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| Status |
Public on Sep 07, 2022 |
| Title |
Parental_Neo6_CENPA_ChIP_chip_rep1 |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
CENPA IP from Parental Neo6 cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Parental Human Lymphoblastoid cells
treatment: Untreated
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| Treatment protocol |
Normally growing cells
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| Growth protocol |
Human/Hamster hybrid cell lines GM10253A and HybNeo3 were grown in RPMI 1640 with L-Glutamine, 10% FCS and penicillin and streptomycin. Human parental lymphoblastoid cell line was grown in RPMI 1640 with L-Glutamine, 20% FCS and penicillin and streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was done as described in Kimura et al. (2008) Cell Structure and function, except that a Soniprep 150 (Sanyo) was used for sonication. In brief, cells (5-6 x 10^6 in 10 cm dishes) were cross-linked with 10 ml 1% formaldehyde (Sigma) in medium for 5 min at room temperature and then incubated in 10 ml 200mM glycine in medium for 5 min. Cells were rinsed twice with PBS and incubated with 7 ml lysis buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl and 0.5% NP-40) for 10 min at room temperature with mild rotation. This lysis buffer was then aspirated off and cells were scraped into 1 ml lysis buffer and centrifuged at 3000 rpm for 3 min at 4°C. Cell pellet was resuspended in 100 μl SDS lysis buffer (50 mM Tris-HCl (pH 7.5), 10 mM EDTA and 1% SDS) and mixed by pipetting. 400 μl ChIP dilution buffer (50 mM Tris-HCl (pH 7.5), 167 mM NaCl, 1.1% Triton X-100, 0.11% sodium deoxycholate and protease inhibitor cocktail (complete EDTA-free; Roche)) was added before sonication (fifteen times for 20 seconds at 2 μm). After centrifugation at 13,000 rpm for 15 min at 4°C to remove the insoluble material the supernatant was removed to a new 1.5 ml tube and the volume made up to 500 μl with ChIP dilution buffer. 50 μl was removed as input for ChIP and the rest of the sample was incubated with antibody-bound Dynabeads overnight at 4°C with rotation. Dynabeads were prepared in advance by taking 50 μl of beads and washing three times with 500 μl cold RIPA-150 mM NaCl buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.11% sodium deoxycholate and protease inhibitor cocktail). Beads were then incubated with 500 μl cold RIPA-150 mM NaCl buffer plus antibody for 2 hr at 4°C with rotation. Beads were then washed three times with 500 μl cold RIPA-150 mM NaCl buffer and were then ready for overnight incubation with the ChIP sample. Beads were washed sequentially with 1 ml cold RIPA- 500 mM NaCl (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.11% sodium deoxycholate and protease inhibitor cocktail) and twice with 1 ml TE (10 mM Tris-HCl (pH 8.0) and 1 mM EDTA). DNA was eluted by the addition of 200 μl ChIP direct elution buffer (10mM Tris-HCl (pH8.0), 300 mM NaCl, 5mM EDTA and 0.5% SDS) and incubated overnight at 65°C. Samples were then treated with DNase-free RNase (Roche; 5 μg.ml-1; 37°C; 30 min) and proteinase K (250 μg.ml-1; 55°C; 1 hr). DNA was extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and ethanol precipitated with carrier (1μl glycogen, Invitrogen) on dry ice for 30 min. Following 70% ethanol wash the DNA pellet was resuspended in 20 μl water and quantified using a NanoDrop.
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| Label |
Cy3
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| Label protocol |
For microarray hybridization, immunoprecipitated DNA was amplified using whole-genome amplification (Sigma) and 500 ng DNA was random prime labeled (ENZO) with Cy3 (Sample DNA) or Cy5 (Input DNA) and purified on a MinElute PCR purification column (Qiagen).
