Illumina HiSeq 2500 sequencing; ChIP-seq of SMRT in CD8α+ DCs at 0hr and 6hr CpG stimulation
ChIP-seq of SMRT in CD8α+ DCs at 0hr and 6hr CpG stimulation
For SMRT ChIP assays, 40x106 CD8α+ cDC1 MutuDCs were seeded in 15 cm2 plates and cross-linked with 1% formaldehyde (sigma) at room temperature followed by quenching using 2.5 M 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 1x PBS at 2,000 rpm for 10 min at 4°C and the cell pellets were stored at −80°C The cells were lysed in nuclei extraction buffer (50mM HEPES-NaOH pH 7.5, 140mM NaCl, 1mM EDTA pH 8.0, 10% glycerol, 0.5% NP-40, 0.25% TritonX-100) supplemented with a protease inhibitor tablet (Roche) and phosphatase inhibitors (5mM NaF, 1mM β-glycerol phosphate and 1mM sodium orthovanadate) for 10 min at 4°C while shaking to isolate the nuclei. The isolated nuclei were then washed using protein extraction buffer (200mM NaCl, 1mM EDTA pH 8.0, 0.5mM EGTA pH 8.0, 10mM Tris-HCl pH 8.0) supplemented with a protease inhibitor tablet (Roche) and phosphatase inhibitors (5mM NaF, 1mM β-glycerol phosphate and 1mM sodium orthovanadate) at room temperature for 10 min. Washed nuclei were resuspended in chromatin extraction buffer (1mM EDTA pH 8.0, 0.5mM EGTA pH 8.0, 10mM Tris-HCl pH 8.0 and 1% TritonX-100) supplemented with protease and phosphatase inhibitor tablets (Roche) and incubated for 20 min on ice. The chromatin was fragmented using a Bioruptor (Diagenode) sonicator for 80 min using high amplitude and 30s ON & 30s OFF cycles to obtain 200-500 bp-sized fragments. A cooling unit was used to circulate the cold water during sonication to avoid decrosslinking because of overheating. The fragmented chromatin was centrifuged at 17,000xg for 10 min and then clear supernatant was collected in chilled 15ml falcon tubes. The DNA concentration of the chromatin was estimated using a NanoDrop and the sonicated chromatin was diluted with ChIP dilution buffer (1mM EDTA pH 8.0, 10mM Tris-HCl pH 8.0 and 1% TritonX-100 containing protease and phosphatase inhibitors) to get 100 µg/ml of chromatin for each IP. BSA and ssDNA (Salmon Sperm DNA) -preblocked protein-A sepharose (80 µl/IP) beads were added to the samples and incubated for 2h to remove non-specific- binding chromatin. To the supernatant, 5 µl/IP rabbit polyclonal anti-SMRT antibody (Abcam, cat no.: ab-24551) was added to immuno-precipitate the chromatin complex at 4°C overnight. After the overnight incubation, 50µl blocked beads were added to each sample and incubated for 90 min at 4°C to pull down the respective antibody-chromatin complexes. The beads were then washed four times with low salt wash buffer (20mM Tris-Cl pH 8.0, 150mM NaCl, 2mM EDTA pH 8.0, 0.1% SDS, 1% TritonX-100) followed by two washes with high salt wash buffer (20mM Tris-Cl pH 8.0, 500mM NaCl, 2mM EDTA pH 8.0, 0.1% SDS, 1% TritonX100), lithium chloride wash buffer (10mM Tris-Cl pH 8.0, 0.25 M LiCl, 1mM EDTA pH 8.0, 1% NP-40, 1% sodium deoxycholate) and tris-EDTA (TE) buffer (10mM Tris-Cl pH 8.0, 1mM EDTA pH 8.0). After removing the wash buffer completely, protein-bound chromatin complexes were eluted from beads for 30 min using elution buffer (100mM sodium bicarbonate and 1% SDS in milliQ water). The eluted chromatin was then reverse-crosslinked by incubating the eluted supernatant at 65°C overnight on a heat block after adding 8µl of 5M NaCl. The next day, DNA was purified from the reversecrosslinked chromatin by proteinase and RNase digestion followed by purification using Qiagen DNA purification columns. The purified DNA was eluted in 50µl of Qiagen elution buffer Multiplex libraries were prepared using barcoded adapters (see Table SE1 below) for each sample following an Illumina recommended protocol with slight modifications. In brief, ChIP-DNA fragments were end-repaired using an End-IT DNA end repair kit (Epicentre Technologies, Madison, WI, USA) followed by the addition of an „A‟ base and ligation of bar-coded adapters. The ligated DNA fragments were separated on a 2% agarose gel to select 200-400 bp-sized DNA fragments and DNA was subsequently isolated from gel slices using a Qiagen gel extraction kit. The gel-extracted DNA was then amplified for 17 cycles by PCR using high fidelity Phusion hot start polymerase (NEB). The concentration and quality of purified amplified DNA were estimated respectively using a Qubit dsDNA high sensitivity kit (Invitrogen) and a Bioanalyzer 2100 (Agilent)