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| Status |
Public on Nov 12, 2017 |
| Title |
wt(RPB1) SER2P ChIP-Seq |
| Sample type |
SRA |
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| Source name |
exponential growing culture
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
background: BY4741
genotype: wt
strain name: YF336
growth media: YPD
chip antibody: anti-Ser2P
sequence barcode: ATATAGGA+T
s.pombe spike-in: yes
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| Treatment protocol |
Cells were crosslinked with formaldehyde to a final concentration of 1%. Cells were incubated for 20 min at room temperature and the reaction quenched for 5 minutes with 3M glycine. Cell pellets were recovered and washed with FA buffer (50 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 1mM EDTA,1% Triton X-100 and 0.1% Na Deoxycholate), 0.1% SDS.
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| Growth protocol |
Cells were grown in indicated media at 25 C to an OD of 0.500.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed in FA buffer 0.5% SDS using glass beads. Cross-linked extracted were sonicated using a Misonix 300 with cup horns, with 10s on/off pulses for a total sonication time of 20 min. Soluble chromatin was recovered by centrifugation and protein concentration quantified by Bradford assay. A total of 700 ug of chromatin was used per immunoprecipitation. Where S.pombe “spike-in” was used, 10 % S.pombe chromatin prepared similarly was added to the immunoprecipitation. The indicated antibody and 10 ul of Protein-G sepharose beads were added, followed by overnight incubation at 4 C. Beads were washed once with FA buffer, 0.1% SDS, 275 mM NaCl, once with FA buffer, 0.1% SDS, 500 mM NaCl, once with 10 mM Tris-HCl, pH 8.0, 0.25 M LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% Na Deoxycholate, and once with TE (10 mM Tris-HCl, pH 8.0, 1mM EDTA). Immunoprecipitated material was eluted with 50 mM Tris-HCl 7.5, 10 mM EDTA, 1% SDS by incubating at 65 C for 20 min. Beads were washed once with TE, and the supernatant added to eluted material. Recovered immunoprecipitate was de-crosslinked with 40 ug of Pronase 1 hour at 42 C and overnight at 65 C. Samples were treated with 5 ug RNAse A and phenol-chloroform/chloroform extracted followed by precipitation with LiCl, 40 ug Glycogen, and 100 % EtOH. Precipitated DNA was resuspended in appropriate volume of water and quantified using Qubit assay.
5 ng of immunoprecipitated DNA was used for ChIP-Seq library production. Immunoprecipitated DNA was end repaired using T4 DNA polymerase, T4 PNK and DNA Polymerase I, Large (Klenow) fragment. A single adenosine “tail” was added to the 3’ end of fragments using Klenow (3’ to 5’ exo minus) and adapters containing inline multiplexing barcodes were added using T4 Quick DNA ligase. Adapter ligated DNA fragments between 200 and 500 bp were gel purified and PCR amplified with 16 cycles. Library quality was assessed using Agilent 2100 Bioanalyzer. Equal molarity of each library was mixed.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Raw fastq files were demultiplexed using Sabre (https://github.com/najoshi/sabre) allowing one mismatch in the barcode.
Demultiplexed reads were aligned to S.cerevisiae genome, with bowtie1.1.1, reads with multiple alignments removed. S.pombe spiked-in samples were aligned first to S.pombe genome with bowtie1.1.1, S.pombe unaligned reads were aligned to S.cerevisiae genome, with reads with multiple alignments removed.
Genome_build: S.cerevisiae version R64-1-1, S.pombe version ASM294v2.31
Supplementary_files_format_and_content: bedgraph files were generated with macs2.0 callpeak function with extsize of 150, number of duplicates 1, normalized for SPMR
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| Submission date |
Oct 16, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Luis Soares |
| E-mail(s) |
luis_soares@hms.harvard.edu
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| Phone |
6173881077
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| Organization name |
Harvard Medical School
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| Department |
BCMP
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| Lab |
Buratowski
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| Street address |
240 Longwood Ave, Building C1-207
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| City |
Boston |
| State/province |
Massachusetts |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL13821 |
| Series (1) |
| GSE95356 |
Determinants of H3K4me pattern establishment |
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| Relations |
| BioSample |
SAMN07788241 |
| SRA |
SRX3287335 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2813907_wt_RPB1_ser2_2.bedgraph.gz |
15.9 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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