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| Channel 2 |
| Source name |
Input from Parental cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Parental Human Lymphoblastoid cells
treatment: Untreated
|
| Treatment protocol |
Normally growing cells
|
| Growth protocol |
Human/Hamster hybrid cell lines GM10253A and HybNeo3 were grown in RPMI 1640 with L-Glutamine, 10% FCS and penicillin and streptomycin. Human parental lymphoblastoid cell line was grown in RPMI 1640 with L-Glutamine, 20% FCS and penicillin and streptomycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was done as described in Kimura et al. (2008) Cell Structure and function, except that a Soniprep 150 (Sanyo) was used for sonication. In brief, cells (5-6 x 10^6 in 10 cm dishes) were cross-linked with 10 ml 1% formaldehyde (Sigma) in medium for 5 min at room temperature and then incubated in 10 ml 200mM glycine in medium for 5 min. Cells were rinsed twice with PBS and incubated with 7 ml lysis buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl and 0.5% NP-40) for 10 min at room temperature with mild rotation. This lysis buffer was then aspirated off and cells were scraped into 1 ml lysis buffer and centrifuged at 3000 rpm for 3 min at 4°C. Cell pellet was resuspended in 100 μl SDS lysis buffer (50 mM Tris-HCl (pH 7.5), 10 mM EDTA and 1% SDS) and mixed by pipetting. 400 μl ChIP dilution buffer (50 mM Tris-HCl (pH 7.5), 167 mM NaCl, 1.1% Triton X-100, 0.11% sodium deoxycholate and protease inhibitor cocktail (complete EDTA-free; Roche)) was added before sonication (fifteen times for 20 seconds at 2 μm). After centrifugation at 13,000 rpm for 15 min at 4°C to remove the insoluble material the supernatant was removed to a new 1.5 ml tube and the volume made up to 500 μl with ChIP dilution buffer. 50 μl was removed as input for ChIP and the rest of the sample was incubated with antibody-bound Dynabeads overnight at 4°C with rotation. Dynabeads were prepared in advance by taking 50 μl of beads and washing three times with 500 μl cold RIPA-150 mM NaCl buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.11% sodium deoxycholate and protease inhibitor cocktail). Beads were then incubated with 500 μl cold RIPA-150 mM NaCl buffer plus antibody for 2 hr at 4°C with rotation. Beads were then washed three times with 500 μl cold RIPA-150 mM NaCl buffer and were then ready for overnight incubation with the ChIP sample. Beads were washed sequentially with 1 ml cold RIPA- 500 mM NaCl (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.11% sodium deoxycholate and protease inhibitor cocktail) and twice with 1 ml TE (10 mM Tris-HCl (pH 8.0) and 1 mM EDTA). DNA was eluted by the addition of 200 μl ChIP direct elution buffer (10mM Tris-HCl (pH8.0), 300 mM NaCl, 5mM EDTA and 0.5% SDS) and incubated overnight at 65°C. Samples were then treated with DNase-free RNase (Roche; 5 μg.ml-1; 37°C; 30 min) and proteinase K (250 μg.ml-1; 55°C; 1 hr). DNA was extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and ethanol precipitated with carrier (1μl glycogen, Invitrogen) on dry ice for 30 min. Following 70% ethanol wash the DNA pellet was resuspended in 20 μl water and quantified using a NanoDrop.
|
| Label |
Cy5
|
| Label protocol |
For microarray hybridization, immunoprecipitated DNA was amplified using whole-genome amplification (Sigma) and 500 ng DNA was random prime labeled (ENZO) with Cy3 (Sample DNA) or Cy5 (Input DNA) and purified on a MinElute PCR purification column (Qiagen).
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| Hybridization protocol |
Labeled DNA was diluted in hybridization buffer (Agilent) and hybridized to arrays for 24 h at 65°C. Slides were washed according to the manufacturer’s instructions. Agilent-053809 (7MB human chr3) and Agilent -077398 (22MB human chr3) arrays were used.
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| Scan protocol |
Arrays were scanned on a Nimblegen Microarray scanner at 2 μm resolution generating a TIFF file.
|
| Description |
Parental Human Lymphoblastoid cells with one canonical chromosome 6 (HSA6) and one chromosome 6 with a neocentromere (Neo6)
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| Data processing |
Spot signal intensity was extracted from the TIFF files using Agilent Feature Extraction software and were pre-processed in R using the RINGO bioconductor package to give the raw Cy5 and Cy3 signal intensities for each spot. Individual Cy5 and Cy3 channels were normalized to each other and between arrays and then normalised and scaled using the standard Bioconductor LIMMA package. Normalisation was as follows: loess normalisation for H3K27ac, H3K4me2, H3K9me2, H3K9me3, H4K20me1, H3K27me3 and H3K36me3 ChIps, vsn normalisation for RNA pol II and CTCF ChIps and nimblegen normalisation for CENPA and CENPC ChIPs.
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| Submission date |
Sep 06, 2022 |
| Last update date |
Sep 07, 2022 |
| Contact name |
Catherine Naughton |
| E-mail(s) |
[email protected]
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| Organization name |
University of Edinburgh
|
| Department |
MRC Human Genetics Unit
|
| Lab |
Nick Gilbert Lab
|
| Street address |
Crewe Road
|
| City |
Edinburgh |
| ZIP/Postal code |
EH4 2XU |
| Country |
United Kingdom |
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| Platform ID |
GPL31869 |
| Series (2) |
| GSE195885 |
Human centromere repositioning activates transcription and opens chromatin fibre structure [Agilent ChIP-chip] |
| GSE195886 |
Human centromere repositioning activates transcription and opens chromatin fibre structure |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM6552577_255380910029_2015-09-29_15-34_Area1_532_635_ChIP_1105_Oct12_1_3.txt.gz |
18.7 Mb |
(ftp)(http) |
TXT |
| Processed data are available on Series record |
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