GSM7979177: Input; Mus musculus; ChIP-Seq
The cells were washed in RPMI1640 containing cocktail of protease and phosphatase inhibitors: 100 uM PMSF, 1 mM sodium ortho-vanadate, 2mM sodium fluoride and 1.25mM beta-glycero-phosphate. Crosslinking of the chromatin was achieved by resuspending the cells in RPMI1640 at 2.106/mL density and adding 37% formaldehyde (1% final formaldehyde concentration) and incubated in 37°C water bath for 5min. The crosslinking reaction was terminated by adding glycine to a final concentration of 125mM using 2.5M stock solution. The cells were washed in PBS and stored at -80°C for at least overnight. The cells were re-suspended at a concentration of 40.106 cell/mL in sonication buffer (2% SDS, 10 mM EDTA, 50mM Tris-Cl at pH=8, and containing the cocktail of protease and phosphatase inhibitors as mentioned above) and subjected to ultrasound sonication to obtain DNA fragments of average length of 250bp. Following the sonication, the cells were diluted in a dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-Cl pH=8, 167 mM NaCl) and incubated with 5ug of an antibody per sample for 2.5h at 4°C, after with Protein A on beads was added to each sample and the incubation continued for another 2.5h at 4°C. The beads were washed with series of buffers, the low salt buffer (0.1% SDS, 1%, 2 mM EDTA, 20 mM Tris-Cl, pH=8, 150 mM NaCl), high-salt buffer (0.1% SDS, 1%, 2 mM EDTA, 20 mM Tris-Cl, pH=8, 500 mM NaCl), LiCl buffer (250 mM LiCl, 1% deoxycholate, 1% NP-40, 1mM EDTA, 10 mM Tris-Cl pH=8), TE buffer (10 mM Tris-Cl pH=8, 1mM EDTA)  and finally the elution buffer (0.5% SDS, 5 mM EDTA, 10 mM Tris-Cl, pH=8, 300 mM NaCl) to elute the protein-chromatin complexes off the beads. The samples were heated overnight at 65°C to reverse the crosslinking, followed by addition of Protease K and RNase A and subsequent overnight incubation at 55°C to destroy remaining protein and RNA. The massive parallel sequencing was performed on ABI SOLiD 5500 platform. First the sonicated ChIP DNA samples were quantified by Quant-iTTM dsDNA High-Sensitivity Assay Kit (Thermo Fisher) according to the manufacturer's protocol. Next the libraries for high throughput sequencing were prepared using Fragment Library Preparation kit for 5500 Series SOLiD Systems (Applied Biosystems) according to the manufacturer's protocol, briefly, the DNA fragments were first blunt-ended and phosphorylated at the 5'end. Next the DNA fragment size-selection was performed on Agencourt AMPure® XP beads and separated on 2% agarose gel. The fragments of 250bp bp size were selected by gel excision. Then, the dA-tail was ligated onto the size-selected DNA using A-tailing enzyme and using T4 DNA ligase the P1-T and Barcode-T adaptors and barcodes were ligated onto the DNA fragments. These adaptors/barcodes are designed to be compatible with the reverse-read sequencing on 5500 Series SOLiD™ sequencers. Prior to ligation-mediated PCR the sample was incubated at 72°C for 20 minutes in PCR mix to let the DNA diffuse out of the gel and to perform nick translation on non-ligated 3′-ends of DNA fragments. Finally, the libraries were amplified using Platinum® PCR amplification kit and the final size-distribution was checked using Agilent Technologies 2100 Bioanalyzer™.  To sequence the libraries an amplification on P1 beads was performed in an emulsion PCR (ePCR), followed by enrichment of the templated beads. Templated bead preparation was performed using the SOLiD® EZ Bead™ System (Applied Biosystems) according to the manufacturers' protocol. Emulsion was dispensed into 96-well plate and cycled for 60 cycles. After amplification emulsion was broken with butanol, beads were enriched for template positive beads, 3′-end extended and covalently attached onto sequencing slides. Physically separated samples were deposited on one sequencing slide and sequenced using standard settings on the 5500 Series SOLiDTM sequencer