3CHTGTS_IGL_RAD21_V-16h_Rep2
3C-HTGTS was performed on RAD21-degron cells treated with 0.5 M NEG or dTAGV-1 for 2 hours, 6 hours or 12 hours. Briefly, 3 million cells were crosslinked with 2% (v/v) formaldehyde for 10 min at room temperature and quenched with glycine to a final concentration of 125 mM. Cells were lysed in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, and protease inhibitors (Roche, #11836153001). Nuclei were digested overnight at 37C with 350 units of either NlaIII (NEB, #R0125), followed by ligation under dilute conditions at 16C overnight. Crosslinks were reversed, and samples were treated with Proteinase K (Roche, #03115852001) and RNase A (Invitrogen, #8003089) prior to DNA precipitation. The resulting 3C libraries were sonicated using a Diagenode Bioruptor sonicato. LAM-HTGTS libraries were subsequently prepared and analyzed as described in the HTGTS-Rep-Seq to determine VH utilization frequencies.