MEFs were grown in 25 ml of culture medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 10–4 M β-mercaptoethanol, nonessential amino acids, L-glutamine, and antibiotics at standard concentrations). Confluent 15-cm plates provided about 15 to 35 million cells. Formaldehyde (1% final concentration) was added directly into the medium, and the dishes were incubated at room temperature for exactly 2 min (for native ChIP [N-ChIP] assay) or 10 min (for cross-linking and ChIP [X-ChIP]) on an orbiting platform. Cross-linking was stopped with a 0.125 M final concentration of glycine solution. Cells were washed twice with 20 ml of ice-cold phosphate-buffered saline and scraped off the plate with a rubber policeman; cells were then pelleted by centrifugation for 3 min at 250 x g and resuspended in 750 µl of ChIP lysis buffer (1% sodium dodecyl sulfate, 10 mM EDTA, and 50 mM Tris-HCl, pH 8.1) containing protease inhibitors (Complete protease inhibitor cocktail tablets, catalog number 1697498; Roche Applied Science). Sonication was carried out on ice in 1.7-ml Eppendorff tubes using a Branson Sonifer with a micro tip three or four times (N-ChIP or X-ChIP, respectively) for 10 s at 40% output. The sheared chromatin was centrifuged at 13,000 rpm for 15 min at 4°C. The supernatant was transferred to a fresh tube. An aliquot of the chromatin preparation was reverse cross-linked, proteinase K digested, phenol-chloroform extracted, precipitated, and resuspended in Tris-EDTA buffer. DNA concentration was determined using a UV spectrophotometer. One microgram of DNA was resolved on a 1.5% agarose gel to check sonication efficiency. Fragment sizes ranged from 0.2 to 1.5 kb, with a peak at 0.8 kb. Chromatin preparations were diluted to 0.4 µg/µl with ChIP lysis buffer containing protease inhibitors and were snap-frozen in liquid nitrogen in small aliquots and stored at –80 °C. 10 µl (4 µg) of chromatin and 4 µg of specific antibody was used for each immunoprecipitation. The immunoprecipitated chromatin was eluted from 40 µl of protein A/G agarose beads and reverse cross-linked, and DNA was isolated with a QIAquick kit, (Qiagen, Germany) and eluted in 100 µl of Elution Buffer. RNA was isolated from MatDup.dist7 and PatDup.dist7 MEFs using RNA-Bee according to manufacturer’s instructions (Tel-Test). The pellet was dissolved in DEPC water containing RNasin (Promega) and 10 mM DTT. Contaminating DNA was removed with the DNA-free Kit (Ambion). ds cDNA was prepared from 10 g of MatDup.dist7 and PatDup.dist7 MEF total RNA according to NimbleGen Arrays User’s Guide (http://www.nimblegen.com/products/lit/exp_userguide_v3p2.pdf) using SuperScript® Double-Stranded cDNA Synthesis Kit (Invitrogen). The ds cDNA was purified using QIAquick PCR Purification Kit (Qiagen) for hybridization. For MIRA The methylated fraction of sonicated genomic DNA was captured using recombinant MBD3L1 and MBD2b proteins.
parental duplication of distal chr7: PatDup.dist7 epigenetic feature: none, input DNA cell type: MEF
Growth protocol
Standard
Extracted molecule
genomic DNA
Extraction protocol
MEFs were grown in 25 ml of culture medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 10–4 M β-mercaptoethanol, nonessential amino acids, L-glutamine, and antibiotics at standard concentrations). Confluent 15-cm plates provided about 15 to 35 million cells. Formaldehyde (1% final concentration) was added directly into the medium, and the dishes were incubated at room temperature for exactly 2 min (for native ChIP [N-ChIP] assay) or 10 min (for cross-linking and ChIP [X-ChIP]) on an orbiting platform. Cross-linking was stopped with a 0.125 M final concentration of glycine solution. Cells were washed twice with 20 ml of ice-cold phosphate-buffered saline and scraped off the plate with a rubber policeman; cells were then pelleted by centrifugation for 3 min at 250 x g and resuspended in 750 µl of ChIP lysis buffer (1% sodium dodecyl sulfate, 10 mM EDTA, and 50 mM Tris-HCl, pH 8.1) containing protease inhibitors (Complete protease inhibitor cocktail tablets, catalog number 1697498; Roche Applied Science). Sonication was carried out on ice in 1.7-ml Eppendorff tubes using a Branson Sonifer with a micro tip three or four times (N-ChIP or X-ChIP, respectively) for 10 s at 40% output. The sheared chromatin was centrifuged at 13,000 rpm for 15 min at 4°C. The supernatant was transferred to a fresh tube. An aliquot of the chromatin preparation was reverse cross-linked, proteinase K digested, phenol-chloroform extracted, precipitated, and resuspended in Tris-EDTA buffer. DNA concentration was determined using a UV spectrophotometer. One microgram of DNA was resolved on a 1.5% agarose gel to check sonication efficiency. Fragment sizes ranged from 0.2 to 1.5 kb, with a peak at 0.8 kb. Chromatin preparations were diluted to 0.4 µg/µl with ChIP lysis buffer containing protease inhibitors and were snap-frozen in liquid nitrogen in small aliquots and stored at –80 °C. 10 µl (4 µg) of chromatin and 4 µg of specific antibody was used for each immunoprecipitation. The immunoprecipitated chromatin was eluted from 40 µl of protein A/G agarose beads and reverse cross-linked, and DNA was isolated with a QIAquick kit, (Qiagen, Germany) and eluted in 100 µl of Elution Buffer. RNA was isolated from MatDup.dist7 and PatDup.dist7 MEFs using RNA-Bee according to manufacturer’s instructions (Tel-Test). The pellet was dissolved in DEPC water containing RNasin (Promega) and 10 mM DTT. Contaminating DNA was removed with the DNA-free Kit (Ambion). ds cDNA was prepared from 10 g of MatDup.dist7 and PatDup.dist7 MEF total RNA according to NimbleGen Arrays User’s Guide (http://www.nimblegen.com/products/lit/exp_userguide_v3p2.pdf) using SuperScript® Double-Stranded cDNA Synthesis Kit (Invitrogen). The ds cDNA was purified using QIAquick PCR Purification Kit (Qiagen) for hybridization. For MIRA The methylated fraction of sonicated genomic DNA was captured using recombinant MBD3L1 and MBD2b proteins.
Label
Cy3
Label protocol
According to standard NimbleGen Protocol
Hybridization protocol
According to NimbleGen Kit
Scan protocol
Follow NimbleScan's default using Agilent Scanner
Description
chip_id: = 9690102
Data processing
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction.
