Rag1KO DN cells were obtained from thymus of 3-4 week old mice.
Extracted molecule
genomic DNA
Extraction protocol
5 million cells were fixed with 1% formaldehyde in culture medium for 10 min at room temperature followed by quenching with 0.125 M glycine for 5 min. The cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH8.0) for at least 30 min on ice. The crosslinked chromatin was sheared to an average size of 400 bp using a BiorupterTM sonicator (Diagenode). 5 µg of antibody was incubated with Dynabeads ProteinA beads (Invitrogen at 4C for 2-4 hours . The chromatin (from 5 million cells) was diluted 10-fold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA 16.7 mM Tris pH8.10, 167 mM NaCl) and added to the anitbody conugated to the Dynabeads overnight at 4C. After incubation, the immune complexes were collected by centrifugation and washed with the following buffers each for 3 min at 4C;low salt (150 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), high-salt buffer (500 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), LiCl buffer (250 mM LiCl, 50 mM Tris pH8.0, 0.5% Na deoxycholate, 1% NP40) and TE (10 mM Tris pH8.0, 1 mM EDTA). The protein-DNA complexes were eluted from the beads in 250 µl elution buffer (1% SDS, 100 mM NaHCO3) at 65C overnight followed by the addition of proteinaseK to 500 µg/ml and incubaton at 56C for 1 hour. Genomic DNA was isolated using QIAquick PCR purification column and subject to whole genome amplication (Sigma).
Label
Cy5
Label protocol
1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
Rag1KO DN cells were obtained from thymus of 3-4 week old mice.
Extracted molecule
genomic DNA
Extraction protocol
5 million cells were fixed with 1% formaldehyde in culture medium for 10 min at room temperature followed by quenching with 0.125 M glycine for 5 min. The cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH8.0) for at least 30 min on ice. The crosslinked chromatin was sheared to an average size of 400 bp using a BiorupterTM sonicator (Diagenode). 5 µg of antibody was incubated with Dynabeads ProteinA beads (Invitrogen at 4C for 2-4 hours . The chromatin (from 5 million cells) was diluted 10-fold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA 16.7 mM Tris pH8.10, 167 mM NaCl) and added to the anitbody conugated to the Dynabeads overnight at 4C. After incubation, the immune complexes were collected by centrifugation and washed with the following buffers each for 3 min at 4C;low salt (150 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), high-salt buffer (500 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), LiCl buffer (250 mM LiCl, 50 mM Tris pH8.0, 0.5% Na deoxycholate, 1% NP40) and TE (10 mM Tris pH8.0, 1 mM EDTA). The protein-DNA complexes were eluted from the beads in 250 µl elution buffer (1% SDS, 100 mM NaHCO3) at 65C overnight followed by the addition of proteinaseK to 500 µg/ml and incubaton at 56C for 1 hour. Genomic DNA was isolated using QIAquick PCR purification column and subject to whole genome amplication (Sigma).
Label
Cy3
Label protocol
1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
Hybridization protocol
The labeled ChIP DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in 45 ul of buffer containing 20% formamide, 1.2 M betaine, 0.1 ug/ul herring sperm DNA and 10 ug of human COT1 DNA (Invitrogen). Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in 42C 0.2% SDS/0.2x SSC, room temperature 0.2x SSC, and 0.05x SSC. Hybridization buffers and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html)