title: ChIP-seq of H3K27ac in Control and NCoR1 KD CD8α+ DCs at 0hr, 2hr and 6hr of TLR stimulation
design: ChIP-seq of H3K27ac in Control and NCoR1 KD CD8α+ DCs at 0hr, 2hr and 6hr of TLR stimulation
library_construction_protocol: The CD8α+ lines (mutuDC) were cultured and stable Empty (Control) and NCoR1 shRNA vector (NCoR1 KD) transduced cells were generated as described in previous studies (Fuertes Marraco et al., 2012; Pigni et al., 2018). Two  independent biological replicate of Control and NCoR1 KD cells were treated with CpG-B, poly(I:C) (pIC) and combined CpG+pIC for 2hr and 6hr. Cells were further taken for H3K27ac ChIP-seq. The ChIP experiments were performed as per the Mayer's Lab Protocol. The cells were cross-linked using 1% formaldehyde (Sigma) for 10 min at room temperature followed by quenching the reaction using 2.5 M glycine (Sigma) for 10 min. The cells were lysed in the FARHAM lysis buffer and centrifuged at 2000rpm at 4°C for 8 min. The chromatin was fragmented using a Bioruptor (Diagenode) sonicator for 30 min using high amplitude and 30s ON & 30s OFF cycles to obtain 200-500 bp size fragments. The concentration of the chromatin was estimated using a NanoDrop (Thermo) and the chromatin was diluted with a RIPA buffer prepared without protease inhibitor to make 125μg/ml of chromatin for each IP. 30ul of Dyna Magnetic beads (Anti-rabbit) were taken and added to 1ml tube for each IP. 3ul of rabbit monoclonal anti-H3K27ac antibody (Abcam, cat no: ab-177178), were added and incubated at 4°C overnight on a rocker shaker. Next day, the beads were washed six to seven times with LiCl buffer (1% NP-40, 100mM Tris HCl (pH 7.5), 500mM LiCl, 1% Sodium Deoxycholate) followed by two washes with TE buffer (10nM Tris HCl (pH 7.5), 0.1mM EDTA (pH 8)). After removing the wash buffer completely, protein-bound chromatin complexes were eluted from beads for 30 min using 200ul of elution buffers. The eluted chromatin was reverse-crosslinked by overnight incubation on the shaker using 8ul of 5M NaCl. Next day DNA was purified from the reverse cross-linked chromatin by proteinase-K and RNase digestion followed by purification using PCR purification kit (Qiagen). H3K27ac ChIP sample library preparation was performed using an NEB ChIP library preparation Kit