GSM1574788: RUNX1ETO_progenitors_expansion_ChIP-Seq; Mus musculus; ChIP-Seq
For chromatin immunoprecipitation the cells were fixed with 1% formaldehyde (Thermo Scientific, 28906) for 10 minutes at room temperature. Then it was quenched with 1/10th volume of 2M glycine for 5 min followed by two washes with PBS. The PBS was completely removed and the pellet was stored at -80ºC till used. All the solutions used in ChIP (except the wash and elution buffers) contained 1x Halt protease inhibitor cocktail (Thermo Scientific 87786). Before ChIP, the cells were thawed on ice and resuspended in ice-cold Buffer A (10 mM Hepes, pH 8.0, EDTA  pH 8.0, 0.5 mM EGTA, pH 8.0, 0.25 % TritonX100). The cells were shaken for 10 minutes at room temperature and pelleted at 500g for 10 minutes. The supernatant was decanted, pellet was resuspended in ice-cold buffer B (10 mM Hepes, pH 8.0, 200 mM NaCl, 1 mM EDTA  pH 8.0, 0.5 mM EGTA, pH 8.0, 0.01 % TritonX100) and incubated at room temperature for 10 minutes. After pelleting, the cells were resuspended in ChIP buffer 1 (150 µl for 2 million cells, 25 mM Tris 1 M pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 1% TritonX-100 and 0.25 % SDS), and sonicated at for 20-25 cycles (30 seconds on and 30 seconds off).  Two volumes of ice-cold  Chip dilution buffer II (25 mM Tris pH 8.0, 150mM NaCl, 2 mM EDTA pH 8.0, 1% TritonX-100, 7.5% Glycerol)  was added to the tube and centrifuged for 10 minutes. The supernatant was collected without disturbing the pellet. About 5% of the chromatin was stored in a separate tube for input.  Antibody coated Dynabeads (Invitrogen) were prepared as per manufacturer’s instructions by incubating the beads overnight with the antibody. Before mixing with the sonicated chromatin, the antibody was removed and the beads were washed twice with PBS.  The sonicated chromatin was added to the beads and rotated at 25 rpm at 4ºC for 2-3 hours. After incubation, the beads were separated from the chromatin suspension by keeping the tubes on a magnetic stand and the supernatant was removed.  The beads were washed once with ice-cold low salt buffer (20 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 1% TritonX-100, 0.1% SDS), twice with high salt buffer (20 mM Tris pH 8.0, 500 mM NaCl, 2 mM EDTA pH 8.0, 1% TritonX-100, 0.1% SDS), twice with LiCl buffer (10 mM Tris pH 8.0, 250 mM lithium chloride, 1 mM EDTA pH 8.0, 0.5% NP40, 0.5% sodium-deoxycholate) and once with TE-NaCl buffer (TE pH 8.0 containing 50 mM sodium chloride). All the washings were done at rotating the tubes for 5-10 minutes at room temperature with cold wash solutions. The chromatin was eluted twice from the beads using elution buffer (100 mM NaHCO3, 1% SDS) and shaking at 1500 rpm for 15 minutes at room temperature. After adding 4 μl 5M sodium chloride and proteinase K, the pooled eluate was incubated at 65ºC overnight to reverse the crosslinks. The DNA was recovered by adding 1.8 volumes of Ampure beads (Beckman Coulter A63881). The beads were washed twice with 80% ethanol and the chromatin immunoprecipitated DNA was eluted from the Ampure beads in 0.1X TE. The library preparation was done using the TruSeq ChIP sample prep kit (Illumina 15034288), with minor modifications. The size of the DNA fragments excised from gel after PCR, varied from 200-700 bp. Libraries were validated by doing qPCR for positive and negative control regions and libraries with no or very low signals from negative controls were chosen for sequencing. The DNA quality assessed by running 1 μl of the library on an Agilent Technologies 2100 Bioanalyser and the library concentration was determined by library quantification kit (Kapa Biosystems, Illumina KK4835). The libraries were pooled and subjected to massively parallel DNA sequencing on an Illumina Genome Analyzer.
GEO
GSM1574788