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| Status |
Public on Mar 02, 2016 |
| Title |
Input for 309M3-B and 309M3-A |
| Sample type |
SRA |
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| Source name |
HEK293, input
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
chip antibody: none
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| Growth protocol |
The HEK293 cells were grown in the DMEM media (Thermo) with 10% FBS and Penicillin-Streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Native ChIP experiments using HEK293 cells were performed as previously described (Brand M. et al., Nat. Protoc., 3, 398-409 (2008) and Ruthenburg, A.J. et al., Cell, 145, 692-706 (2011)) except for the following modifications. Micrococcal nuclease-digested nuclei from HEK293 cells were purified using hydroxyapatite chromatography with stepwise elution in 50, 100, 200 and 500 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl, 1 mM EDTA and 200 uM PMSF. The purity of nucleosomes in elution fractions was examined by SDS-PAGE and high-purity fractions were used for ChIP. The antibody-coated beads were prepared by incubating 132 uL of Dynabeads M280 streptavidin (Life Technology) and 3.3 ug of a biotinylated Fab antibody (molar equivalent to 1 ug of IgG antibody) for 1hr at 4 ˚C. Excess biotin-binding sites of streptavidin were blocked with biotin. The antibody-coated beads were incubated with 10 ug of purified nucleosomes at 4˚C for overnight. Washing, elution and DNA purification were performed as previously described (Brand M. et al., Nat. Protoc., 3, 398-409 (2008)).
Library preparation for sequencing was performed using TruSeq ChIP sample preparation Kit, Set A (Illumina) according to the manufacturer’s protocol.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Sample 1
Cell culture
This is input sequencing for Samples 2-5.
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| Data processing |
Data analysis was performed with Galaxy (Blankenberg et al., 2010; Giardine et al., 2005; Goecks et al., 2010).
Raw reads in FastQ fromat were first submitted to FastQ Groomer.
Reads were mapped with Bowtie2 (Langmead et al., 2009) (sensitive preset option, end-to-end alignment), to human (hg19) reference genomes.
Resulting SAM files were then filtered using SAMtools (Li et al., 2009). Reads that were unmapped were removed from the set by this data analysis pipeline.
To remove noise coming from low-quality reads and contaminants, as well as to mask repeatable genomic sequences, reads with mapping quality lower 13 (p-value < 0.05) were removed.
BEDTools (Quinlan and Hall, 2010) were used to create genome coverage bedgraphs, which were converted into bigWig format.
Genome_build: GRCh37 (hg19)
Supplementary_files_format_and_content: bigWig (Score); Score : Genome coverage.
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| Submission date |
Mar 04, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Takamitsu Hattori |
| Organization name |
The University of Chicago
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| Department |
Department of Biochemistry and Molecular Biology
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| Lab |
Koide lab
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| Street address |
900 E. 57th Street
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE66530 |
ChIP-seq validation of recombinant antibodies to histone post-translational modifications |
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| Relations |
| BioSample |
SAMN03387302 |
| SRA |
SRX897637 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1624500_Hek_309M3_Input_S1_mQ13.bigwig |
209.8 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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