AGM cells were crosslinked in 0.5% formaldehyde for 10 min at room temperature followed by quenching with 0.125 M glycine for 5 min. The cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH8.0) for 30 min on ice. The crosslinked chromatin was sheared to an average size of 1000 bp by ten 30-second pulses using a Dr. Hielscher sonicator (GmbH). The lysate was then centrifuged to remove cell debris, and the chromatin was visualized in an agarose gel. The chromatin was diluted 10-fold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA 16.7 mM Tris pH8.10, 167 mM NaCl) and pre-cleared with 25 µl protein A sepharose beads (pre-absorbed with salmon sperm DNA and BSA) for 2 h at 4C. The chromatin supernatant was then incubated overnight with 2 µg of the different antibodies at 4C. After incubation with protein A sepharose beads (50 µl) for 2h at 4C, the immune complexes were collected by centrifugation and washed with the following buffers each for 10 min at 4C; RIPA buffer (150 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), high-salt buffer (500 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS, 1% NP40) LiCl buffer (250 mM LiCl, 50 mM Tris pH8.0, 0.5% Na deoxycholate, 1% NP40) and 2x TE (20 mM Tris pH8.0, 2 mM EDTA). The protein-DNA complexes were eluted from the beads in 500 µl elution buffer (1% SDS, 100 mM NaHCO3) for 1 h at RT. After centrifugation the soluble chromatin was incubated overnight at 65C, followed by a 2 hour treatment with proteinase K to 500 µg/ml at 55C. Genomic DNA was isolated from the precipitated material as well as from the sheared chromatin input (1/100 of the material used for ChIP) by phenol extraction and ethanol precipitation.
Label
Cy3
Label 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
AGM cells were crosslinked in 0.5% formaldehyde for 10 min at room temperature followed by quenching with 0.125 M glycine for 5 min. The cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH8.0) for 30 min on ice. The crosslinked chromatin was sheared to an average size of 1000 bp by ten 30-second pulses using a Dr. Hielscher sonicator (GmbH). The lysate was then centrifuged to remove cell debris, and the chromatin was visualized in an agarose gel. The chromatin was diluted 10-fold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA 16.7 mM Tris pH8.10, 167 mM NaCl) and pre-cleared with 25 µl protein A sepharose beads (pre-absorbed with salmon sperm DNA and BSA) for 2 h at 4C. The chromatin supernatant was then incubated overnight with 2 µg of the different antibodies at 4C. After incubation with protein A sepharose beads (50 µl) for 2h at 4C, the immune complexes were collected by centrifugation and washed with the following buffers each for 10 min at 4C; RIPA buffer (150 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), high-salt buffer (500 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS, 1% NP40) LiCl buffer (250 mM LiCl, 50 mM Tris pH8.0, 0.5% Na deoxycholate, 1% NP40) and 2x TE (20 mM Tris pH8.0, 2 mM EDTA). The protein-DNA complexes were eluted from the beads in 500 µl elution buffer (1% SDS, 100 mM NaHCO3) for 1 h at RT. After centrifugation the soluble chromatin was incubated overnight at 65C, followed by a 2 hour treatment with proteinase K to 500 µg/ml at 55C. Genomic DNA was isolated from the precipitated material as well as from the sheared chromatin input (1/100 of the material used for ChIP) by phenol extraction and ethanol precipitation.
Label
Cy5
Label 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
Hybridization protocol
Scanned on an Agilent G2565AA scanner.
Scan protocol
Images were quantified using Agilent Feature Extraction Software (version A.8.5.1.1).
Data processing
Agilent Feature Extraction Software (v 8.5.1.1) was used for background subtraction and LOWESS normalization. Probes are are divided in to two different chips; chip-1 and chip-2.
