title: ChIP-seq of NCoR1 in CD8α+ DCs at 6hr of stimulation
design: ChIP-seq of NCoR1 in CD8α+ DCs at 6hr of stimulation
library_construction_protocol: The CD8α+ lines (MutuDC1940) were cultured as described in previous studies. (Fuertes Marraco et al., 2012; Pigni et al., 2018).The cells were treated with poly(I:C) and combined CpG+pIC+IFNγ for 6hr prepared for the NCoR1 ChIP experiment.  The ChIP for NCoR1 and TFs was performed according to the method  optimized previously by Raghav and colleagues (Raghav and Deplancke, 2012). For ChIP assays, 30 x 106 CD8a+ cDC1 DCs were seeded in 15 cm2 plates and prepared for ChIP before and after 6h of pIC and CpG+pIC+IFNg stimulation. The cells were cross-linked using 1% formaldehyde (Sigma) for 10 min at room temperature followed by quenching the reaction using 2.5M glycine (Sigma) for 10 min. The plates were placed on ice and the cells were scraped and collected in 50 ml conical tubes. The cells were then washed three times using cold 1 x PBS and cell pellets were stored at -80°C. At the day of ChIP experiment, the cells were thawed on ice followed by lysis in nuclei extraction buffer (50 mM HEPESNaOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 10% glycerol, 0.5% NP-40, 0.25% TritonX-100) supplemented with protease and phosphatase inhibitors (Roche) for 10min at 4°C on rocker shaker. The prepared nuclei were then washed using protein extraction buffer (200 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 10 mM Tris-HCl pH 8.0) supplemented with a protease and phosphatase inhibitors (Roche) at room temperature for 10 min. Washed nuclei were resuspended in chromatin extraction buffer (1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 10 mM Tris-HCl pH 8.0 and 1% TritonX-100) supplemented with protease and phosphatase inhibitors (Roche) and incubated for 20 min on ice for equilibration. 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. A cooling unit was used to circulate the cold water during sonication to avoid de-crosslinking because of overheating. After sonication, chromatin length was checked in agarose gel. The fragmented chromatin was centrifuged at 10,000 rpm for 5 min and then clear supernatant was collected in 15 ml conical tubes. The DNA concentration of the chromatin was estimated using a NanoDrop (Thermo) and the chromatin was diluted with ChIP dilution buffer (1 mM EDTA pH 8.0, 10 mM Tris-HCl pH 8.0 and 1% TritonX-100 containing protease and phosphatase inhibitors) to use 150 µg/ml of chromatin for each IP. BSA and ssDNA (Salmon Sperm DNA) pre-blocked protein-A sepharose (80 µl/IP) beads were added to the samples on ice and incubated for 2h to remove non-specific-binding chromatin. To the supernatant, 5 µl of rabbit polyclonal anti-NCoR1 (Abcam, cat no: ab-24552) was added to immuno-precipitate the chromatin complex at 4°C overnight on rocker shaker.After the overnight incubation, 50 µl blocked protein-A sepharose beads were added to each sample and incubated for 2.5h at 4°C to pull down the respective antibodychromatin complexes. The beads were then washed three times with low salt wash buffer (20 mM Tris-Cl pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 0.1% SDS, 1%  TritonX-100) followed by two washes with high salt wash buffer (20 mM Tris-Cl pH 8.0, 500 mM NaCl, 2 mM EDTA pH 8.0, 0.1% SDS, 1% TritonX- 100), lithium chloride wash buffer (10 mM Tris-Cl pH 8.0, 0.25 M LiCl, 1 mM EDTA pH 8.0, 1% NP-40, 1% sodium deoxycholate) and Tris-EDTA (TE) buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA pH 8.0). After removing the wash buffer completely, protein-bound chromatin complexes were eluted from beads for 30 min using elution buffer (100 mM sodium bicarbonate and 1% SDS in milliQ water). The eluted chromatin was the reverse-crosslinked by incubating the eluted supernatant at 65°C overnight on a heat block after adding 8 µl of 5 M 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). The purified DNA was eluted in 40 µl of elution buffer. ChIP-seq library were prepared using 30 µl ChIP-DNA according to ChIP-seq library preparation recommended protocol (NEB).