sample type: Elute from a Smad2 chromatin immunoprecipitation cell type: R1 ES cell
Treatment protocol
Undifferentiated ES cells under normal culture conditions
Growth protocol
Murine R1 ES cells were cultured under typical feeder free ES cell conditions (DMEM supplemented with Knockout serum replacement (Invitrogen Gibco), leukemia inhibitory factor (LIF), non-essential amino acids, GlutaMax-I (Invitrogen Gibco), β-mercaptoethanol and penicillin/streptomycin.
Extracted molecule
genomic DNA
Extraction protocol
R1 ES cells (5x107 – 1x108 cells) were chemically cross-linked by addition of one tenth volume of freshly-prepared 11% formaldehyde solution to cell media for 15 min at room temperature. Cells were rinsed twice with 1xPBS, harvested using a silicon scraper, frozen in liquid nitrogen and stored at -80°C prior to use. Cells were resuspended, lysed (1% SDS, 50 mM Tris pH 8.0, 5 mM EDTA, proteinase inhibitors), and sonicated to obtain DNA fragments of about 300-500bp length on average. Samples were then centrifuged at 14,000 rpm for 10 min. The supernatant was diluted (20 mM Tris pH 8.0, 2 mM EDTA, 1% Trion X-100, 150 mM NaCl, proteinase inhibitors) and pre-absorbed by 50 ul protein A beads (Zymed) and then incubated with 10 ug antibodies (anti-Smad1/5) overnight at 4°C. The immunocomplex was collected with 100 ul protein A beads by 3 hour co-incubation and then washed sequentially with TSE I (0.1% SDS, 20 mM Tris pH 8.0, 2 mM EDTA, 1% Trion X-100, 150 mM NaCl, proteinase inhibitors), TSE II (0.1% SDS, 20 mM Tris pH 8.0, 2 mM EDTA, 1% Trion X-100, 500 mM NaCl, proteinase inhibitors), LiCl buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.25 mM LiCl, 0.1% NP-40, 1% deoxycholate sodium) and TE (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0). The bound immunocomplex was eluted with 400 ul fresh elution buffer (25 mM Tris pH 8.0, 10 mM EDTA, 0.5% SDS) by heating at 65°C with occasional vortex for 15 min and crosslinking was reversed by overnight incubation at 65°C. Whole cell extract (WCE) DNA (Input fraction reserved from the sonication step) was also treated for crosslinking reversal. Immunoprecipitated DNA and WCE DNA were then purified by treatment with RNaseA, proteinase K and multiple phenol:chloroform:isoamyl alcohol extraction.
Label
Cy5
Label protocol
DNA was labeled with CGH labeling kits (Invitrogen)
sample type: Input for Smad2 chromatin immunoprecipitation cell type: R1 ES cell
Treatment protocol
Undifferentiated ES cells under normal culture conditions
Growth protocol
Murine R1 ES cells were cultured under typical feeder free ES cell conditions (DMEM supplemented with Knockout serum replacement (Invitrogen Gibco), leukemia inhibitory factor (LIF), non-essential amino acids, GlutaMax-I (Invitrogen Gibco), β-mercaptoethanol and penicillin/streptomycin.
Extracted molecule
genomic DNA
Extraction protocol
R1 ES cells (5x107 – 1x108 cells) were chemically cross-linked by addition of one tenth volume of freshly-prepared 11% formaldehyde solution to cell media for 15 min at room temperature. Cells were rinsed twice with 1xPBS, harvested using a silicon scraper, frozen in liquid nitrogen and stored at -80°C prior to use. Cells were resuspended, lysed (1% SDS, 50 mM Tris pH 8.0, 5 mM EDTA, proteinase inhibitors), and sonicated to obtain DNA fragments of about 300-500bp length on average. Samples were then centrifuged at 14,000 rpm for 10 min. The supernatant was diluted (20 mM Tris pH 8.0, 2 mM EDTA, 1% Trion X-100, 150 mM NaCl, proteinase inhibitors) and pre-absorbed by 50 ul protein A beads (Zymed) and then incubated with 10 ug antibodies (anti-Smad1/5) overnight at 4°C. The immunocomplex was collected with 100 ul protein A beads by 3 hour co-incubation and then washed sequentially with TSE I (0.1% SDS, 20 mM Tris pH 8.0, 2 mM EDTA, 1% Trion X-100, 150 mM NaCl, proteinase inhibitors), TSE II (0.1% SDS, 20 mM Tris pH 8.0, 2 mM EDTA, 1% Trion X-100, 500 mM NaCl, proteinase inhibitors), LiCl buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.25 mM LiCl, 0.1% NP-40, 1% deoxycholate sodium) and TE (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0). The bound immunocomplex was eluted with 400 ul fresh elution buffer (25 mM Tris pH 8.0, 10 mM EDTA, 0.5% SDS) by heating at 65°C with occasional vortex for 15 min and crosslinking was reversed by overnight incubation at 65°C. Whole cell extract (WCE) DNA (Input fraction reserved from the sonication step) was also treated for crosslinking reversal. Immunoprecipitated DNA and WCE DNA were then purified by treatment with RNaseA, proteinase K and multiple phenol:chloroform:isoamyl alcohol extraction.
Label
Cy3
Label protocol
DNA was labeled with CGH labeling kits (Invitrogen)
Hybridization protocol
Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequential.
Scan protocol
Scanned on an Agilent G2565BA scanner and probe features were extracted using Feature Extraction Software (version 10.0)
Description
SMAD2 binding targets in murine R1 ES cells (1/2)
Data processing
Log ratios were calculated by limma (Bioconductor package) and underwent Lowess normalization.
