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| Status |
Public on Sep 10, 2021 |
| Title |
spt6-1004 Spt5-V5 ChIP-Seq replicate 3 |
| Sample type |
SRA |
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| Source name |
log phase S. cerevisiae
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: FY3334
temperature: 30C
genotype: spt6-1004
chip target: Spt5-V5
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| Growth protocol |
S.cerevisiae cells were grown to OD600 ≈ 0.6 (~2x10^7 cells/ml) in YPD at 30ºC
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq was performed as described previously (Gopalakrishnan et al. 2019, Nucleic acids research) and is reproduced here. Cultures were cross-linked by the addition of formaldehyde to a final concentration of 1% followed by incubation with shaking at room temperature for 20 minutes. Glycine was added to a final concentration of 125 mM and the incubation was continued for 10 minutes. The cells were pelleted and washed twice with cold 1x TBS (100mM Tris, 150 mM NaCl, pH 7.5) and once with cold water. The cell pellets were then suspended in 800 µl cold LB140 buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1x cOMPLETE Protease Inhibitor tablet (Roche)). One ml of glass beads was added and the cells were lysed by bead beating for 8 minutes at 4oC with incubation on ice for 3 minutes after every one minute. The lysate was collected and centrifuged at 12,500 rpm for 5 minutes and the resulting pellet was washed once with 800 µl cold LB140 buffer. The pellet was resuspended in 580 µl cold LB140 buffer and sonicated in a QSonica Q800R machine for 20 minutes (30 seconds on, 30 seconds off, 70% amplitude). The sonicated samples were centrifuged at 12,500 rpm for 30 minutes and the resulting supernatant was taken for the immunoprecipitation step. 300-500 µg of S. cerevisiae chromatin was mixed with 33-55 µg (10%) of S. pombe chromatin and the volume was brought up to 800 µl with WB140 buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate). This was used as the input for the immunoprecipitation reaction. Antibody was added to the input and the samples incubated overnight at 4oC with end-over-end rotation. Fifty µl of Protein G sepharose beads (GE healthcare) pre-washed twice in WB140 was added to the IPs and samples were incubated for 4 hours at 4oC with end-over-end rotation. . The beads were washed twice with WB140, twice with WB500 (50 mM HEPES-KOH, pH 7.5, 500 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate), twice with WBLiCl (10 mM Tris pH 7.5, 250 mM LiCl, 1mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate) for two minutes each and once with TE (10 mM Tris pH 7.4, 1 mM EDTA) for five minutes. The immunoprecipitated material was eluted twice with 100 µl TES (50 mM Tris pH 7.4, 1 mM EDTA, 1% SDS) at 65oC for 30 minutes. The eluates were incubated at 65oC overnight to reverse the crosslinking. Two hundred µl of TE was added to the eluates followed by RNase A/T1 to a final concentration of 0.02 µg/µl. The samples were incubated at 37oC for 2 hours. Proteinase K was added to a final concentration of 0.4 mg/ml and samples were incubated at 42oC for 2 hours. DNA was purified using Zymo DCC (for ChIP-Seq).
The library preparation was done as described in Wong et al., 2013, Current protocols in molecular biology
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
ChIP-Seq of Spt5-V5 in spt6-1004 S. cerevisiae cells grown at 30oC
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| Data processing |
Reads were trimmed using cutadapt (base quality threshold of 20)
Reads were aligned to a combined S. cerevisiae + S. pombe genome using Bowtie2
Average fragment size of DNA molecules sequenced was determined by cross-correlation (done in R using the SPP package)
Reads mapping to S. cerevisiae genome were separated from the reads mapping to the S. pombe genome
Coverage files (wig format) were produced using the igvtools count function, extending reads by (length of fragment sizes - average read length) for each library.
wig format files were converted to the bedgraph format
Spike in normalization was done as described previously (Orlando et al., 2014), correcting for variations in the input samples.
Genome_build: Saccharomyces cerevisiae S288C R64-2-1, Schizosaccharomyces pombe ASM294v2
Supplementary_files_format_and_content: The bedGraph files contain spike-in normalized coverage of DNA fragments obtained after immunoprecipitation. Average fragment length determined using cross-correlation
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| Submission date |
Apr 12, 2021 |
| Last update date |
Sep 10, 2021 |
| Contact name |
Fred Winston |
| E-mail(s) |
[email protected]
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| Organization name |
Harvard Medical School
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| Department |
Genetics
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| Street address |
77 Avenue Louis Pasteur
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL19756 |
| Series (1) |
| GSE171953 |
The histone chaperone Spt6 is required for normal recruitment of the capping enzyme Abd1 to transcribed regions |
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| Relations |
| BioSample |
SAMN18718140 |
| SRA |
SRX10583009 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5238741_v5-spt6-3_Scer_normSI.bedgraph.gz |
4.6 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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