The C2C12 murine myoblast cell line (obtained from ATCC)
Growth protocol
C2C12 cells were grown in DMEM supplemented with 10% fetal bovine serum. Differentiated cells were obtained by culturing confluent C2C12 cells in DMEM supplemented with 2% horse serum. Differentiated cells were separated from undifferentiated cells using diluted trypsin.
Extracted molecule
genomic DNA
Extraction protocol
Differentiated C2C12 cells were cross-linked by addition of formaldehyde (to 1% final concentration) to separated differentiated myotubes. Cross-linking was allowed to proceed at room temperature for 10 min and was terminated with glycine (final concentration 0.125 m). Cells were washed with PBS. Cells were collected by centrifugation and were rocked in buffer containing 50 mm HEPES (pH 7.5), 140 mm NaCl, 1 mm EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, and protease inhibitors (1 mm AEBSF, 1 mm benzamidine, 50 μg/ml TLCK, 50 μg/ml TPCK, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A), at 4°C for 10 min. Cells were collected by centrifugation at 4K rpm for 10 min, and the resulting pellet was resuspended in 200 mm NaCl, 1 mm EDTA, 0.5 mm EGTA, 10 mm Tris (pH 8), and protease inhibitors and incubated 10 min at room temperature. Nuclei were collected by centrifugation at 4K rpm for 10 min, resuspended in sonication buffer (1 mm EDTA, 0.5 mm EGTA, 10 mm Tris at pH 8, and protease inhibitors), and sonicated on ice to an average length of 350 bp
Label
Cy3
Label protocol
input samples were amplified by LMPCR and 1 microgram of LM-PCR product was labeled with Cy3 using Invitrogen CHG kit (18095-011). labeling reaction were cleaned up by invitrogen bind and elute CGH columns (18095-011).
Channel 2
Source name
Elute from a mouse Sin3a chromatin immunoprecipitation
The C2C12 murine myoblast cell line (obtained from ATCC)
Growth protocol
C2C12 cells were grown in DMEM supplemented with 10% fetal bovine serum. Differentiated cells were obtained by culturing confluent C2C12 cells in DMEM supplemented with 2% horse serum. Differentiated cells were separated from undifferentiated cells using diluted trypsin.
Extracted molecule
genomic DNA
Extraction protocol
Differentiated C2C12 cells were cross-linked by addition of formaldehyde (to 1% final concentration) to separated differentiated myotubes. Cross-linking was allowed to proceed at room temperature for 10 min and was terminated with glycine (final concentration 0.125 m). Cells were washed with PBS. Cells were collected by centrifugation and were rocked in buffer containing 50 mm HEPES (pH 7.5), 140 mm NaCl, 1 mm EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, and protease inhibitors (1 mm AEBSF, 1 mm benzamidine, 50 μg/ml TLCK, 50 μg/ml TPCK, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A), at 4°C for 10 min. Cells were collected by centrifugation at 4K rpm for 10 min, and the resulting pellet was resuspended in 200 mm NaCl, 1 mm EDTA, 0.5 mm EGTA, 10 mm Tris (pH 8), and protease inhibitors and incubated 10 min at room temperature. Nuclei were collected by centrifugation at 4K rpm for 10 min, resuspended in sonication buffer (1 mm EDTA, 0.5 mm EGTA, 10 mm Tris at pH 8, and protease inhibitors), and sonicated on ice to an average length of 350 bp
Label
Cy5
Label protocol
IP samples were amplified by LMPCR and 1 microgram of LM-PCR product was labeled with Cy5 using Invitrogen CHG kit (18095-011). labeling reaction were cleaned up by invitrogen bind and elute CGH columns (18095-011).
Hybridization protocol
Hybridization onto the mouse promoter array, which includes 244K features distributed onto two slides (Agilent –G4490), washing and scanning were carried out according to the manufacturers instructions.
Scan protocol
According to Agilent Scanning Software
Description
Sin3a genome-wide binding profiles in Differentiated C2C12 cells by chip on chip
Data processing
Log ratios were calculated by the Agilent Feature Extraction software and automatically underwent Lowess normalization.
