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| Status |
Public on May 01, 2013 |
| Title |
CTCFbiotag/Mus musculus:activated B/mZF4/rep_d |
| Sample type |
SRA |
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| Source name |
murine primary activated B cells
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| Organism |
Mus musculus |
| Characteristics |
experiment type: ChIP-Seq
chip antibody: Streptavidin beads
chip antibody manufacturer: Invitrogen
chip antibody catalog #: 11205D
activation protocol: LPS/IL4
precipitated ctcf genotype: mZF4
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| Treatment protocol |
B lymphocytes were isolated from spleens of wild-type C57BL6 male mice by immunomagnetic depletion using CD43 MACS beads (Miltenyi Biotec). Purified cells were cultured at 0.3x10^6 cells per ml in B cell media (Advanced RPMI 1640, 10% FCS, 1x antibiotic-antimycotic, 1% glutamine, 50uM 2-beta-mercaptoethanol, and 10mM Hepes). Cells were pre-activated overnight in the presence of 0.5 ug/ml of antiCD180 (RP105) antibody (RP/14, BD Pharmingen). At 0, 8, and 24h cells were transduced with Vector1 (pMy-CTCFbiotag-T2A-mOrange) and Vector2 (pMy-BirA-T2A-eGFP) by centrifugation for 90min at 2500rpm, at 32C. B cell media was supplemented with 50 ug/ml of LPS (Escherichia coli 0111:B4; Sigma-Aldrich), 2.5 ng/ml of IL-4 (Invitrogen), and 0.5 ug/ml of antiCD180. At 32h cells were diluted to 0.1x10^6 cells per ml. 72h after 1st infection, B cells were harvested and GFP/mOrange double positives were cell sorted using a BD FACSAria III (Becton, Dickinson and Company). The percentage of double positive cells was 30-40%.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells (10-20 x 10^6 cells) were crosslinked for 10min at 37C with 1% (vol/vol) formaldehyde, followed by quenching with 0.125 M glycine (final concentration). Crosslinked cell samples were then sonicated with a Covaris sonicator to obtain DNA fragments 200-300 bp in length. Biotinylated samples were incubated with 40ul of Dynabeads M-280 Streptavidin Beads (Invitrogen) overnight at 4C in RIPA buffer (10 mM Tris, pH 7.6, 1 mM EDTA, 0.1% (wt/vol) SDS, 0.1% (wt/vol) sodium deoxycholate and 1% (vol/vol) Triton X-100). Beads were washed twice with Wash buffer1 (2% (vol/vol) SDS), once with Wash buffer2 (0.1% (vol/vol) deoxycholate, 1% (vol/vol), once with Wash buffer3 (250 mM LiCl, 0.5% (vol/vol) NP-40, 0.5% (vol/vol) deoxycholate, 1 mM EDTA and 10 mM Tris-HCl (pH 8.1)) and then twice with TE buffer (10 mM Tris-HCl (pH 7.5) and 1 mM EDTA). ChIP DNA was then extracted for 4h at 65C in Tris-EDTA buffer with 0.3% (wt/vol) SDS and proteinase K (1 mg/ml). Library preparation followed standard protocols
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
***Short read processing and alignment***: Fastq files were generated from the Illumina Real Time Analysis output using CASAVA with default settings. The 36 nt or 50 nt reads were then aligned to the mm9 genome with 'bowtie --sam --all --strata --best -m1 -n2 -l50'. The result of this alignment strategy is to only include reads that have a unique alignment in the top stratum of alignments where the stratum is defined by the number of mismatches. Up to two mismatches over the whole read length were allowed. ***Density tracks (wiggle format) for visualization***: Density tracks were generated using custom software based on the samtools library that counted the number of reads per 100 nt window across the genome and normalized to window size and library size to obtain densities in units of reads per million per kb (RPKM). A redundancy of up to 5 reads was allowed in the density tracks. Tracks were smoothed in the UCSC browser for display.
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| Submission date |
May 01, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Seolkyoung Jung |
| Organization name |
NIH
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| Department |
NIAMS
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| Lab |
biodata mining and discovery section
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| Street address |
10 Center Dr
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| City |
bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE33819 |
A genome-wide map of CTCF multivalency redefines the CTCF code |
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| Relations |
| BioSample |
SAMN02116124 |
| SRA |
SRX273240 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1132337_W_r120428A_l2i10_aB180_CTCFZNF4_bioCTCF_rep4.wig.gz |
38.5 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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