 rpm, then cooled to 4 ˚C overnight. The digested and ligated chromatin was decrosslinked at 65 ˚C in the presence of proteinase K, then DNA was purified using a DNeasy Blood and Tissue Kit (Qiagen). Digestion and ligation was assessed by D1000 TapeStation (Agilent). The protocol used for the generation of the 3C libraries was similar to other 3C-based methods. Briefly, cells were crosslinked with 2% formaldehyde, quenched with cold glycine, washed in phosphate buffered saline, resuspended in cold lysis buffer and snap frozen to -80. Cells were thawed on ice, pelleted and resuspended with 0.25% SDS and restriction enzyme buffer and incubated at 37 °C for 1h at 1400 rpm on a Thermomixer, followed by a further incubation of 1h after the addition of Triton X-100 (final concentration 1.67%). An overnight digestion was performed using DpnII (final concentration 500U/ml; 37 °C / 1400 rpm). The digested chromatin was ligated overnight using T4 DNA HC ligase (final concentration 10U/ml; 16 °C / 1400 rpm). The samples were de-crosslinked overnight with Proteinase K at 65 °C, followed by a 30 min incubation at 37 °C with RNase. A Phenol/Chloroform extraction was performed, followed by an ethanol precipitation and wash. Digestion efficiencies were assessed by gel electrophoresis and RT-PCR, which showed digestion efficiencies higher than 80%. The DNA contents of the DpnII 3C libraries were quantitated using a Qubit fluorometer. 2 ug DNA was adapter ligated using a strategy based on sonication combined with the NEBNext Ultra II DNA Library Prep kit. The quality of the libraries was assessed using the Agilent ScreenTape system. Biotinylated capture oligonucleotides were designed to the ends of the viewpoint fragments. 700 ng of the adapter ligated libraries was hybridized with the biotinylated capture oligonucleotides, using the Nimblegen SeqCap reagents and an adapted protocol. The quality of the resulting captured library was assessed using the Agilent ScreenTape system. The libraries were sequenced using the Illumina NextSeq (150bp paired end reads) platform.