The cells were recovered by trypsinization and used for RNA preparation.
Growth protocol
ES cells were cultured in Glasgow Minimum Essential Medium (GMEM, Sigma) supplemented with 10% KNOCKOUT Serum Replacement (Invitrogen), 10 micro-M human adrenocorticotrophic hormone (Kurabo), 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, and 2000 U/ml leukemia inhibitory factor, on gelatinized culture dishes without feeder cells.
Extracted molecule
genomic DNA
Extraction protocol
Approximately 3-5 x 10^6 cells were collected and washed twice with ice-cold PBS. The cells were suspended in 50 ul of 0.3 M sucrose containing buffer 1 (60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 0.5mM dithiothreitol, 0.5 % Protease Inhibitor Cocktail (SIGMA P8340) , 15 mM Tris-HCl pH7.5). The cells were lysed by adding 50 ul of 0.8 % NP-40 and 0.3 M sucrose containing buffer 1 and put on ice for 10 min. The lysate was layered over 0.8 ml of 1.2 M sucrose containing buffer 1. The nuclear pellets were collected by centrifuge 8.5 k rpm 10 min, and resuspended in 250 ul of micrococcal nuclease (MNase) digestion buffer (4 mM MgCl2, 1 mM CaCl2, 0.32 M sucrose, 0.5 % Protease Inhibitor Cocktail (SIGMA P8340) , 50 mM Tris-HCl pH7.5). MNase digestion was performed at 37 degrees C for 20 min with 0.8 U MNase (Takara), then stopped by addition of EDTA to final 20 mM. The chromatin extract was obtained as the supernatant of centrifugation at 13 k rpm 10 min. 60 x 2 (duplicate), 30 and 80 ul of the chromatin extracts were used for immunoprecipitation for H3K4me2, H3K9me2 and H3K27me3, respectively, and remained extract was served for the input sample (MNase digested input DNA). 60 x 2 (duplicate), 30, 80 ul of Dynabeads M-280 Sheep anti-Mouse IgG (Invitorogen), pre-equilibrated with incubation buffer (50 mM NaCl, 5 mM EDTA, 0.1 % NP-40, 0.1 mM PMSF, 20 mM Tris-HCl pH 7.5), were incubated overnight with 3, 1.5 and 4 ug of mouse monoclonal antibodies against H3K4me2 (Monoclonal antibody institute, MABI0303), H3K9me2 (Monoclonal antibody institute, MABI0307) and H3K27me3 (Abcam, ab6002), respectively, in 200 ul of incubation buffer. Beads-antibody complexes were collected and resuspend in 540 x 2 (duplicate), 270 and 720 ul, for H3K4me2, H3K9me2 and H3K27me3, respectively, of incubation buffer and incubated 4 hours with the chromatin extract. The immune complexes were sequentially washed with 75, 125 and 175 mM NaCl containing buffer A (10 mM EDTA, 0.1 % NP-40, 50 mM Tris-HCl pH 7.5). Both input and immunoprecipitated samples were treated with RNase and proteinase K, and purified with MinElute Reaction Cleanup kit (Qiagen).
Label
Cy5
Label protocol
Without amplification, ChIP DNA and input MNase digested DNA were labeled with Cy5 and Cy3, respectively, using the Genomic DNA Enzymatic Labeling Kit (Agilent) according to the manufacturer's instructions (G4481-90010 Agilent Mammalian ChIP-on-chip Protocol Version 10.11). Purification of labeled DNA was performed using the Amicon Ultra-0.5, Ultracel-30, 30kDa (Millipore). Quantity and dye incorporation of labeled DNA were checked using the NanoDrop ND-1000 Spectrophotometer (Thermo) and the 2100 Bioanalyzer (Agilent).
Channel 2
Source name
MNase digested input DNA from mouse wild-type (WT) ES cells for H3K9me2 ChIP, repalicate 2
cell line: Line J1 genotype/variation: wild-type cell type: embryonic stem (ES) cells chip antibody: none, MNase digested input DNA
Treatment protocol
The cells were recovered by trypsinization and used for chromatin IP.
Growth protocol
ES cells were cultured in Glasgow Minimum Essential Medium (GMEM, Sigma) supplemented with 10% KNOCKOUT Serum Replacement (Invitrogen), 10 micro-M human adrenocorticotrophic hormone (Kurabo), 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, and 2000 U/ml leukemia inhibitory factor, on gelatinized culture dishes without feeder cells.
