|
Status |
Public on Sep 07, 2022 |
Title |
HybNeo3_bTMP_chip_amanitin_rep2 |
Sample type |
genomic |
|
|
Channel 1 |
Source name |
bTMP-IP from HybNeo3 cells
|
Organisms |
Homo sapiens; Cricetulus griseus |
Characteristics |
cell type: HybNeo3 cells treatment: 5h alpha-amanitin treatment
|
Treatment protocol |
Cells were untreated or treated with alpha-amanitin (5h)
|
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 parenral lymphoblastoid cell line was grown in RPMI 1640 with L-Glutamine, 20% FCS and penicillin and streptomycin.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Cells were treated with 500 μg.ml-1 of bTMP in PBS for 20 min at room temperature in the dark. bTMP was UV cross-linked to DNA at 360 nm for 10 min. DNA was purified from cells using SDS and proteinase K digestion and extracted using phenol-chloroform-isoamyl alcohol (25:24:1). DNA was fragmented by sonication (thirteen times for 30s at 2 μm). Biotin incorporation into DNA was detected by dot blotting using alkaline phosphatase−conjugated avidin as a probe. The bTMP−DNA complex in TE was immunoprecipitated using avidin conjugated to magnetic beads (Dynabeads MyOne Streptavidin Invitrogen, 65001) for 2 h at room temperature and then overnight at 4°C. Beads were washed sequentially for 5 min each at room temperature with TSE I (20 mM Tris-HCl, pH 8.1, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100 and 0.1% SDS), TSE II (20 mM Tris-HCl, pH 8.1, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100 and 0.1% SDS) and buffer III (10 mM Tris-HCl, pH 8.1, 0.25 M LiCl, 1 mM EDTA, 1% NP40 and 1% deoxycholate). Beads were then washed twice with TE buffer for 5 min. To extract DNA and to release psoralen adducts, the samples were boiled for 10 min at 90°C in 50 μl of 95% formamide with 10 mM EDTA. Samples were then made up to 200 μl with water, and the DNA was purified using a Qiagen MinElute PCR purification kit. bTMP bound DNA was amplified using whole-genome amplification (Sigma) prior to microarray hybridization.
|
Label |
Cy3
|
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).
|
|
|
Channel 2 |
Source name |
Input from HybNeo3 cells
|
Organisms |
Homo sapiens; Cricetulus griseus |
Characteristics |
cell type: HybNeo3 cells treatment: 5h alpha-amanitin treatment
|
Treatment protocol |
Cells were untreated or treated with alpha-amanitin (5h)
|
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 parenral lymphoblastoid cell line was grown in RPMI 1640 with L-Glutamine, 20% FCS and penicillin and streptomycin.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Cells were treated with 500 μg.ml-1 of bTMP in PBS for 20 min at room temperature in the dark. bTMP was UV cross-linked to DNA at 360 nm for 10 min. DNA was purified from cells using SDS and proteinase K digestion and extracted using phenol-chloroform-isoamyl alcohol (25:24:1). DNA was fragmented by sonication (thirteen times for 30s at 2 μm). Biotin incorporation into DNA was detected by dot blotting using alkaline phosphatase−conjugated avidin as a probe. The bTMP−DNA complex in TE was immunoprecipitated using avidin conjugated to magnetic beads (Dynabeads MyOne Streptavidin Invitrogen, 65001) for 2 h at room temperature and then overnight at 4°C. Beads were washed sequentially for 5 min each at room temperature with TSE I (20 mM Tris-HCl, pH 8.1, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100 and 0.1% SDS), TSE II (20 mM Tris-HCl, pH 8.1, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100 and 0.1% SDS) and buffer III (10 mM Tris-HCl, pH 8.1, 0.25 M LiCl, 1 mM EDTA, 1% NP40 and 1% deoxycholate). Beads were then washed twice with TE buffer for 5 min. To extract DNA and to release psoralen adducts, the samples were boiled for 10 min at 90°C in 50 μl of 95% formamide with 10 mM EDTA. Samples were then made up to 200 μl with water, and the DNA was purified using a Qiagen MinElute PCR purification kit. bTMP bound DNA was amplified using whole-genome amplification (Sigma) prior to microarray hybridization.
|
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).
|
|
|
|
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) arrays were used.
|
Scan protocol |
Arrays were scanned on a Nimblegen Microarray scanner at 2 μm resolution generating a TIFF file.
|
Description |
Chinese hamster/human hybrid retaining the following human chromosomes ; 4, 6, 8, 11, 13, 18 ,X and a 3 carrying a neocentromere (Neo3).
|
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 nimblegen normalised and scaled using the standard Bioconductor LIMMA package. bTMP_normlized_matrix: Normalized Log2 (bTMP/Input)
|
|
|
Submission date |
Feb 01, 2022 |
Last update date |
Sep 07, 2022 |
Contact name |
Catherine Naughton |
E-mail(s) |
Catherine.Naughton@ed.ac.uk
|
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 |
|
|
Platform ID |
GPL31869 |
Series (2) |
GSE195884 |
Human centromere repositioning activates transcription and opens chromatin fibre structure [Agilent bTMP-chip] |
GSE195886 |
Human centromere repositioning activates transcription and opens chromatin fibre structure |
|