cell line: Ring1A(-/-);Ring1B(fl/fl);R26::CreERT2 cell type: ES cells treatment: OHT-treated: Ring1A/B-dKO
Treatment protocol
ES cells were treated with 1 % formaldehyde/PBS for 10 min at room temperature. Cells were washed with PBS, collected and resuspended in swelling buffer [20 mM Hepes (pH 7.8), 1.5 mM MgCl2, 10 mM KCl, 0.1 % NP-40, and 1 mM DTT] by pipetting and then kept on ice for 10 min. After Dounce homogenizing 10-20 times, the cells were centrifuged and then the pellets were resuspended in RIPA buffer [20 mM Tis-HCl (pH 8.0), 1 mM EDTA, 140 mM NaCl, 1 % Triton X-100, 0.1 % SDS, and 0.1 % deoxycholic acid] containing protease inhibitors and sonicated into fragments with an average length of 0.3-0.5 kb. After centrifugation, the supernatants were subjected to IP with specific antibodies as previously described (Orlando et al., 1997)
Growth protocol
ES cells were cultured in DMEM with 20 % fetal bovine serum, MEM nonessential amino acids (Invitrogen), sodium pyruvate (Invitrogen), L-glutamine (Invitrogen), 2-mercaptoethanol (Sigma), and ESGRO (Chemicon) either on irradiated MEF as feeder layers or directly on gelatin-coated surfaces.
Extracted molecule
genomic DNA
Extraction protocol
ES cells were subjected to ChIP assay using antibodes. Catalogue number of antibodies were Millipore 07-449 for H3K27me3, Millipore 05-678 for H2AK119u1, Abcam ab18255 for H2A and Sigma F1804 for Flag tag. Antibody against Ring1B, equivalent to MBL D139-3, was developed by our laboratory using published protocol by Atsuta et al (Hybridoma, 2001). Purified immunoprecipitated and input DNA was subjected to T7 RNA polymerase-based amplification as described previously (van Bakel et al., 2008).
Label
Cy3
Label protocol
Agilent mammalian ChIP-on-chip protocol (ver.9.0)
Channel 2
Source name
Immunoprecipitated DNA from Ring1A(-/-);Ring1B(fl/fl);R26::CreERT2 ES cells 2 days after the start of OHT treatment
ES cells were treated with 1 % formaldehyde/PBS for 10 min at room temperature. Cells were washed with PBS, collected and resuspended in swelling buffer [20 mM Hepes (pH 7.8), 1.5 mM MgCl2, 10 mM KCl, 0.1 % NP-40, and 1 mM DTT] by pipetting and then kept on ice for 10 min. After Dounce homogenizing 10-20 times, the cells were centrifuged and then the pellets were resuspended in RIPA buffer [20 mM Tis-HCl (pH 8.0), 1 mM EDTA, 140 mM NaCl, 1 % Triton X-100, 0.1 % SDS, and 0.1 % deoxycholic acid] containing protease inhibitors and sonicated into fragments with an average length of 0.3-0.5 kb. After centrifugation, the supernatants were subjected to IP with specific antibodies as previously described (Orlando et al., 1997)
Growth protocol
ES cells were cultured in DMEM with 20 % fetal bovine serum, MEM nonessential amino acids (Invitrogen), sodium pyruvate (Invitrogen), L-glutamine (Invitrogen), 2-mercaptoethanol (Sigma), and ESGRO (Chemicon) either on irradiated MEF as feeder layers or directly on gelatin-coated surfaces.
Extracted molecule
genomic DNA
Extraction protocol
ES cells were subjected to ChIP assay using antibodes. Catalogue number of antibodies were Millipore 07-449 for H3K27me3, Millipore 05-678 for H2AK119u1, Abcam ab18255 for H2A and Sigma F1804 for Flag tag. Antibody against Ring1B, equivalent to MBL D139-3, was developed by our laboratory using published protocol by Atsuta et al (Hybridoma, 2001). Purified immunoprecipitated and input DNA was subjected to T7 RNA polymerase-based amplification as described previously (van Bakel et al., 2008).
Label
Cy5
Label protocol
Agilent mammalian ChIP-on-chip protocol (ver.9.0)
Hybridization protocol
Agilent mammalian ChIP-on-chip protocol (ver.9.0)
Scan protocol
ChIP-v1_10_Apr08, Agilent DNA Microarray Scanner G2505C, Agilent Scan Control (Ver. A.8.4.1)
Description
H2A in Ring1A/B-dKO ESCs (OHT+ day2)_Promoter 1
Data processing
Scanned images were quantified with Agilent Feature Extraction software under standard conditions.
