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| Status |
Public on Dec 11, 2015 |
| Title |
GM12878_CTCF |
| Sample type |
SRA |
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| Source name |
GM12878
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| Organism |
Homo sapiens |
| Characteristics |
cell line: GM12878
factor: CTCF
antibody: anti-CTCF antibody (Abcam, ab70303)
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| Treatment protocol |
Approximately 100-200 million cells were harvested and fixed by 30 ml of 1.5 mM EGS (ethylene glycol bis[succinimidylsuccinate]) in PBS buffer for 45 minutes at room temperature. Next, formaldehyde was added to final concentration of 1% to cross-link the cells for another 20 minutes at room temperature and then neutralized by using glycine. The cross-linked cells were lysed by cell lysis buffer and nuclear lysis buffer.
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| Growth protocol |
GM12878 and HeLa cells (ATCC®) were cultured following manufacture’s instruction.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was obtained and subjected to fragmentation with an average length of 300 bp by sonication. The anti-PolII monoclonal antibody 8WG16 (Covance, MMS-126R) and anti-CTCF antibody (Abcam, ab70303) were used to enrich PolII and CTCF-bound chromatin fragments, respectively.
ChIP DNA on beads was used for ChIA-PET library preparation. After performing the end-repair and A-tailing using T4 DNA polymerase (NEB) and Klenow enzyme, the ChIP DNA ends were proximity-ligated by the single biotinylated bridge-linker with the 3' nucleotide T over-hanging on both strands. Proximity ligation DNA was reverse cross-linked and fragmented and added sequencing adaptors simultaneously by using Tn5 transposase (Nextera kit, Illumina). DNA fragments contained the bridge-linker at ligation junctions were captured by Streptividin beads, and used as templates for PCR amplification. These DNA products were then subjected to size-selection and paired-end sequencing (2×150 bp) using Illumina Hi-Seq 2500.
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| Library strategy |
ChIA-PET |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Combined
ChIA-PET
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| Data processing |
Data processing of ChIA-PET reads was performed as previously described in Li et al., 2012 with slight modifications.
Pair-end read (PET) sequences were scanned for the bridge linker sequence and only PETs with the bridge linker were used for downstream processing. After trimming the linkers, the sequences flanking the linker were mapped to the human reference genome (hg19) using bwa-mem (Li and Durbin, 2010) and only uniquely aligned (MAPQ ≥ 30) PETs were retained. PCR duplicates were removed using the MarkDuplicates tool of the Picard Tools library (http://broadinstitute.github.io/picard/). Each PET was categorized as either a self-ligation PET (two ends of the same DNA fragment) or inter-ligation PET (two ends from two different DNA fragments in the same chromatin complex) by evaluating the genomic span between the two ends of a PET.
PETs with a genomic span less than 8 kb are classified as self-ligation PETs and are used as a proxy for ChIP fragments since they are derived in a manner analogous to ChIP-Seq mapping for protein binding sites. PETs with a genomic span greater than 8 kb are classified as inter-ligation PETs and represent the long-range interactions of interest. To accurately represent the frequency of interaction between two loci and to define the interacting regions, both ends of inter-ligation PETs were extended by 500 bp along the reference genome, and PETs overlapping at both ends (with extension) were clustered together as one PET cluster.
ChIP-nexus sequencing raw reads (including the fixed and random barcodes) were first reduced for PCR redundancy, and then the fixed and random barcodes (read positions 1-9) were trimmed. The remaining sequences were aligned to human genome assembly hg19 by bwa-mem (Li and Durbin, 2010) and only uniquely aligned (MAPQ ≥ 30) PETs were retained.
The sequence alignment results were used as input to MACE (v1.2) (Wang et al., 2014) to identify RAD21 and SMC3 occupancy borders (footprints).
genome build: hg19
processed data files format and content: Tab-delimited file documenting the genomic coodinates and frequency of each interaction PET cluster. For ChIP-nexus, the processed data is the MACE processed genome coverage profile in bigwig format.
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| Submission date |
Sep 09, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Oscar J. Luo |
| E-mail(s) |
Oscar.Luo@jax.org
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| Organization name |
The Jackson Laboratory for Genomic Medicine
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| Street address |
10 Discovery Dr
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| City |
Farmington |
| State/province |
CT |
| ZIP/Postal code |
06030 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE72816 |
CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription |
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| Relations |
| BioSample |
SAMN04041571 |
| SRA |
SRX1210750 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1872886_GM12878_CTCF_PET_clusters.txt.gz |
1.8 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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