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| Status |
Public on Aug 28, 2013 |
| Title |
Mock-p65_ChIPSeq |
| Sample type |
SRA |
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| Source name |
B_lymphocytes
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| Organism |
Homo sapiens |
| Characteristics |
cell line: immortalized Namalwa B cell line (ATCC CRL-1432)
infection: mock
chip-antibody: NFκB p65 (Abcam, ab7970)
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| Treatment protocol |
Infection of cells with Sendai virus, Cantell strain was performed at 5 plaque-forming units per cell (pfu/cell) in serum-free media. After 1 hr, cells were lightly centrifuged, washed, and re-suspended in growth media supplemented with 2% CCS. For mock-infection, cells were subjected to all manipulations with PBS substituted for virus inoculum.
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| Growth protocol |
Human immortalized Namalwa B cell line (ATCC CRL-1432) were cultured in RPMI 1640 medium (Life Technologies) containing 10% cosmic calf serum (Hyclone), glutamine, 1 mM sodium pyruvate (Cellgro), and 1% penicillin/streptomycin (Life Technologies).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with antibody.
Purified immunoprecipitated DNA was prepared for sequencing according to the 5500 Series SOLiD Systems Fragment Library Preparation protocol. Fragmented DNA was prepared by blunt-ending the DNA, size selecting DNA between 100-250 bp using Agencourt AMPure XP Reagent beads, and adding a dA-tail to allow for directional ligation. Following the addition of a single adenine nucleotide overhang, a 1:20 dilution of the appropriate adaptor pair was used in the ligation step. After ligation, the DNA library was size selected to a narrow range of fragment sizes by using Agenoncourt AMPure XP Reagent (~200-300 bp, which represents shear fragments between 100 and 200 nt in length and ~100 bp of primer sequence). A subsequent PCR with limited (14-17) amplification was conducted and a final size selection was carried out prior to e-PCR and paired-end sequencing on the ABI SOLiD series 5500 DNA sequencing platform.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB 5500xl Genetic Analyzer |
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| Data processing |
All sequence data were with aligned the human hg19 assembly using BioScope v1.3.1.
The reference genome was converted to colorspace for appropriate alignment with colorspace reads, initially locating perfect, full-length 50 bp matches for each read. If a perfect match could not be found, then 25 bases were aligned with 2 colorspace mismatches allowed, starting at position 0 (i.e. the first sequenced base). The alignment was then extended base-by-base toward the 3' end. If the alignment failed within the first 25 bp of the read, then 25 bases of the read were aligned, with 2 allowed colorspace mismatches, starting at position 15 of the read (i.e. the 16th sequenced base), and extended toward the 5' end, and forward toward the 3' end until the best alignment is obtained.
Operating under the default parameters and using the sequenced input DNA as the control, MACS software was used to identify binding sites (or peaks) in each sample.
Subsequently, we further filtered peaks that are contained in signal artifact regions, as specified by the set of blacklists used for Grch37/hg19 (ftp://encodeftp.cse.ucsc.edu/users/akundaje/rawdata/blacklists/hg19/).
Data analysis was performed using HOMER, a software suite for ChIP-Seq analysis. Each ChIP-Seq experiment was normalized to a total of 107 uniquely mapped tags by adjusting the number of tags at each position in the genome to the correct fractional amount given the total tags mapped. This normalization was used for all downstream analysis.
Genome_build: Grch37
Supplementary_files_format_and_content: bigWig and BED files. Both file types we made using the software suite HOMER. (For details see http://biowhat.ucsd.edu/homer/)
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| Submission date |
Mar 07, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Jonathan E Freaney |
| E-mail(s) |
jfreaney@u.northwestern.edu
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| Organization name |
Northwestern University
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| Street address |
2200 Campus Drive
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| City |
Evanston |
| State/province |
Illinois |
| ZIP/Postal code |
60208 |
| Country |
USA |
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| Platform ID |
GPL16288 |
| Series (1) |
| GSE44939 |
Genome-wide maps of virus-induced transcription factors and transcription machinery at steady state and after virus infection |
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| Relations |
| SRA |
SRX247150 |
| BioSample |
SAMN01940774 |
| Named Annotation |
GSM1094196_Mock-p65-Tags.ucsc.bigWig |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1094196_Mock-p65-Tags.ucsc.bigWig |
210.1 Mb |
(ftp)(http) |
BIGWIG |
| GSM1094196_p65_Mock_Peaks.bed.gz |
10.2 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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