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| Status |
Public on Oct 30, 2012 |
| Title |
Hog-HA 0' Rep2 |
| Sample type |
SRA |
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| Source name |
Yeast
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
background strain: BY4741
genotype: Hog1-6HA::HIS3
chip antibody: anti-HA 12CA5
condition: not exposed to osmostress
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| Treatment protocol |
Exposed to osmostress (0.4M NaCl) for 5 minutes for Hog1 immunoprecipitation (anti-HA 12CA5) and for 10 minutes for RNA Pol II immunoprecipitation (8WG16, Covance)
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| Growth protocol |
Yeast cultures were grown to early log phase.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Samples were initially quantified with the double-strand specific Quanti-iT dsDNA HS Assay Kit (Invitrogen) to make sure enough starting material had been provided (usually 10ng). Following end-repair of DNA by incubation with T4 DNA polymerase and Klenow fragment (NEB), DNA was purified with a QIAquick PCR purification kit (Qiagen). Thereafter, 3’-adenylation was performed by incubation with dATP and 3’à5’ exo- Klenow fragment (NEB). DNA was purified using MinElute spin columns (Qiagen) and double-stranded adapters (Adapter1/Adapter2) were ligated to the DNA using rapid T4 DNA ligase (NEB). The sample was purified again using a MinElute spin column and run on a 2% agarose gel and fragments in the size range of interest (aproximately 250 bp plus 65 bp of adapters for usual ChIP samples and 150 bp for MNAse digested ChIP samples) were excised with a sterile single-use scalpel and recovered from the gel by QIAquick gel extraction. Then, Adapter-ligated fragments were enriched, and adapters were extended, by selectively amplifying with an 18-cycle PCR reaction using Phusion DNA polymerase (Finnzymes), and primers 1.1 and 2.0 (in this step adapters are extended to a total of 112 bp). Finally, the quality of libraries was confirmed on the Agilent Technologies 2100 Bioanalyzer, and by cloning into a blunt TOPO vector. Six colonies were sequenced by conventional Sanger method to verify correct adapter ligation and sequence match.
Libraries were quantified by TaqMan Universal PCR Master Mix, No AmpErase kit (Applied Biosystems-Roche) and specific oligos and probe. DNA was loaded into a lane of a single-read flow cell for cluster generation using a single-read cluster generation kit v4 (Illumina). During this process, DNA molecules are immobilized on the surface of the flow cell, amplified in situ to create same-sequence containing clusters, and following surface blocking and DNA denaturation, binding of the sequencing primer was performed. The flow cell was then mounted on a Genome Analyzer IIx instrument for sequencing, and 36 sequencing cycles were performed, using v4 SBS kits sequencing kits. Each flow cell we run on the Genome Analyzer IIx contains a PhiX control lane (loaded at a concentration of 4pM), used to monitor run quality.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Reads were mapped using GEM mapper version build 539 with default parameters
Calculated heights using Pyicos convert version 1.0.6c default parameters
Calculated enrichment using Pyicos enrichment version 1.0.6c
Genome_build: sarCer2
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| Submission date |
Oct 11, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Francesc Posas |
| Organization name |
UPF
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| Street address |
Dr. Aiguader
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| City |
Barcelona |
| ZIP/Postal code |
08003 |
| Country |
Spain |
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| Platform ID |
GPL13272 |
| Series (1) |
| GSE41494 |
Hog1 bypasses stress-mediated down-regulation of transcription by PolII redistribution and chromatin remodeling |
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| Relations |
| SRA |
SRX193517 |
| BioSample |
SAMN01760927 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1018224_100810_lane7_hog1_HA_5b_unambiguous.bed.gz |
172.9 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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