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| Status |
Public on May 07, 2016 |
| Title |
human1_RS_K27me3_ChIP |
| Sample type |
SRA |
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| Source name |
adult testis
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| Organism |
Homo sapiens |
| Characteristics |
tissue: testis
cell type: round spermatids
chip antibody: H3K27me3 (abcam, #ab6002)
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| Growth protocol |
Human testis samples were obtained from patients undergoing vasectomy reversals at the Infertility Clinic of St. Louis.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Pachytene spermatocyte and round spermatid fractions were collected by StaPut (Bellve et al 1993). Purity was assessed under phase optics and was consistently >95% for each desired cell type. Cells were washed once in PBS and then split into two aliquots. One aliquot (for ChIP) was fixed in 1% formaldehyde then quenched with 2.5M glycine, while the second (for RNA) was left unfixed. Both fixed and unfixed aliquots were snap frozen in liquid nitrogen, then stored at -80C. ChIP was performed as previously described (Lesch et al 2013).
ChIP-seq libraries were prepared using a TruSeq ChIP sample prep kit (Illumina) according to the manufacturer’s instructions, except that size selection was performed after instead of before PCR amplification.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Image analysis and base calling was done using the standard Illumina pipeline for HiSeq2500.
Data was quality-filtered using fastq_quality_filter from the FASTX toolkit with the following parameters: -q 20 -p 80. For libraries with >50% adapter reads (one library total: human_PS_K4me3_ChIP), reads containing only adapter sequence were discarded using fastx_clipper.
For ChIP-seq, alignment was done using Bowtie 1.1.1 with the following parameters: -k 1 -m 1 -n 1 -l 40. For RNA-seq, alignment was done using TopHat 2.0 with default settings, with Ensembl transcripts as a reference (-G parameter).
For ChIP-seq, peaks were called using MACS 1.4 at p < 10^-5 (-p 1e-5), with default parameters.
For ChIP-seq, raw counts in the +/- 2kb intervals surrounding the transcription start sites were calculated using htseq-count with the following settings: -m intersection-nonempty -s no.
genome build: hg19
processed data files format and content: All files are tab-delimited text (.txt). For RNA-seq, FPKM is reported for each Ensembl (build 75) transcript and gene. For ChIP-seq, raw counts normalized to (aligned, non-duplicated) library size are reported for the +/- 2kb region surrounding each Ensembl (build 75) transcript.
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| Submission date |
May 04, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Bluma J Lesch |
| Organization name |
Yale University
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| Department |
Genetics
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| Street address |
333 Cedar Street
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| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06525 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE68507 |
Cross-species comparison of epigenetic poising in the mammalian germ line |
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| Relations |
| BioSample |
SAMN03481897 |
| SRA |
SRX994301 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1673961_human1_RS_K27me3_ChIP_counts.txt.gz |
284.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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