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| Status |
Public on May 31, 2017 |
| Title |
ChIPseq.YFP_HumanPRDM9.antiH3K4me3.ProtocolN |
| Sample type |
SRA |
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| Source name |
HEK293T cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T (ATCC CRL-3216)
transfected with: YFP_HumanPRDM9
chip antibody: H3K4me3 (Abcam ab8580)
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| Treatment protocol |
A transfection cocktail was prepared for each bottle by adding 0.5 mg of chloroform-purified construct DNA to 50 ml of serum-free DMEM (1X NEAA, 2 mM L-glut) and 1 mg polyethylenimine, followed by a 10-minute incubation, and then addition of 375 ug of kifunensine. After the cells reached 75% confluence, the growth medium was removed from each roller bottle and replaced with 200 ml low-serum DMEM (2% FCS, 1X NEAA, 2 mM L-Glut) and 50 ml transfection cocktail.
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| Growth protocol |
Cells were grown in DMEM media (10% FCS, 1X NEAA, 2 mM L-Glut, Sigma D6546) in 200-ml roller bottles at 37C with 5% CO2
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed as described in Gupta-Hinch et al. 2014
Libraries were prepared for sequencing using standard Illumina protocols by the Oxford Genomics Centre.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1000 |
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| Description |
Uniquely Mapped Fragments: 77625060
Multiplex Group: 1
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| Data processing |
ChIP-seq: Reads were aligned to hg19 using BWA (v0.7.0-r313, option -q 10) followed by Stampy (v1.0.23_(r2059), option --bamkeepgoodreads). PCR duplicates, reads not mapped in a proper pair, reads with an insert size larger than 10 kb, and read pairs for which neither read had a mapping quality score greater than 0 were removed using samtools (v0.1.19-44428cd). For samples with only one replicate, fragments were split at random into two equally-sized pseudo-replicates. Fragment coverage from each replicate was then computed at each position in the genome using in-house code and the samtools (v0.1.19-44428cd) and bedtools (v2.23.0, genomecov -d) packages. Peaks were called using a custom peak calling algorithm (described in Gupta-Hinch et al. 2014). Enrichments and p-values are reported for the base with the maximum likelihood value within each peak region.
ATAC-seq: Reads were mapped to hs37d5 using BWA (v0.7.0-r313) followed by Stampy (v1.0.23_(r2059), with option --bamkeepgoodreads). PCR duplicates, mtDNA-mapped reads, reads not mapped in a proper pair, reads with mapping quality equal to 0, and pairs with an insert size larger than 2 kb were removed using samtools (v0.1.19-44428cd). Fragments were split by size into inter-nucleosome (51-100 bp) and mono-nucleosome fragments (180-247 bp), and the position of the central base in each fragment was reported.
RNA-seq: We created a custom reference sequence by merging the hs37d5 reference with the construct and vector sequences transfected into our cells. Data were analyzed using the Tuxedo software package. Reads were mapped and processed using TopHat (version 2.0.13, options --mate-inner-dist=250 --mate-std-dev 80 --transcriptome-index=Ensembl.GRCh37.genes.gtf); followed by Cufflinks, CuffQuant, and CuffDiff (version 2.2.1); then analyzed using CummeRbund.
Genome_build: hg19 (ChIP-seq), hs37d5 (ATAC-seq), hs37d5+ (RNA-seq)
Supplementary_files_format_and_content: bedgraph files contain fragment coverage data for each base across the 22 autosomes and X chromosome. For ChIP-seq samples, these values were used as inputs for de novo peak calling and force-calling. ChIP-seq peak call files are in tab-delimited text format, with one peak per rown and column headers indicating the contents of each column. For ATAC-seq samples, fragment coverage data were split into subsets representing internucleosome fragments (51-100 bp long) and mononucleosome fragments (180-247 bp long), and only the position of the central base of each fragment is reported. For RNA-seq samples, the final CuffDiff output file (tab-delimited diff format) is provided.
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| Submission date |
May 30, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Nicolas Altemose |
| Organization name |
University of California, Berkeley
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| Department |
Bioengineering
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| Lab |
Aaron Streets
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| Street address |
327 Stanley Hall
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| City |
Berkeley |
| State/province |
CA |
| ZIP/Postal code |
94720 |
| Country |
USA |
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| Platform ID |
GPL15433 |
| Series (1) |
| GSE99407 |
Mapping PRDM9 binding and its effects in transfected HEK293T cells |
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| Relations |
| BioSample |
SAMN07176248 |
| SRA |
SRX2867674 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2643614_ChIPseq_FragmentDepth.YFP_HumanPRDM9.antiH3K4me3.ProtocolN.q1.rmdup.hg19.bedgraph.gz |
612.8 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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