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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 06, 2015 |
| Title |
Raji CIITA Boss Lab |
| Sample type |
SRA |
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| Source name |
Raji cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Raji cells
chip antibody: CIITA (Brown, JA et al. Nucleic acids research, 1998. 26:4128-36)
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| Growth protocol |
Raji and RJ2.2.5 cells were cultured in RPMI supplemented with 5% FCS, 5% FBS, and 100 U/ml Penecillin/Streptomycin
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
10 ng of CIITA ChIP DNA was End Repaired using 2U T4 DNA polymerase, 0.5U Klenow, and 5U T4 DNA Polynucleotide kinase for 30 min at 20° in NEB T4 DNA Ligase buffer supplemented with 0.4 mM DNTPs. A-tailing was performed using 5U Klenow (3’->5’) exo- for 30 min at 37° in NEB buffer 2 supplemented with 0.2 mM dATP. 250 μM annealed adaptors were ligated onto A-tailed DNA using 2000U Quick Ligase for 20 min at room temperature in Quick Ligase buffer. Adaptor ligated DNA was converted to double stranded DNA by 5 cycles of PCR using 2U of Phusion Polymerase in GC Buffer supplemented with 0.4 mM DNTPs and 0.5 mM Adaptor Primers. Double-stranded adaptor-ligated DNA was size selected on a 2% agarose gel by excising a 200-400 bp band. Size selected DNA was PCR amplified for 8 additional cycles to generate sequencing libraries.
Chromatin immunoprecipitation (ChIP) assay was performed as previously described (Scharer et al. Cancer Research, 69:709-717). Briefly, the indicated cells were fixed in 1% formaldehyde for 10 minutes, nuclei isolated, and sonicated to an average chromatin fragment size of 200-600 bp. 30 mg chromatin was immunoprecipitated with 5 μg anti-CIITA, anti-H3K27ac or anti-H3K4me3 antibody over night at 4 degrees. Antibody-chromatin complexes were captured with Protein A magnetic beads (Invitrogen), cross-links reversed, and DNA purified. Input fraction was isolated after sonication and prior to the IP.
See each sample for speicific extract 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 |
Raw sequencing reads were mapped to the hg19 version of the mouse genome with Bowtie using the default options
For CIITA ChIPseq peak calling HOMER software (Heinz et al. Mol Cell, 2010. 38:576-589) was used with the options "-style factor"
For ATACseq paired-end and single end fastq files from the same cell type were merged into one bigWig file
Genome_build: hg19
Supplementary_files_format_and_content: bigWig files are normalized to reads per million (rpm) using R/Bioconductor. Bed files contain the genomic locations of significantly enriched peaks as determined by HOMER
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| Submission date |
Feb 04, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Chris Scharer |
| E-mail(s) |
cdschar@emory.edu
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| Organization name |
Emory University
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| Department |
Microbiology and Immunology
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| Lab |
Chris Scharer
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| Street address |
1510 Clifton Rd, Suite 3086A
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| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30322 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE52941 |
CIITA regulated genes in human B cells |
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| Relations |
| BioSample |
SAMN03329459 |
| SRA |
SRX865410 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1602232_Raji.CIITA.BossLab.bigWig |
118.5 Mb |
(ftp)(http) |
BIGWIG |
| GSM1602232_Raji.CIITA.BossLab.peaks.bed.gz |
28.7 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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