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| Status |
Public on Apr 15, 2017 |
| Title |
mock for CgPho2 ChIP w/ CgPho4 in high Pi media |
| Sample type |
SRA |
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| Source name |
OD600=0.3~0.5 log-phase yeast culture
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| Organism |
Nakaseomyces glabratus |
| Characteristics |
strain: CG43
condition: Synthetic media with 10 mM inorganic phosphate
type: ChIP (mock)
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| Treatment protocol |
300 mL of the culture was filtered and washed with 100 mL of synthetic media containing 10 mM inorganic phosphate, after which the cells were released into 300 mL of fresh synthetic media with 10 mM inorganic phosphate and grown for 1 hr prior to extraction.
After the sample specific treatment, 300 mL of OD600=0.3-0.5 cell cultures was cross-linked by adding 8.33 mL of 37% formaldehyde (final formaldehyde concentration = 1%) and incubated with swirling for 10 minutes at room temperature. The sample was then quenched by adding 2.5 M glycine solution to a final concentration of 125 mM, and incubated with swirling for 5 minutes at room temperature. Cells were then collected by centrifugation and washed two times with ice-cold TBS.
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| Growth protocol |
Cells were pre-grown in 10 mM Pi Synthetic Complete (SC) medium to saturation, diluted 1:100 and regrown to saturation. The culture is then diluted to OD600 ~ 0.1 in fresh 10 mM Pi SC medium and grown to OD600 ~ 0.3-0.5.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The cross-linked cell sample was resuspended in ~1 mL of ChIP lysis buffer (50 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate), and mechanically lysed using a mini-Beadbeater-24 (BioSpec) with 0.5 mm Zirconia beads (BioSpec), using the following setting: 12 cycles of 30s bead beating, with 1 min on ice-water bath in between. The lysed sample was fragmented by sonication using Covaris E220 Adaptive Focus system (setting: Duty Factor: 10%; Peak Incident Power: 175; Cycles/Burst: 200; Time:150s). Afterwards, the sample was centrifuged to remove the unsoluable fraction. The supernatant containing the solubalized chromatin was subjected to immunoprecipitation. Briefly, the supernatant was incubated with either Anti-FLAG M2 magnetic beads (Sigma-Aldrich) or Anti-V5 antibody (Abcam #15828) depending on the epitope tag that was used. After 2 hours of incubation at 4C with rotation, the V5-tagged samples were additionally incubated with Dynabeads Protein-G (Thermo-Fisher Scientific #10003D). In a traditional ChIP experiment, what follows are the washing and de-crosslinking, prior to DNA extraction. In ChIP-exo, however, the washing steps are combined with part of the library construction steps. A detailed account for the protocol can be found in (Rhee and Pugh, 2012), and is also available from Active Motif ChIP-exo vA4 manual.
As mentioned above, the ChIP washing step is combined with part of the library construction steps in ChIP-exo. A brief description of the steps is provided here: with the chromatin still bound to the beads, the DNA was end-polished and P7-exo adapters are ligated onto the blunt ends. The nicked DNA was repaired and then digested by lambda and RecJf exonulceases to excise DNA in a 5’ to 3’ direction, trimming up to the site of the cross-linking and selectively eliminating the P7 adapter at the 5 ́ end. Following cross-link reversal and elution from the beads, the DNA was made double-stranded by P7 primer extension and a P5- exo adapter was added to the exonuclease-treated ends. The DNA library was PCR amplified and size selected before it was subjected to high-throughput sequencing.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
F6
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| Data processing |
Basecalling using bcl2fastq-1.8.3
Sequencing reads were aligned to the corresponding species reference genome using Bowtie (v1.1.1) with the option ‘-m 1 --best –strata’
The resulting SAM files were sortedand indexed using Samtools (v1.2)
Peak calling for CgPho4 was performed using GEM v2.6 with the option '--smooth 3 --fold 1.5', using mock samples as the control, and using the Read Distribution for ChIP-exo file downloaded from the authors' website. GEM didn't work for CgPho2 due to lack of strong sequence motif. Instead, we used MACS2 with the option "-s 50 --bw -q 0.01 --keep-dup auto --slocal 1000 -B --verbose 4 -m 2 100 --call-summits”.
To generate a visual fold enrichment profile for each ChIP experiment (including the mock samples), we used MACS2's "build signal track" feature to calculate a fold enrichment value at each position of the genome, using the input sample as the control. The resulting "BedGraph" format file was converted to "WIG" format using "bedgraph_to_wig.pl" downloaded from https://gist.github.com/svigneau/8846527. The WIG file was then imported into MochiView v1.6 for visualization.
Genome_build: C. glabrata genome version s02-m02-r09
Supplementary_files_format_and_content: Peaklist
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| Submission date |
Apr 14, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Bin He |
| E-mail(s) |
emptyhb@gmail.com, bin-he@uiowa.edu
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| Organization name |
the University of Iowa
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| Department |
Biology
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| Lab |
Gene Regulatory Evolution
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| Street address |
129 East Jefferson St., Biology Building 450
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| City |
Iowa City |
| State/province |
IA |
| ZIP/Postal code |
52242 |
| Country |
USA |
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| Platform ID |
GPL22622 |
| Series (2) |
| GSE97798 |
Evolution of Reduced Co-Activator Dependence Led to Target Expansion of a Starvation Response Pathway [Cgla ChIP-seq] |
| GSE97801 |
Evolution of Reduced Co-Activator Dependence Led to Target Expansion of a Starvation Response Pathway |
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| Relations |
| BioSample |
SAMN06756868 |
| SRA |
SRX2737273 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2577555_F6_mock_CgPho2_PHO4_hiPi_CCGTCC.wig.gz |
9.2 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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