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| Status |
Public on Apr 19, 2023 |
| Title |
Hu3110_v_1_Input |
| Sample type |
SRA |
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| Source name |
Hu3110
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: Hu3110
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| Treatment protocol |
ChIP-seq: T0 means vegetaive cell, we harvest them when cells entry to log-phase in PMG media; T1D meas quiescent cells which are cells entred to log-phase and were switched to PMG-N media and continue grown for 1 day.
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| Growth protocol |
ChIP-seq: Hu3110 smt0 iec1D::ura4 pht1-myc and the Hu3112 smt0 pht1-myc strains are the cells were used in ChIP-seq assay, they were grown in PMG media.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Wild type and mutant strains were grown in a 200 ml liquid PMG+N medium using a shaking incubator (200 rpm at 30o C) to reach between 1.0x10^6 and 10x10^6 cells/ml. For each culture 100 ml was removed for the T0 timepoint, and the rest of the culture was washed with pre-warmed PMG-N and incubated for 24 hours in 500 ml of pre-warmed PMG-N using a shaking incubator (200 rpm at 30o C). For RNA extraction, cells were washed with ice-cold PBS and resuspended in 500 ¼l of ice-cold RNA extraction buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 2% Triton X-100, 1% SDS, 100 mM NaCl). Then we added 500 µl of Phenol (acidic phenol pH 4.5, Sigma) and 500 µl of glass beads (acid washed, Sigma). The tubes were vortexed vigorously and incubated at 65¡C for 45-60 min. Next, the tube was placed on ice for 5 min. and centrifuged (1300 g, 5 min, 4¡C). The upper aqueous part was collected and transferred to a tube with 500 µl of chloroform (Sigma Aldrich), vortexed and centrifuged (1300 g, 5 min, 4 ¡C). The upper phase was collected and subjected to RNA precipitation at -20o C overnight. The precipitated RNA was washed once with 70% ethanol and dissolved in 30 ¼l H2O.
RNA-seq: To remove rRNA, 3 µg of purified total RNA was treated with Ribominus Eukaryote System v.2 kit (Ambion, Thermo Fisher Scientific). To generate sequencing libraries, a total of 100 ng of rRNA-depleted stocks and Illumina Stranded mRNA Prep Ligation kit (Illumina) were used. To quantify the samples, Qubit (HS dsDNA) was used, and samples were sequenced using an Illumina Nextseq 2000 platform (P3 100 cycle kit, 58 + 58 cycles, paired-end sequencing) at the BEA facility (Huddinge, Swe den) following the manufacturer's instruction.
ChIP-seq: Log phase cells grown in PMG or PMG-N media were harvested and cross-linked by 1% formaldehyde for 30 min, and then 125 mM Glycine was added to quench the crosslinking for 5 min. After three times washing with cold PBS, the cells pellet was resuspended in ChIP lysis buffer with 0.5mm Zirconia/Silica Beads, and then lysed in FastPrep machine for 7 times at max power 6.5. Sonication was done by using Bioruptor® Pico for 10 cycles, and then chromatin concentration was measured with Qubit dsDNA HS assay kit. Immunoprecipitation was performed with 20 µg sheared chromatin, 40 ng spike-in chromatin (activemotif 53083), 1.6 µl Spike-in antibody (activemotif 61686) and 6 µl anti-c-Myc antibody (Sigma-Aldrich, M4439). After three times washing with low salt wash buffer, high salt wash buffer and LiCl wash buffer successively, ChIP-DNA was extracted by ChIP DNA Clean & Concentrator kit (ZYMO RESEARCH, D5205) and DNA concentration measured by Qubit dsDNA HS assay kit.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 2000 |
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| Data processing |
RNA-seq:
Bcl files were converted and demultiplexed to fastq using the bcl2fastq (v2.20.0.422) program
STAR (2.7.9a) was used to index the Schizosaccharomyces_pombe (ASM294v2) genome and ERCC spike ins and then map the fastq files.
featureCounts (v1.5.1) was used to count mapped reads in annotated exons using gene annotations from Ensembl (Schizosaccharomyces_pombe.ASM294v2.35.gff3).
ChIP-Seq:
Bcl files were converted and demultiplexed to fastq using the bcl2fastq (v2.20.0.422) program
STAR (2.7.9a) was used to index the Schizosaccharomyces_pombe (ASM294v2) genome and Drosophila melanogaster reference genome (BDGP6) and then map the fastq files.
bedtools (v2.30.0) was used to generate bedgraph files scaled according to the Drosophila spike in counts and bedGraphToBigWig (v4) to convert them to big wig format
Assembly: RNA-seq: Schizosaccharomyces_pombe (ASM294v2)
Assembly: ChIP-seq: Schizosaccharomyces_pombe (ASM294v2) and Drosophila melanogaster reference genome (BDGP6)
Supplementary files format and content: RNA-seq: Tab delimited text files that contain gene or ERCC raw counts, one column for each sample.
Supplementary files format and content: ChIP-seq: bigwig
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| Submission date |
Mar 16, 2023 |
| Last update date |
Apr 19, 2023 |
| Contact name |
Anastasios Erik Damdimopoulos |
| Organization name |
Karolinska Institute
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| Department |
Department of Biosciences and Nutrition
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| Lab |
Bioinformatics and Expression Analysis Core Facility
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| Street address |
Hälsovägen 7c
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| City |
Stockholm |
| State/province |
Huddinge |
| ZIP/Postal code |
141 83 |
| Country |
Sweden |
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| Platform ID |
GPL30658 |
| Series (1) |
| GSE200378 |
An essential role for the Ino80 chromatin remodeling complex in regulation of gene expression during cellular quiescence |
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| Relations |
| BioSample |
SAMN33782559 |
| SRA |
SRX19691610 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7101424_Hu3110_v_1_Input.bigwig |
36.3 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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