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| Status |
Public on Oct 26, 2020 |
| Title |
CS170_H3K9MFlag_FlagChIP |
| Sample type |
SRA |
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| Source name |
CS170
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: hht3-K9M-Flag
media: YEA
antibody: A2220, M2 Flag agarose
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| Growth protocol |
Cells were grown in YEA until mid-log phase
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Log-phase yeast cells were incubated at 18 ̊C for 2 hours and then fixed for 30 minutes in 3% freshly made formaldehyde. The crosslinking reaction was stopped by the addition of 2.5 M Glycine to make a final concentration of 0.125 M. The cells were pelleted and washed with PBS (phosphate buffered saline) before resuspended in ChIP lysis buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1% Triton X-100, 0.1% Deoxycholate supplemented with cOmplete protease inhibitors cocktail (Roche)). Ice cold glass beads were added and the mixtures were vigorously disrupted in a bead- beater. The lysates were collected and subjected to sonication to reduce chromatin size to 500– 1000 base pairs. The cleared cell lysates were incubated with anti-Flag (Sigma) over night at 4 ̊C. Protein G beads were then added to isolate the antibodies and associated chromatin fragments. The beads were then washed with ChIP lysis buffer twice, ChIP lysis buffer containing 0.5 M NaCl, Wash buffer (10 mM Tris, pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% Deoxycholate, 1 mM EDTA), and TE (50 mM Tris pH 8.0, 1 mM EDTA). The bound chromatin fragments were eluted with TES (50 mM Tris pH 8.0, 1 mM EDTA, 1% SDS) and the crosslinking was reversed by incubating at 65 ̊C overnight. The protein DNA mixture were then subjected to proteinase K treatment and phenol:chloroform extraction before the DNA was precipi- tated by ethanol.
Libraries were prepared according to TruSeq ChIP sample preparation guide (Illumina)
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
ChIP-seq
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| Data processing |
The raw reads were trimmed by Trimmomatic (v0.35) to remove remaining adapter contaminants and low-quality regions. The trimmed reads were aligned to the S. pombe reference genome (Ensembl assembly version: ASM294v2.29) by bwa (v0.7.12-r1039) (http://bio-bwa.sourceforge.net/). The read-mapping files were further processed by Samtools (v1.2) picard-tools (v2.0.1) (http://broadinstitute.github.io/picard/) and GATK (v3.5) for indexing, sorting, PCR duplicates removal, and local-realignment around Indels. The per-based mapping depth was calculated by bedtools (v2.25.0) and the sliding window analyses were further performed using a 100-bp window size and 50-bp step size.
Genome_build: ASM294v2.29
Supplementary_files_format_and_content: tdf: coverage tracks
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| Submission date |
Oct 07, 2020 |
| Last update date |
Oct 27, 2020 |
| Contact name |
Songtao Jia |
| E-mail(s) |
kb2830@columbia.edu
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| Organization name |
Columbia University
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| Department |
Biological Sciences
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| Street address |
116th & Broadway
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| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10027 |
| Country |
USA |
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| Platform ID |
GPL17225 |
| Series (1) |
| GSE159192 |
Mechanism for the selective sequestration of a histone H3K9 methyltransferase at heterochromatin by the H3K9M mutation |
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| Relations |
| BioSample |
SAMN16391713 |
| SRA |
SRX9258729 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4822364_RES15-16-P9-ChIP-cs170-flag-ChIP_Run1.coverage.tdf |
28.6 Mb |
(ftp)(http) |
TDF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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