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| Status |
Public on Jul 30, 2019 |
| Title |
H3K56ac/H3_Input_spt16-197_rep2 |
| Sample type |
SRA |
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| Source name |
Yeast cell extract (MATα, leu2Δ1, spt16-197)
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
spike-in: Schizosaccharomyces pombe (yFR2050 strain)
strain name: yFR973
genotype: MAT{alpha}, leu2{delta}1, spt16-197
chip antibody: none
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| Treatment protocol |
For both S. cerevisiae and S. pombe strains, cultures were crosslinked with 1% formaldehyde (Fisher Scientific, BP531-500) at room temperature for 30 min. Crosslinking was quenched with 125 mM glycine.
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| Growth protocol |
Cells were grown at 30°C and 200 rpm in YPD (yeast extract-peptone-2% glucose) medium as follow. 50 mL of YPD medium was inoculated at OD600 0.1 from an overnight preculture in the same medium and allowed to grow until it reached OD600 ~0.5. Cell cultures were then switched to 37°C and allowed to grow for additional 80 min before crosslinking, at which point they had reached ~0.9 OD600. The S. pombe strain (yFR2050) used as spike-in control was grown in similar conditions as the S. cerevisiae strains but omitting the switch to 37°C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Crosslinked cells were collected by centrifugation and washed twice with 1 X TBS (20 mM Tris-HCl pH 7.5, 150 mM NaCl). Cells were then resuspended in 700 uL Lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 1 mM PMSF, 1 mM Benzamidine, 10 μg/mL Aprotinin, 1 μg/mL Leupeptin, 1 μg/mL Pepstatin). The same number of OD600 units was used for all samples. A fixed amount of crosslinked S. pombe cells (representing 10% of the S. cerevisiae cells) was added to the S. cerevisiae cells prior to cell lysis. This spike-in control was used to normalise samples. Cells were lysed by bead beating and the lysate was sonicated with a Model 100 Sonic dismembrator equipped with a micro probe (Fisher Scientific), 4 x 20 sec at output 7 Watts, with a 1 min break between sonication cycles. Soluble fragmented chromatin was recovered by centrifugation. 20 uL of the chromatin sample were saved for control of the average chromatin fragment size. For H3K4me3 and H4 immunoprecipitations, 25% (150 uL) of the chromatin sample, completed to 600 uL with Lysis buffer, was used. For H3K56ac and H3 immunoprecipitations, 50% (300 uL) of the chromatin sample, completed to 600 uL with Lysis buffer, was used. 6 uL (1%) of the diluted chromatin samples were saved as Input sample. 12.5 uL of Protein G Dynabeads (Thermo Fisher Scientific, 10004D) pre-coupled with the indicated rabbit polyclonal antibody were added to the 600 uL of diluted chromatin sample and incubated overnight at 4°C. Beads were washed twice with Lysis buffer, twice with Lysis buffer 500 (Lysis buffer + 360 mM NaCl), twice with Wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EDTA) and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Immunoprecipitated chromatin was eluted and reverse-crosslinked with 50 uL TE/SDS (TE + 1% SDS) by incubating overnight at 65°C. Input and fragment size control samples were also added with 50 uL TE/SDS and incubated overnight at 65°C to reverse crosslinking. Eluted chromatin as well as Input and fragment size control samples were treated with RNase A (345 uL TE, 3 uL RNAse A 10 mg/mL, 2 uL Glycogen 20 mg/mL) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 uL 10% SDS, 7.5 uL Proteinase K 20 mg/mL) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 100 % EtOH. Precipitated DNA was resuspended in 50 uL of TE and subjected to a 1.7X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions. Samples were eluted with 40 uL of elution buffer (10 mM Tris pH 8.0). The sample used for determining the average chromatin fragment size was analysed on an Agilent 2100 Bioanalyzer instrument using Agilent High Sensitivity DNA Kit. DNA concentration of the ChIP and Input samples was determined by qPCR using a standard curve made with the fragment size control sample. 3-10 ng of ChIP and Input DNA were used for ChIP-seq library preparation as follow. The ends of DNA were repaired using T4 DNA polymerase (NEB, M0203L) and T4 polynucleotide kinase (NEB, M0201S) at 12˚C for 30 min. Repaired DNA was then subjected to a 1.7X cleanup using KAPA Pure Beads before dA tailing using Klenow Fragment (3'→5' exo-) (NEB, M0212M) at 37˚C for 30 min. After a 1.8X cleanup with KAPA Pure Beads, the A-tailed DNA was ligated to index adapters (Roche, SeqCap Adapter kit A, 12 adapters (07141530001) and SeqCap Adapter kit B, 12 adapters (07141548001)) using T4 DNA ligase (ThermoFisher, 15224041) at room temperature for 60 min. The ligated DNA was then subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.52X-1X) leading to fragments in the 200-600 bp range. Libraries were PCR amplified with 12-13 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and cleaned up using 1X KAPA Pure Beads. Libraries were qualified on Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA Kit and quantified by qPCR using NEBNext Library Quant Kit for Illumina. Equal molarity of each library was pooled (12 libraries per pool) and subjected to sequencing on an Illumina HiSeq 2500 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp single-end reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Adapter 4 from SeqCap Adapter kits A and B (Roche, 07141530001 and 07141548001)
Biological replicate 2 of 2. Input signal of spt16-197 temperature-sensitive cells treated 80 minutes at 37°C. This Input DNA was obtained from the same yeast extract used to perform H3K56ac and H3 ChIP-seq.
