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| Status |
Public on Apr 08, 2021 |
| Title |
HF-2927_ChIP-free-chia-pet |
| Sample type |
SRA |
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| Source name |
Glioblastoma neurosphere cell suspensions
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| Organism |
Homo sapiens |
| Characteristics |
source: Glioblastoma
ip antibody: NA
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| Growth protocol |
Tumor specimens were dissociated and cultured as neurospheres in DMEM/F12 medium (11330-032, Gibco) supplemented with N-2 supplement (17502-048, Gibco) and growth factors (EGF and FGF-basic). Cells with passages between 15 and 26 were used for experiments.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
10 million cells were dual-crosslinked with 1.5mM EGS (21565, Thermo Fisher) for 45 min followed by 1% formaldehyde (F8775, Sigma) for 20 min at room temperature and then quenched with 0.125 M Glycine (G8898, Sigma) for 10 min. The crosslinked cells were washed with PBS twice and lysed in 100 ul 0.55% SDS with incubation at room temperature, 62°C and 37°C sequentially for 10 min each, which was followed by 37°C for 30 min with addition of 25 ul 25% Triton-X 100 to quench SDS and 37°C overnight with addition of 50 ul AluI (R0137L, NEB), 50 μl 10 × CutSmart buffer and 275 μl H2O to fragmentize the chromatin. The pelleted digested nuclei were resuspended in 500 ul A-tailing solution containing 50 μl 10 × CutSmart buffer, 10 μl BSA (B9000S, NEB), 10 μl 10 mM dATP (N0440S, NEB), 10 μl Klenow (3’- 5’ exo-) (M0202L, NEB), and 420 μl H2O with 1 hr incubation at room temperature and then subjected to proximity ligation by adding 200 μl 5 × ligation buffer (B6058S, NEB), 6 μl biotinylated bridge linker (200 ng/ul), 10 μl T4 DNA ligase (M0202L, NEB) and 284 μl H2O and incubating at 16°C overnight. The ligated chromatins were then sheared by sonication and decrosslinked.
The decrosslinked DNA were tagmented using the Nextera DNA Sample Preparation Kit (Illumina). The linker ligated ChIA-PET constructs were selected using streptavidin magnetic beads. The magnetic beads were blocked with sheared gDNA prior to the selection. The streptavidin selected constructs were amplified with PCR and purified.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
ChIAPET
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| Data processing |
ChIA-PET data was processed by ChIA-PET Utilities (CPU) (https://github.com/cheehongsg/CPU). CPU (options: stat -s -p -T 18 -t 16) removed the adaptors, and grouped the reads into three categaries: PET reads, single tag reads and no-linker reads. Tags identified (>=18bp) and reads without linkers were mapped to hg19 using BWA alignment and mem according to the tag/read length. CPU further separated the mapping results into uniquely-mapped,multiply-mapped, and unmapped classes with the pairing consideration (options: pair -S -t 16) and removed duplicates generated by PCR amplification (dedup -g -t 16). Next, the non-redundant uniquely-mapped reads from 3 categaries were merged. CPU furhter clustered the non-redundant uniquely-mapped pair-end tags and grouped them as intra-chromosomal PET clusters (cis-interactions) and inter-chromosomal PET clusters (trans-interactions) (options: cluster -s 8000 -M -B 1000 -5 5,-20 -3 5,480 -t 16 -O).
Illumina RTA-1.18 software used for basecalling
Genome_build: hg19
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| Submission date |
Apr 22, 2020 |
| Last update date |
Apr 08, 2021 |
| Contact name |
Chia-Lin Wei |
| E-mail(s) |
[email protected]
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| Organization name |
University of Washington
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| Department |
The Northwest Genomics Center (NWGC)
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| Lab |
Genome Sciences
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| Street address |
3720 15th Ave NE
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195-5065 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE124769 |
Oncogenic extrachromosomal DNA functions as mobile enhancers to globally amplify chromosomal transcription |
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| Relations |
| BioSample |
SAMN14671411 |
| SRA |
SRX8158042 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4491245_HF-2927_ChIP-free_ChIAPET.trans.chiasig.interactions.txt.gz |
571.5 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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