GSM9104778: HiC_WT; Mus musculus; Hi-C
Hi-C libraries were prepared using a modified three-enzyme Hi-C (3e Hi-C) protocol (Ren, G. el al. Mol Cell 2017). Wild-type (WT) and CD8CreSatb1fl/fl (KO) mice were infected with LCMV Clone 13 and sacrificed at day 21 post-infection. Antigen-specific CD8⁺ T cells were sorted, and 300,000 to 500,000 cells per sample were collected for Hi-C. Cells were crosslinked with 1% formaldehyde for 10 minutes at 25 °C and quenched with 2.5 M glycine. Cells were crosslinked with 1% formaldehyde for 10 minutes at 25 °C and quenched with 2.5 M glycine. Fixed cells were lysed in 10 mL of ice-cold lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 0.2% NP-40) supplemented with protease inhibitors and incubated at 4 °C for 1 hour. Nuclei were pelleted and permeabilized in 0.1% SDS in CutSmart buffer (NEB) at 65 °C for 10 minutes, followed by quenching with Triton X-100 to a final concentration of 1%. Chromatin was digested with the restriction enzymes FatI, CviQI, and BfaI (New England Biolabs) at 37 °C for 20 minutes. Digestion was stopped by two washes with buffer containing 10 mM NaCl, 1 mM EDTA, and 0.1% Triton X-100. DNA ends were filled in using Klenow enzyme in the presence of dCTP, dGTP, dTTP, and biotin-14-dATP to label ligation junctions. Proximity ligation was performed using T4 DNA ligase under dilute conditions to promote intranuclear ligation. After reverse crosslinking, DNA was sheared to 200–700 bp fragments using a Diagenode Bioruptor. Fragmented DNA was end-repaired, and biotinylated ligation products were purified using Dynabeads MyOne Streptavidin C1 beads (Invitrogen). Sequencing libraries were constructed by ligating Illumina-compatible paired-end adapters and performing PCR amplification. Size selection (200–700 bp) was performed using E-gel electrophoresis, and libraries were sequenced on an Illumina NovaSeq 6000 platform with 50 bp paired-end reads.