GSM5201400: Capture C_Tn-2 (CD4+CD25-CD62L+CD44- Tn cells sorted from male B6 mice); Mus musculus; OTHER
10 million mouse primary T cells were fixed in 1% formaldehyde for 10 min at RT with gentle rotation. The reaction was quenched by 0.125 M glycine followed. Cells were then resuspended in 5 mL lysis buffer and incubated on ice for 20 min. Cells were pelleted and resuspended in 1 mL of 1x DpnII digestion buffer and transferred to a 2 mL tube for homogenization followed with centrifugation at 14,000 rpm for 5 min at 4°C. Complete homogenization was confirmed under microscope and the supernatant was removed. The remaining pellets were subjected to DpnII (NEB) digestion, T4 DNA ligation (Thermo Fisher Scientific), and de-crosslinking. The resulting 3C library DNA was precipitated, and a small portion was separated by electrophoresis with an agarose gel to assess the digestion and ligation efficiency. 5 µg of the 3C DNA library was sheared to approximately 200 bp followed by end-repair, adaptor ligation, and PCR enrichment of adaptor ligated DNA. Two rounds of captures were conducted at 47°C by using 2 µg of DNA, 5 µg mouse Cot-1 DNA (Thermo Fisher Scientific), biotinylated DNA oligonucleotide probes, 1 µl xGen Universal Blocking Oligo i7, and 1 µl xGen Universal Blocking Oligo i5 (IDT DNA Technologies). Amplicon sequencing