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| Status |
Public on Jul 09, 2019 |
| Title |
IP_Rpc2_WT |
| Sample type |
SRA |
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| Source name |
yeast culture
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: FBY2210
ip tag: TAP
chip target: Rpc2
genotype: leu1-32 ura4-D18 his3-D1 ade6-M216
chip antibody: Pan Mouse IgG Dynabeads (Life Technologies, 11041)
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| Growth protocol |
Cells were grown at 30°C in complete YES medium.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
1,5.10^8 cells were cross-linked with 1% formaldehyde (Sigma) at 18°C for 30 minutes. After 3 washes with cold PBS, the cells were frozen in liquid nitrogen. Frozen cells were then lysed in cold lysis buffer (Hepes-KOH 50mM [pH 7.5], NaCl 140mM, EDTA 1mM, Triton 1%, Na-deoxycholate 0.1%, PMSF Phenylmethanesulfonyl fluoride 1 mM) with glass beads using a Precellys 24 mill (Bertin Technology). To fragment the chromatin, the lysates were sonicated at 4°C using a Covaris S220 or Diagenode Bioruptor sonicator. Immuno-precipitation was done overnight at 4°C using Pan Mouse IgG Dynabeads (Life Technologies, 11041) for TAP-tagged proteins. The immunoprecipitated complexes were washed for 5’ successively with: Wash I buffer (20mM Tris pH 8, 150 mM NaCl, 2mM EDTA, 1% Triton-X100, 0.1% SDS), Wash II buffer (20mM Tris pH 8, 500mM NaCl, 2 mM EDTA, 1% Triton-X100, 0.1% SDS) and Wash III buffer (20mM Tris pH 8, 1mM EDTA, 0.5% Na-deoxycholate, 1% Igepal, 250mM LiCl). After two additional washes in Tris EDTA pH 8, the beads were resuspended in 10% Chelex resin (Biorad) and incubated at 98°C for 10’. After addition of 2 μL of 10 mg/mL proteinase K, the mixture was incubated at 43°C for 1 hour, then for another 10’ at 98°C. After centrifugation, the supernatant was collected and analyzed by qPCR in a thermocycler Rotor Gene (Qiagen).
DNA libraries for ChIP-seq experiments were prepared as described previously (Lemay et al., 2016) using the SPARK DNA Sample Prep Kit Illumina Platform (Enzymatics) according to the manufacturer’s instructions.
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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 |
Briefly, the raw reads were trimmed using Trimmomatic version 0.32 (Bolger et al., 2014) with parameters ILLUMINACLIP:2:30:15 LEADING:30 TRAILING:30 MINLEN:23, and quality inspection was conducted using FastQC version 0.11.4 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
The trimmed reads from all data sets were aligned using BWA version 0.7.12-r1039 (Li and Durbin, 2010) with the algorithm mem and the parameter -split onto the sequence of the S. pombe ASM294v2. Note that no filtering on mapQ was performed in order to avoid discarding the signal at regions of the genome that are duplicated (BWA is randomly assigning the reads), but only primary alignments were kept and we generated mappability tracks for various read length to help the interpretation of particular regions.
Signal density files in BedGraph format were then generated using BEDTools genomecov version 2.17 (Quinlan and Hall, 2010) with default parameters, then converted in uniform 10 nt bins WIG files for further normalization steps (inspired by the script bedgraph_to_wig.py [https://gist.github.com/svigneau/8846527]).
Each signal density file was scaled based on sample's sequencing depth, then the signal of the input data set was subtracted from its corresponding IP data set.
The normalized WIG files were then encoded in bigWig format using the Kent utilities (Rhead et al., 2010). Visual inspection of the data was performed using an AssemblyHub on the UCSC Genome Browser (Casper et al., 2018).
Genome_build: ASM294v2
Supplementary_files_format_and_content: IP: bigwig files of the normalized IP signal after substraction of the WCE signal; WCE: bigwig files of the normalized WCE signal
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| Submission date |
May 03, 2019 |
| Last update date |
Jul 09, 2019 |
| Contact name |
François Bachand |
| E-mail(s) |
francois.bachand2@usherbrooke.ca
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| Organization name |
Université de Sherbrooke
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| Department |
Biochimie
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| Street address |
3201 Jean Mignault
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| City |
Sherbrooke |
| State/province |
Québec |
| ZIP/Postal code |
J1E 4K8 |
| Country |
Canada |
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| Platform ID |
GPL20584 |
| Series (1) |
| GSE130709 |
Sen1 is required for robust RNA Polymerase III transcription termination |
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| Relations |
| BioSample |
SAMN11571087 |
| SRA |
SRX5787087 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3748091_IP_Rpc2_WT.bw |
5.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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