title: GSM4773984: B(HT-1); Gallus gallus; OTHER
library_construction_protocol: cells were washed twice in Wash Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail) by gentle pipetting. 10 µL of concanavalin A coated magnetic beads (Bangs Laboratories) were added per sample and incubated at room temperature (RT) for 15 min. The bead-bound cells were resuspended in  Dig-wash Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail; 0.05% Digitonin) containing 2 mM EDTA and a 1:50 dilution of the appropriate primary antibody. Primary antibody incubation was performed on a rotating platform for 2 hours overnight at 4 °C. Then, the secondary antibody was diluted 1:50 in Dig-Wash buffer and cells were incubated at RT for 30 min. The pA-Tn5 adapter complex was added to the cells with gentle vortexing, and incubated at RT for 1 h. Next, cells were resuspended in Tagmentation buffer (10 mM MgCl2 in Dig-med Buffer) and incubated at 37 °C for 1 h. Then, DNA extraction was performed with proteinase K digestion and the following purification with Ampure XP beads (Beckman Counter). Cut&Tag libraries were amplified with NEBNext HiFi 2× PCR Master mix (New England Biolabs, Inc., USA). Libraries were qualified using Agilent 2100 bioanalyzer and then sequenced in a NovaSeq platform (Illumina). CUT&Tag