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| Status |
Public on Apr 13, 2022 |
| Title |
WT_H3K4me3_eSPAN_S60 |
| Sample type |
SRA |
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| Source name |
whole cell
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| Organism |
Saccharomyces cerevisiae W303 |
| Characteristics |
treatment: MNase
strains genetic background: W303
genotype: WT
chip antibody: anti-H3K4me3 (ab8580, Abcam)
spike-in: none
tissue: whole cell
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| Growth protocol |
Yeast cells were grown in rich medium (1% yeast extract, 2% peptone) supplemented with 2% glucose (YPD) or, for galactose-induction experiments, with 2% raffinose (YPRaff). Exponentially growing cells were arrested at G1 phase with 5 ug/ml alpha-factor (EZBiolab) for 3 hours (YPD) or 3.5 hours (YPRaff). Degradation of AID-tagged proteins was achieved with the addition of 1 mM 3-indolacetic acid (IAA, I2886 Sigma) into the media. Induction of proteins under the GAL1 promoter was achieved with the addition of 2% galactose into YPRaff.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
All ChIP and eSPAN assays were performed as described previously (Yu et al., 2014). For H3K4me3 and H3K56ac ChIP, chromatin was pelleted, washed twice with NP buffer (1.6 M sorbitol, 2 mM CaCl2, 5mM MgCl, 50 mM NaCl, 14 mM b-mercaptoethanol, 10 mM Tris-HCl (pH 7.4), 0.075% NP-40, 5 mM spermidine), resuspended in the same buffer, and digested with MNase (9013-53-0, Worthington Biochemical) for 20 min at 37C yielding a majority of mono- and di-nucleosome fragments. Digestion was stopped with the addition of 5 l 0.5 M EDTA, 1/5 volume of 5xChIP lysis buffer, and incubating 10 min on ice. For RNA Pol II CTD-Ser5p ChIP, chromatin was sheared by sonication using a Bioruptor Pico (15 seconds ON, 30 seconds OFF, 10 cycles)(Diagenode) to yield fragments of an average size of 300 bp. Sonicated and MNase-digested chromatin was cleared by centrifugation and immunoprecipitated with the appropriate antibody (anti-H3K4me3 (ab8580, Abcam), anti-H3K56ac (38), or anti-RPB1 CTD-Ser5p (04-1572, Millipore)) and protein G Sepharose beads (17-0618-02, GE Healthcare). After washing the beads extensively, DNA from both input and ChIP was recovered with the Chelex-100 protocol (39) and used for BrdU IP/eSPAN or purified with MinElute PCR Purification kit (28004, Qiagen). Single-stranded DNA sequencing libraries were prepared using the Accel-NGS 1S Plus DNA library kit (10096, Swift Biosciences). Input and ChIP DNA was used for BrdU immunoprecipitation to prepare BrdU IP and eSPAN samples, respectively. Recovered DNA from the Chelex-100 extraction was incubated at 100C for 5 min and immediately chilled on ice for 5 min. BrdU immunoprecipitation was performed by diluting DNA 10-fold with BrdU IP buffer (PBS, 0.0625% Triton X-100(v/v), 6.7 g/ml Escherichia coli tRNA, 0.17 g/ml BrdU antibody (555627, BD Bioscience)) and incubated for 2 hours at 4C. Protein G Sepharose beads were added to each sample for 1 additional hour at 4C. Beads were washed extensively and DNA was recovered with TE + 1% SDS at 65C for 15 min and purified with MinElute PCR Purification kit (28004, Qiagen). Single-stranded DNA sequencing libraries were prepared using the Accel-NGS 1S Plus DNA library kit (10096, Swift Biosciences).
ChIP-seq libraries were prepared and deep sequencing was performed by Columbia University Genome Center.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Raw reads were trimmed to remove sequencing adaptors and low-quality reads using Trim Galore (version 0.6.7) with default parameters.
Sequence reads were mapped back to the yeast reference genome (sacCer3) using Bowtie2 software. Only consistent pair-end reads were chosen for further analysis.
For BrdU-IP-ssSeq and eSPAN strand bias calculations, consistent paired-end reads mapped to Watson (W) and Crick (C) strands of the reference genome were separated with the bamtobed function from BEDTools and in-house Perl programs. Genome-wide reads coverage was calculated by deepTools. Strand bias was calculated in 10bp bins from these separated Watson and Crick reads using the formula Bias = (W − C)/(W + C) across the whole genome.
MACS2 was used to perform BrdU peak calling (--broad-cutoff 0.001) in the BrdU-IP-ssSeq samples relative to the corresponding control samples (input).
For spike-in normalization, we first followed the same procedure as above to align FASTQ files to the S. pombe reference genome. S. cerevisiae genome coverage at each base pair was multiplied by scaling factor based on S. pombe reads as described previously (Jeronimo et al., 2019).
Genome_build: S. cerevisiae (UCSC sacCer3) and S. pombe (Downloaded from https://www.pombase.org/ on March 10th 2018)
Supplementary_files_format_and_content: bigwig files were generated for plus strand, minus strand and both strands, scores represent the normalization reads density.
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| Submission date |
Sep 15, 2021 |
| Last update date |
Apr 14, 2022 |
| Contact name |
Zhiguo Zhang |
| E-mail(s) |
zz2401@cumc.columbia.edu
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| Phone |
212-851-4936
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| Organization name |
Columbia University
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| Department |
Pediatric and Genetics and Development
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| Lab |
Irving Cancer Research Center
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| Street address |
1130 St. Nicholas Avenue
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
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| Platform ID |
GPL27477 |
| Series (1) |
| GSE184188 |
H3K4me3 recognition by the COMPASS complex guides de novo histone methylation to restore the symmetric distribution of H3K4me3 following DNA replication |
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| Relations |
| BioSample |
SAMN21441818 |
| SRA |
SRX12195096 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5579764_Alb484_WT_H3K4me3_eSPAN_S60.bw |
33.6 Mb |
(ftp)(http) |
BW |
| GSM5579764_Alb484_WT_H3K4me3_eSPAN_S60_minus.bw |
17.6 Mb |
(ftp)(http) |
BW |
| GSM5579764_Alb484_WT_H3K4me3_eSPAN_S60_plus.bw |
23.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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