Illumina HiSeq 2000 paired end sequencing; proj022_chipseq
proj022_chipseq
ESCs were grown without feeders, on tissue culture dishes coated wth PBS+1% gelatine, in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies) supplemented with 10% foetal calf serum (FCS, Seralab), 2 mM L-glutamine, 1x non-essential amino acids, 50 mM 2-mercaptoethanol, 50 ug/ml penicillin/streptomycin (Invitrogen) and LIF-conditioned medium, made in house, at a concentration equivalent to 1000 U/ml. (Reference: Nesterova, T. B., Popova, B. C., Cobb, B. S., Norton, S., Senner, C. E., Tang, Y. A., Spruce, T., Rodriguez, T. A., Sado, T., Merkenschlager, M. et al. (2008) 'Dicer regulates Xist promoter methylation in ES cells indirectly through transcriptional control of Dnmt3a, Epigenetics Chromatin 1(1): 2.) 5 x 107 cells were fixed with 2 uM Ethylene glycol bis[succinimidylsuccinate] (ThermoScientific) for 1 h and then 1% formaldehyde for 15 min at RT. The reaction was quenched with glycine (0.125M) and nuclei prepared in LB1 (50 mM HEPES-KOH, pH 7.9, 140 mM NaCl, 1 mM EDTA-NaOH pH 8.0, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100 and 1x complete EDTA-free protease inhibitors (Roche)). Nuclei were washed in LB2 (10 mM Tris-HCl, pH8.0, 200 mM NaCl, 1 mM EDTA-NaOH pH 8.0, 0.5 mM EGTA and 1x complete EDTA-free protease inhibitors) and lysed in LB3 (10 mM Tris-HCl, pH 8, 100 mM NaCl, 1 mM EDTA-NaOH pH 8.0, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine and 1x complete EDTA-free protease inhibitors). Chromatin was sheared in a BioRuptor sonicator (Diagenode) to produce DNA fragments with a length of <1 kb. Triton X-100 was added (1% final) and cell debris cleared by centrifugation before the sample was diluted ten-fold with dilution buffer (1% Triton X-100, 2 mM EDTA-NaOH pH 8.0, 150 mM NaCl, 20 mM Tris-HCl pH 8.0 and 1x complete EDTA-free protease inhibitors). Samples were pre-cleared and incubated with antibodies overnight at 4C, after removal of input aliquots. Antibodies for ChIP-seq were H3K27me3 (Diagenode pAB -069-100), H3K36me3 (Abcam AB9050), anti-Suz12 (Cell Signalling 3737S), anti-Ring1B (Atsuta et al., 2001). Antibody complexes were collected with 30 ul BSA and tRNA blocked rProtein A Sepharose beads and were washed once with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA-NaOH pH 8.0, 150 mM NaCl, 20 mM Tris-Cl pH 8.0), high salt buffer (0.1% SDS, 1% Triton-X-100, 2 mM EDTA-NaOH pH 8.0, 150 mM NaCl, 20 mM Tris-Cl pH 8.0), LiCl buffer (0.25 M LiCl, 1% NP-40, 1% Na-Deoxycholate, 1 mM EDTA-NaOH pH 8.0, 10 mM TrisHCl pH 8.0) and twice with TE buffer. Complexes were eluted from beads by incubation with 100 ul Elution Buffer (1% SDS, 0.1 M NaHCO3) first at 65 degrees C for 5 min, and subsequently for 30 min with vortexing at 25C. NaCl was added to a final concentration of 200 mM and cross-links were reversed overnight at 65C. DNA was recovered after treatment with 100 ug/ml DNase-free RNase A at 42C for 90 min and Proteinase K (0.2mg/ml) for 1 h at 45C using ChIP DNA Clean & concentration columns (Zymo) and eluted in 10 ul volume. Undiluted purified DNA concentration was quantified using the PicoGreen dsDNA Quantitation Kit (Molecular Probes) and post-sonicated to <300 bp. Around 30 ng was sent to the Oxford Genomics Centre and 5 ng was used to prepare libraries using the NEBNext DNA Library Prep Master Mix Set for Illumina.
University of Oxford
E-MTAB-2481:TKO-Ring1B-R1-P1
University of Oxford, Oxford, England, UK
E-MTAB-2481:TKO-Ring1B-R1-P1
Experimental Factor: genotype
Dnmt1/Dnmt3a/Dnmt3b triple knock out
Experimental Factor: immunoprecipitate
Ring1B