NCoR1, SMRT, Polymerase II and input ChIP-Seq
NCoR1, SMRT, Polymerase II and input ChIP-Seq
3T3-L1 cell culture and differentiation: Mouse embryonic fibroblast-adipose like cells (cell line 3T3-L1) obtained from ATCC were maintained in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) containing 10% fetal calf serum (FCS; Amimed), 1X antibiotic solution (Invitrogen) and the cultures were incubated at 37 C and 5% CO2. Cells were sub-cultured in 1:5 into new petri-plates when they were 75-80% confluent. 3T3-L1 pre-adipocytes at 2 days post-confluence were differentiated into adipocytes using differentiation inducing cocktail of 1uM Dexamethasone (Dex), 0.5mM isobutyl-methyl-xanthine (IBMX) and 167nM insulin in DMEM with 10% FCS. After two days of induction with differentiation medium, cells were washed with cell culture grade 1X phosphate buffered saline (PBS) and complete medium containing 167nM insulin was added. Two days thereafter, fresh DMEM medium containing FCS was added to the cells and at day six, cells were stained with oil red-O to estimate the extent of differentiation into mature fat cells. Cells were collected from pre-adipocytes (D0) and at five distinct time points after induction of differentiation (2h, D1, D4, and D6). The cells were washed two times with 1X PBS and cross-linked using 1% formaldehyde for 10 min at room temperature followed by quenching the reaction using 125mM glycine for 5 min. After quenching, the petri-plates were placed on ice, cells were scraped using a cell scraper and collected in falcon tubes. The cells were then washed three times using cold 1X PBS and cell pellets were stored at -80 C until further use. 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 ?-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 ?-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 de-crosslinking 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 ug/ml of chromatin for each IP. BSA and ssDNA (Salmon Sperm DNA) -preblocked protein-A sepharose (80 ul/IP) beads were added to the samples and incubated for 2h to remove non-specific- binding chromatin. To the supernatant, 5 ul/IP rabbit polyclonal anti-SMRT antibody (Abcam, cat no.: ab-24551), anti-NCoR1 (Abcam, cat no.: ab-24552), or RNA Pol-II antibody (Santa Cruz, cat no.: sc9001) was added to immuno-precipitate the chromatin complex at 4 C overnight. After the overnight incubation, 50ul 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% TritonX-100), 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, SMRT-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 8ul of 5M NaCl. The next day, DNA was purified from the reverse-crosslinked chromatin by proteinase and RNase digestion followed by purification using Qiagen DNA purification columns. The purified DNA was eluted in 50ul of Qiagen elution buffer.
Experimental Factor: TIME AFTER INDUCTION
6 day
Experimental Factor: IMMUNOPRECIPITATE
anti-SMRT
Experimental Factor: GENETIC MODIFICATION
none