title: GSM7213919: Treg cells, Foxp3 gRNA, rep2; Mus musculus; Hi-C
library_construction_protocol: A single cell suspension from the thymus or the spleen was prepared, then pre-enriched using EasySep Mouse CD4+ T cell Isolation Kit (STEMCELL Technologies, Cat# 19852), and stained with fluorescent-conjugated primary antibodies on ice for 30 minutes. In situ Hi-C experiments were performed as previously described (Rao et al. Cell 2014) using restriction enzyme MboI (NEB) with minor modifications. Briefly, 0.5×106 cells per each developmental stage from DN to mature thymic Treg cells, and 1×106 splenic Tcon and Treg cells were FACS-sorted and collected for individual biological replicate. Cells were resuspended in RMPI1640 medium at a concentration of 1×106 cells per mL, cross-linked with 1% formaldehyde for 10 minutes at room temperature with rotating, and subsequently lysed and digested with 100 Units of MboI/1 million cells at 37 °C for 2 hours. The following steps including Making of DNA ends, Proximity ligation and Crosslink Reversal. For PLAC-seq experiments, Briefly, 20×106 cells per biological replicate were crosslinked in 1% formaldehyde for 10 minutes at room temperature with rotating. After DNA cutting with the restriction enzyme MboI, marking DNA ends with biotin-14-dATP, and proximity ligation of DNA ends, the resultant chromatin was sonicated to 200-600 bp using Covaris E229 sonicator for 10 min. Sonicated chromatin was pre-cleared with Protein A+G magnetic beads followed by overnight chromatin immunoprecipitations with 5 µg Foxp3 antibody. DNA Shearing and Size Selection, Biotin pull-down and Preparation for Illumine Sequencing, Final amplification and Purification were processed as previously described (Rao et al. Cell 2014). Libraries were prepared using NuGen Ovation Ultralow Library System V2 kit according to the manufacturer's instructions, and sequenced by an Illumina sequencing.