title: GSM5994373: CD4_HIVdelta6_SAHA_H3K27ac_CUTRUN_Rep2; Homo sapiens; OTHER
library_construction_protocol: 3x105 CD4 T cells per antibody condition were harvested, washed, and coupled to Concanavalin A-coated beads for 30 min at 4o C with mixing. Cells were incubated overnight in Digitonin-containing (0.01%) antibody buffer with the indicated antibodies at the following concentrations: IgG 1:50, H3K4me3 1:50, H3K27ac 1:100, H3K27me3 1:17, H3K9ac 1:17, H3K9me3 1:80, RNA pol2 1:17, RNApol2 P-S2 1:50. After incubation, cells were washed twice in cell permeabilization buffer (buffer CP, 0.01% Digitonin) and incubated with pAG-MNase for 15 min at room temperature. Cells were washed twice in buffer CP before CaCl2 was added for a 2h cutting reaction at 4oC with rotation. Stop buffer containing EDTA, RNase A, and E. coli spike-in DNA was added to halt the reaction during a 37° 10-min incubation step. Supernatants were collected and added to DNA binding buffer for subsequent DNA isolation by spin columns. Supernatant DNA yields were eluted from columns in 12μL of Tris-HCl elution buffer and quantified by Qubit High-Sensitivity dsDNA assay. CUT&RUN Next-Generation Sequencing (NGS) library preparation was performed using NEBNext Ultra II DNA library preparation kits (New England Biolabs). DNA end repair and A-tailing was performed followed by adapter ligation to NEBNext hairpin adaptors and linearization of NEBNext adaptors with USER enzyme. Adaptor ligated libraries were amplified with sample-specific barcoded dual-index primers with PCR conditions optimized for amplification of short fragments: 14 cycles 15s at 98°C and 10s at 60°C. Libraries were quantified and examined by Qubit dsDNA assay (ThermoFisher) and capillary electrophoresis (Agilent Tapestation). OTHER: CUT&RUN