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| Status |
Public on Oct 18, 2013 |
| Title |
GM02314_H3K27me3_Abcam_HumanOmni5-4v1_B |
| Sample type |
genomic |
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| Source name |
GM02314_H3K27me3_Abcam
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| Organism |
Homo sapiens |
| Characteristics |
gender: female
tissue: Fibroblasts
population: Caucasian- Irish
assayed molecule: ChIP DNA
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| Biomaterial provider |
BNIGMS Human Genetic Cell Repository
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| Treatment protocol |
After harvesting, cells were cross-linked with 1% formaldehyde at room temperature for 10 min. Cells were then immediately quenched with glycine for 5 min (125 mM glycine per ml of media), and washed twice with ice-cold PBS. Cells were collected after each wash by centrifugation at 2,000g for 5 min. Cell pellets were flash frozen and stored at -80 °C. Frozen pellets were thawed and cells were lysed in Farnhamlysis buffer (5mM PIPES pH8.0, 85mM KCl, 0.5% NP-40 and protease inhibitors) for 10 min on ice. After centrifugation and wash with 1 ml of RIPA buffer containing 50mM TrisHCl pH8, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitors, lysates were then diluted with 500 ul of RIPA buffer. Cells were sonicated in non-stick tubes under conditions optimized to yield soluble chromatin fragments in a size range of 100 to 250 base pairs. Chromatin from 40 million cells was sonicated for 10min using a Branson 250 sonicator at 20% power amplitude (pulses: 10 s on and 30 s off). Lysate was cleared by centrifuging at 12,000g for 10 min at 4 °C to eliminate cellular debris. Chromatin was then flash frozen and stored at -80 °C or used immediately for next step. Before each immunoprecipitation, chromatin was pre-cleared with 50 ul of prewashed ProteinA-magnetic beads (Invitrogen; 100-02D) to avoid non-specific binding. Immunoprecipitation was carried out for 12 hours by rotation at 4 °C in 500 ul of chromatin/RIPA buffer supplemented with protease inhibitor cocktails (Roche; 04 693 159 001) and PMSF. We used 10 to 100 million cells and 2 to 20 ?g of the following antibodies for each assay: H3K4me1 (Diagenode; #pAb-037-050), H3K4me1 (Abcam; H3K4me3 (Diagenode; #pAb-003-050), H3K27ac (Abcam; #ab4729), H3K27me3 (Millipore; #07-449), H3K36me3 (Abcam, #ab9050), RNA Pol II (Abcam; ab5131), RNA Pol II (Covance; 8WG16), Normal IgG (Cell Signaling Technology; #2729). After overnight incubation, samples were rotated with 100 ul of prewashed ProteinA-magnetic beads at 4 °C for 1 h. The beads were then collected by brief centrifugation at 2,000g following by use of magnetic rack. Beads were washed five times with 1 ml of LiCl wash buffer (100mM Tris pH7.5, 500mM LiCl, 1% NP-40, 1% sodium deoxycholate) by resuspending the beads and keeping on ice for 10 min. Bound chromatin was eluted from the beads using 200 ul of Elution buffer (50 mMTris-HCl, pH 8.0, 10 mM EDTA, 1.0% SDS) by incubation at 65 °C for 1 h with vortex every 15 min followed by centrifugation at 14,000g at room temperature for 3 min. The eluted chromatin and the input sample were incubated at 65 °C overnight after adding 0.2M of NaCl to remove crosslink. Samples were then treated with RNase 37 °C for 30 min and digested with proteinase K at 55 °C for 1 h. Immunoprecipitated DNA was purified using QIAquick PCR Purification Kit (QIAGEN; 28104) and eluted in 30 ul.
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| Growth protocol |
All cells were obtained from the Coriell Institute for Medical Research (Camden, NJ). The LCLs were grown in T75 flasks in 1X RPMI 1640 Media (invitrogen, Burlington, ON), with 2 mM L-glutamine, 15% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO2. LCL cells were harvested during log growth phase. The Fibroblast primary cell lines were immortalized as previously desribed (Grundberg et al., PLoS Genet., 2011), and then grown in medium containing a-MEM (SigmaAldrich) supplemented with 2 mmol/l L-Glutamine, 100 U/mL penicillin, 100 mg/ml streptomycin, and 10% fetal bovine serum (SigmaAldrich) at 37°C with 5% CO2. At 70%-80% confluence, fibroblast cells were harvested.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
RNA and gDNA were extracted from cell lysates and we applied a cDNA synthesis protocol as previously described in Moros et al., Genome Biology, 2011 (PMID: 21418647).
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| Label |
Cy3 and Cy5
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| Label protocol |
according to manufacturer's instructions
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| Hybridization protocol |
according to manufacturer's instructions
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| Scan protocol |
according to manufacturer's instructions
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| Description |
NIGMS Family #0056 - Daughter
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| Data processing |
GenomeStudio from Illumina
For convenience, data was compiled based on analysis date into 20 individual sets.
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| Submission date |
Sep 30, 2013 |
| Last update date |
Jan 14, 2014 |
| Contact name |
Tomi Pastinen |
| E-mail(s) |
tomi.pastinen@mcgill.ca
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| Organization name |
McGill University
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| Department |
Human & Medical Genetics
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| Street address |
740 Dr Penfield
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| City |
Montreal |
| State/province |
QC |
| ZIP/Postal code |
H3A0G1 |
| Country |
Canada |
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| Platform ID |
GPL14157 |
| Series (1) |
| GSE51272 |
Genome-wide characterization of allelic chromatin in human fibroblast and lymphoblastoid cell lines by high-density allele-specific analyses |
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| Supplementary data files not provided |
| Processed data are available on Series record |
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