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| Status |
Public on Feb 20, 2018 |
| Title |
RNASeq_NIV_KO_2 |
| Sample type |
SRA |
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| Source name |
Naïve_CD4_T
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6 BCL11B deficient
tissue: Peripheral lymph node
dilution setting: NULL
antibody: NULL
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| Extracted molecule |
total RNA |
| Extraction protocol |
For Hi-C, five thousands to millions cells were stained with Biotin anti-CD45.2 antibody. Cells were bound to biotin binder (Dynabeads Biotin Binder, Cat#11047, Invitrogen) and cross-link with 1% formaldehyde. Biotin binders were blocked by 0.5mM biotin solution. Cells were lysed and digested with CviQ I + CviA II + Bfa I for 20min. For DNase-Seq, 1000 purified cells were lysed in 32ul of lysis buffer (10mM Tris-Cl, pH7.5, 10mM NaCl, 3mM MgCl2) and digested with 8ul of DNase I for 5 min at 37°C. The reaction was stopped by adding 40ul of stop buffer containing 10mM Tris-Cl, pH7.5, 10mM NaCl, 10mM EDTA, 2% SDS, 0.5mg/ml Proteinase K, and 1ng/ul of circular carrier DNA), followed by incubation at 65°C for 1 hour. For MNase-Seq, 100 purified cells were lysed in 32ul of lysis buffer (10mM Tris-Cl, pH7.5, 10mM NaCl, 2mM CaCl2) and digested with 8ul of MNase I for 5 min at 37°C. The reaction was stopped by adding 40ul of stop buffer containing 10mM Tris-Cl, pH7.5, 10mM NaCl, 10mM EGTA, 2% SDS, 0.5mg/ml Proteinase K, and 1ng/ul of circular carrier DNA), followed by incubation at 65°C for 1 hour. For RNA-Seq, 2ng of total RNA were extracted miRNeasy Micro Kit (50) from Qiagen (Cat # 217084).
The Hi-C samples were processed following the Hi-C protocol (PMID 19815776) with modifications briefly described as follows: DNA end were marked by biotin-14-dATP with Klenow(larg) for 1hrs at 37°C. Blunt-end DNA were ligated with T4 DNA Ligase for overnight at 16°C. DNA were reverse cross-linked and purified by phenol/chloroform extraction. Biotin were removed from unligated DNA-ends by T4 DNA polymerase for 2hrs at 12°C. DNA were purified by phenol/chloroform. DNA were sheared to 300-500bps by sonication and flowed by DNA-end repair and add”A”34. Biotin labeled DNA were pull-downed by streptavidin beads and then flowed by illumine adapter ligation and PCR amplification. Library construction protocals for DNase-Seq, MNase-Seq and RNA-Seq follow that described in (PMID 26605532), (PMID 18329373) and (PMID 24056875), respectively.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
Basecalls performed using CASAVA 1.8.4
Sequence reads were mapped to the mouse (mm9) genomes with bowtie (v2-2.0.2; default parameters; PMID 3322381). Only pair-end tags (PETs) mapped with high quality score (MAPQ > 10) were include for the analysis of chromatin interaction. For ChIP-Seq and scDNase-Seq analysis, if multipe reads were mapped to the same genomic site, only one read was kept.
The HOMER (PMID 20513432) package was employed to partition the genome into A and B compartment, at a resolution of 20K bps, with compartment signal across the genome corresponding to the cooridantes of the first principal obtained from PCA analysis on the normalized interaction matrix. Interaction matrices at a resolution of 20Kb were normalized by ICE (PMID 22941365). The gross intearcion of a gene is measured by the PETs with at least one end originated from the gene. PETs with distance less than 4K and longer than 2M were excluded. Also, FitHi-C (PMID: 24501021) was used to filtered PETs potentially orignated from background; potential interactions between any pairs of 2Kb bins were called under p<0.05 and an minimal of 2PETs. Potential HSs from DNase-Seq data were called by SICER (PMID 19505939), with a window size of 100 bps, E-value = 0.001 and no gap. Bcl11b binding sites from ChIP-Seq data were also called by SICER (PMID 19505939) with a window size of 200 bps, E-value = 1, and gap = 400 bps. Gene expression from RNA-Seq was quantified by RPKM with in-house script and RefSeq gene annotation. Nucleosome positioning was called by Danpos (v2.2.1, -m 1, quantile normalization, others default; PMID 23193179).
Genome_build: mm9
Supplementary_files_format_and_content: file name ending with "rpkm.txt": plan text file. Columns from left to right denote RefSeq ID, RPKM, gene transcript size, number of read mapped to exons, a combination of RefSeq ID, gene symbol, etc, and reserved column for future development.
Supplementary_files_format_and_content: file name ending with "scoreisland": plan text output from SICER. Columns from right to left are for chr, start, end, score.
Supplementary_files_format_and_content: file name ending with "positions.xls": plan text file; prediction of nucleosome position from Danpos. Columns from left to right denote chr, start, end, sumit_pos, sumit_value_quantile_normalized, and fuzziness_score.
Supplementary_files_format_and_content: file name ending with "PC1.bedGraph.sort" or "PCA.bedgraph.sort": plan text file; compartment score output from HOMER (PMID 20513432). Columns from left to right include: "chr start end PC1". The file was sorted into the "bedgraph" format acceptable by WahsU epigenome browser (PMID 23629413).
Supplementary_files_format_and_content: file name ending with "mtx.normICE.washU.sort": plain text file recording the number of interacting PETs between pairs of 20K bins, transformed into "longrange" format acceptable by WashU epigenome browser (PMID 20513432). Columns from left to right denotes "chr start end information_about_the_interacting_region ID relative_direction_of_the_interacting_region" (http://wiki.wubrowse.org/Long-range).
Supplementary_files_format_and_content: file name ending with "sbsmp.iii.genWashU.sort": plain text file recording the number of gross interacting PETs of genes, transformed into "longrange" format acceptable by WashU epigenome browser (PMID 20513432). Columns from left to right denotes "chr start end information_about_the_interacting_region ID relative_direction_of_the_interacting_region" (http://wiki.wubrowse.org/Long-range).
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| Submission date |
Jan 17, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Gangqing Hu |
| E-mail(s) |
michael.hu@hsc.wvu.edu
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| Organization name |
West Virginia University
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| Department |
MicroBiology, Immunology, and Cell Biology
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| Lab |
2072A, HSC North, Floor 2
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| Street address |
64 Medical Center Drive
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| City |
Morgantown |
| State/province |
West Virginia |
| ZIP/Postal code |
26506-9177 |
| Country |
USA |
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| Platform ID |
GPL21493 |
| Series (2) |
| GSE79422 |
Integrative analysis of 3D nucleome and chromatin accessibility reveals a chromatin barrier established for T-lineage commitment during early T cell development [Dnase-Seq, HiC-Seq, Mnase-Seq, RNA-Seq] |
| GSE79875 |
Integrative analysis of 3D nucleome and chromatin accessibility reveals a chromatin barrier established for T-lineage commitment during early T cell development |
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| Relations |
| BioSample |
SAMN06237478 |
| SRA |
SRX2499505 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2461029_GB0021.rpkm.txt.gz |
481.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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