Extracted molecule
genomic DNA
Extraction protocol
Approximately 3-5 x 10^6 cells were collected and washed twice with ice-cold PBS. The cells were suspended in 50 ul of 0.3 M sucrose containing buffer 1 (60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 0.5mM dithiothreitol, 0.5 % Protease Inhibitor Cocktail (SIGMA P8340) , 15 mM Tris-HCl pH7.5). The cells were lysed by adding 50 ul of 0.8 % NP-40 and 0.3 M sucrose containing buffer 1 and put on ice for 10 min. The lysate was layered over 0.8 ml of 1.2 M sucrose containing buffer 1. The nuclear pellets were collected by centrifuge 8.5 k rpm 10 min, and resuspended in 250 ul of micrococcal nuclease (MNase) digestion buffer (4 mM MgCl2, 1 mM CaCl2, 0.32 M sucrose, 0.5 % Protease Inhibitor Cocktail (SIGMA P8340) , 50 mM Tris-HCl pH7.5). MNase digestion was performed at 37 degrees C for 20 min with 0.8 U MNase (Takara), then stopped by addition of EDTA to final 20 mM. The chromatin extract was obtained as the supernatant of centrifugation at 13 k rpm 10 min. 60 x 2 (duplicate), 30 and 80 ul of the chromatin extracts were used for immunoprecipitation for H3K4me2, H3K9me2 and H3K27me3, respectively, and remained extract was served for the input sample (MNase digested input DNA). 60 x 2 (duplicate), 30, 80 ul of Dynabeads M-280 Sheep anti-Mouse IgG (Invitorogen), pre-equilibrated with incubation buffer (50 mM NaCl, 5 mM EDTA, 0.1 % NP-40, 0.1 mM PMSF, 20 mM Tris-HCl pH 7.5), were incubated overnight with 3, 1.5 and 4 ug of mouse monoclonal antibodies against H3K4me2 (Monoclonal antibody institute, MABI0303), H3K9me2 (Monoclonal antibody institute, MABI0307) and H3K27me3 (Abcam, ab6002), respectively, in 200 ul of incubation buffer. Beads-antibody complexes were collected and resuspend in 540 x 2 (duplicate), 270 and 720 ul, for H3K4me2, H3K9me2 and H3K27me3, respectively, of incubation buffer and incubated 4 hours with the chromatin extract. The immune complexes were sequentially washed with 75, 125 and 175 mM NaCl containing buffer A (10 mM EDTA, 0.1 % NP-40, 50 mM Tris-HCl pH 7.5). Both input and immunoprecipitated samples were treated with RNase and proteinase K, and purified with MinElute Reaction Cleanup kit (Qiagen).
Label
Cy3
Label protocol
Without amplification, ChIP DNA and input MNase digested DNA were labeled with Cy5 and Cy3, respectively, using the Genomic DNA Enzymatic Labeling Kit (Agilent) according to the manufacturer's instructions (G4481-90010 Agilent Mammalian ChIP-on-chip Protocol Version 10.11). Purification of labeled DNA was performed using the Amicon Ultra-0.5, Ultracel-30, 30kDa (Millipore). Quantity and dye incorporation of labeled DNA were checked using the NanoDrop ND-1000 Spectrophotometer (Thermo) and the 2100 Bioanalyzer (Agilent).
Hybridization protocol
Approximately 4 ug each of Cy3 and Cy5 labeled DNA were hybridized in Agilent hybridization chambers for 40.5-41.0 hours at 65 degrees C and 20 rpm according to the manufacturer's instructions (G4481-90010 Agilent Mammalian ChIP-on-chip Protocol Version 10.11).
Scan protocol
Slide scanning was performed using the Agilent DNA Microarray Scanner G2505C with 5 um resolution and 20-bit dynamic range.
Description
H3K9me2 ChIP-chip data from wild-type ES cells
Data processing
The scanned images were processed with Feature Extraction Software 10.5.1.1 (Agilent) using default parameters (protocol ChIP_107_Sep09 and Grid 027128_D_F_20100129) to generate background subtracted and spatially detrended Processed Signal intensities and log 10 ratios with the linear normalization. Data was processed by a) change log base from 10 to 2, b) removal of data of control probes, c) removal of data of outlier probes (gIsFeatNonUnifOL flag = 1, gIsFeatPopnfOL flag = 1, gIsPosAndSignif flag = 0, rIsFeatNonUnifOL = 1 or rIsFeatPopnfOL = 1 in any of samples).
