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. The lentiviral mammalian vector pLKO.1 containing SMRT, NCoR1 and Kaiso-specific shRNAs (three shRNAs per target were used) along with control shRNA (empty pLKO.1 plasmid or GFP shRNA) were obtained from Sigma. Viral particles containing shRNA expression plasmid were generated in 293T cells using a CalPhos mammalian transfection kit (Clontech) according to . 293T cells were transfected with transfer plasmids containing SMRT or Kaiso-specific shRNAs or control shRNAs along with packaging plasmids (Pcmvr8.74 and PMd2.G) and the next day, the culture medium was refreshed and after 24h, viral particles along with medium were collected in 50 ml falcon tubes. Viral particle-containing medium was filtered with 0.45um centricon syringe filters and preserved at -80 C in small aliquots. 3T3-L1 cells at a density of 5 X 103 cells were transduced with viral particles containing a pool of three shRNA expression plasmids in 10cm petri-plates. After 72h of viral incubation, the medium was changed to a puromycin selection medium (2 ug/ml puromycin in complete DMEM medium) to select the stably transduced cells. After every two days, puromycin selection media was changed and the stably transduced cells were selected for one to two weeks before performing actual experiments. The control shRNA- transduced cells were treated similarly. 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
0 day
Experimental Factor: IMMUNOPRECIPITATE
RNA Pol-II antibody
Experimental Factor: GENETIC MODIFICATION
GFP-shRNA