GSM8490449: rTcon, rep1; Mus musculus; Hi-C
A cell suspension was made from pooled secondary lymphoid organs (spleen, pLNs and mLNs) of Foxp3GFP/LSL mice (cite PMID 15780990; 34426690) by meshing the organs through a 100-μm strainer (Corning, 07-201-432) with a syringe plunger. CD4+ T cells were enriched with the Dynabeads Flowcomp Mouse CD4 Kit (Thermo Fisher, 11461D) according to the manufacturer's instructions, stained with antibodies, washed extensively, resuspended in isolation buffer (PBS with 2% FBS, 10 mM HEPES buffer, 1% l-glutamine and 2 mM EDTA), and sorted on a FACSAria (BD) instrument. Treg cells were sorted as TCRβ+CD4+CD8−NK1.1−Foxp3-GFP+Thy-1.1−, and naïve conventional CD4+ T cells as TCRβ+CD4+CD8−NK1.1−Foxp3-GFP−Thy-1.1−CD44loCD62Lhi. Wannabe Treg cells were sorted from Foxp3GFP/LSL mice as follows: naïve wannabe Treg cells: TCRβ+CD4+CD8−NK1.1−Foxp3-GFP−Thy-1.1+ CD44loCD62Lhi; activated wannabe Treg cells: TCRβ+CD4+CD8−NK1.1−Foxp3-GFP−Thy-1.1+CD44hiCD62L−; Treg cells and conventional CD4+ T cells were sorted from Foxp3DTR mice as follows: naïve Treg cells: TCRβ+CD4+CD8−NK1.1−Foxp3-DTR+ CD44loCD62Lhi; activated Treg cells: TCRβ+CD4+CD8−NK1.1−Foxp3-DTR+ CD44hiCD62L−; naïve conventional CD4+ T cells: TCRβ+CD4+CD8−NK1.1−Foxp3-DTR−CD44loCD62Lhi; activated conventional CD4+ T cells: TCRβ+CD4+CD8−NK1.1−Foxp3-DTR− CD44hiCD62L−. 100,000 cells were sorted for each population per replicate. Hi-C for Tcon and Treg cells: Hi-C was performed as previously described (Viny et al., 2019). Briefly, sorted T cell populations (approximately 1x105) were cross-linked in 1% formaldehyde for 10 minutes and quenched in 125mM glycine. Cross-linked cells were lysed and chromatin was digested using the Arima HiC+ kit (Arima Genomics, San Diego, CA) using protocol adaptations for low cell input. Digested and reverse crosslinked DNA was eluted in 100uL and fragmented to 350 bps using a Covaris LE220Rsc sonicator (Covaris, Woburn, MA). Sheared genomic material was enriched for biotinylated DNA using streptavidin beads followed by library preparation using Arima protocol modifications for Accel-NGS 2S DNA plus library kit (IDT, Coralville, IA). After end repair and ligation, libraries were quantified using the KAPA library quantification kit (Roche, Indianapolis, IN) and PCR amplified for the number of cycles required to generate >4nM per library. Hi-C libraries were sequenced on an Illumina NovaSeq and raw sequencing data in the FASTQ format were obtained.