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| Status |
Public on Feb 27, 2017 |
| Title |
A375, rep2 |
| Sample type |
SRA |
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| Source name |
A375
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Melanoma Cell
tert promoter state: C250T
cell line: A375
passage number: 8-12
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| Treatment protocol |
There was no treatment.
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| Growth protocol |
Cells were cultured with DMEM suplemented with 10%FBS, penicillin and streptomycin using standard tissue culture techniques.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
10 million cells were fixed with 1% formaldehyde and nuclei pellets were isolated after cell lysis with cold lysis buffer (50mM Tris-Hcl pH 7.5, 150mM NaCl, 5mM EDTA, 0.5% NP-40, 1% TritonX-100) supplemented with protease inhibitors. First step digestion was performed over-night at 37°C with 200unit HindIII enzyme. After phenol-chloroform extraction, DNA was ligated over-night at 16°C by T4 DNA ligase. Following de-crosslinking, DNA was processed for second digestion with 50units of DpnII enzyme for over-night at 37°C. After final ligation 4C template DNAs were quantified by Qubit dsDNA High sensitivity kit
4C library was prepared as described previously (Splinter et al. 2012). Tert promoter specific primers with Illimuna Nextera adapters were used for libraries.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Description |
4C-seq (miseq)
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| Data processing |
Library strategy: 4C-Seq
During the analysis of the reads generated by 4C-Seq it was found that multiple fragends were ligated together after the 2nd RE digestion therefore only reads that contain the primer for the tert promoter + HindII cut site (read 2 of the paired end reads) were used.
FastQC v0.11.3 was used to determine the quality of the 4C-Seq libraries and any adaptors were trimmed using scythe v0.991
Tagdust2.3 was used to extract reads that was sandwiched between the bait region (TERT-promoter + HindII) and a DpnII cut site
Extracted reads were aligned to the hg19 genome using bowtie2 with very sensitive parameters in unpaired mode. The aligned reads were filtered for a mapq of 30 and more.
r3CSeq (Thongjuea S et al., Nucleic Acids Research, 2013 ) in batch mode was used to call interactions for A375, BLM
The standard FourCSeq (Klein et al., Bioinformatics, 2015) protocol (with the change of zscore 2 instead of 3) was used to quantify the differential interactions between BLM6 and BLM14 using the aligned reads.
Genome_build: hg19
Supplementary_files_format_and_content: Bed 3 file that contains additional columns for the number of reads, normalized reads per million, p-value and q-value (FDR) generated by r3CSeq for A375, BLM, BLM6 and BLM14. A tab delimited file containing the coordinates of fragments 1Mb around the viewpoint with presence of interaction (TRUE/FALSE) and padjusted value of differential interaction between BLM6 and BLM14.
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| Submission date |
Jan 27, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Melissa Jane Fullwood |
| Organization name |
Cancer Science Institute of Singapore
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| Department |
Centre for Translational Medicine
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| Lab |
Fullwood Lab
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| Street address |
14 Medical Drive, #12-01
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| City |
Singapore |
| ZIP/Postal code |
117599 |
| Country |
Singapore |
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| Platform ID |
GPL15520 |
| Series (1) |
| GSE77265 |
GABP determines the epigenetic status of mutant TERT promoter |
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| Relations |
| Reanalyzed by |
GSE123131 |
| BioSample |
SAMN04442262 |
| SRA |
SRX1548300 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2047013_CHM278_A375rep2_S2_L001_R2_001_r3CSeq_interactions.txt.gz |
403.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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