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| Data processing |
Reads for each biological replicate were independently aligned to the S. cerevisiae (UCSC sacCer3) and S. pombe (Downloaded from https://www.pombase.org/ on March 10th 2018) reference genomes using the short read aligner Bowtie 2 (version 2.2.4) (Langmead and Salzberg, 2012). Coverage for each base pair of the S. cerevisiae genome was computed using genomeCoverageBed from BEDTools (version 2.19.1) (Quinlan and Hall, 2010) and normalized using S. pombe reads as follow. The read density at each position of the S. cerevisiae genome was multiplied by a normalisation factor N defined as: N = 1,000,000 * P / R * C where: 1,000,000 is an arbitrary chosen number used for convenience. R is the total number of reads in the ChIP sample that mapped to the S. pombe genome. C is the total number of reads in the Input sample that mapped to the S. cerevisiae genome. P is the total number of reads in the Input sample that mapped to the S. pombe genome. Simpler normalisation schemes without the use of the Input sample (hence, leaving out C and P) were used before (Bonhoure et al., 2014; Chen et al., 2015b; Hu et al., 2015) and should, in theory, allow for direct comparisons between samples. Such simple scheme, however, assumes that the same number of cells is used as starting material for all ChIP samples. As routinely done when working with yeast, we evaluated cell number using the OD600 of our cultures. In the course of analysing our ChIP-seq data, we noticed that the ratio of the reads mapping to the S. cerevisiae versus the S. pombe genome was systematically lower in the Input material from spt16-197 and spt6-1004 samples compared to the WT sample, suggesting that the number of spt16-197 and spt6-1004 cells used in our experiments was overestimated. We then confirmed that at identical OD600, spt16-197 and spt6-1004 cultures contain fewer cells than their wild type counterparts using dilutions/plating assays (not shown). For FACT, this observation is most likely due to the known fact that spt16-197 cells elicit a delay in G1 (Morillo-Huesca et al., 2010), hence skewing the population for larger cells. Despite this has never been reported before for Spt6, similar reasons may explain the observed spt6-1004 result. To correct for that defect, we used the sequencing of the Input samples and introduced the variables C and P in our scaling factor. Note that the conclusions reached in our analyses were the same, whether we used C and P or not. Replicate samples showed high correlation and were combined using MergeSamFiles from Picard Tools (version 1.56.0) (http://broadinstitute.github.io/picard/) prior to the calculation of the coverage and the normalization.
Genome_build: S. cerevisiae (UCSC sacCer3) and S. pombe (Downloaded from https://www.pombase.org/ on March 10th 2018)
Supplementary_files_format_and_content: Normalized BigWig files (ending in “norm.bigwig”) represent individual replicate ChIP fragment coverage normalized using reads mapped to the S. pombe genome. Combined normalized BigWig files (ending in “comb.bigwig”) represent combined replicates ChIP fragment coverage normalized using reads mapped to the S. pombe genome.
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| Submission date |
Apr 16, 2018 |
| Last update date |
Jul 30, 2019 |
| Contact name |
Francois Robert |
| E-mail(s) |
[email protected]
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| Organization name |
IRCM
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| Lab |
Chromatin and Genomic Expression
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| Street address |
110 av des Pins Ouest
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| City |
Montreal |
| State/province |
QC |
| ZIP/Postal code |
H2W 1R7 |
| Country |
Canada |
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| Platform ID |
GPL17342 |
| Series (2) |
| GSE113213 |
Histone Recycling by FACT and Spt6 during Transcription Prevents the Scrambling of Histone Modifications [ChIP-seq] |
| GSE113270 |
Histone Recycling by FACT and Spt6 during Transcription Prevents the Scrambling of Histone Modifications |
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| Relations |
| BioSample |
SAMN08939459 |
| SRA |
SRX3944379 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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