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| Status |
Public on Jan 09, 2023 |
| Title |
ChIPseq_H3_WT_t2_rep2 |
| Sample type |
SRA |
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| Source name |
BY4741
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: BY4741 (MAT a his3delta1 leu2delta0 met15delta0 ura3delta0)
treatment: time 0 of SECOND Galactose treatment
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| Treatment protocol |
cells were shifted to pre-warmed YPG and then grown at 30°C for 3 hours harvesting at time point 0min, 30 min, 60min and 180 min for the first Galactose treatment. After put in YPD for middle resting for 3 hours, cells were shifted back to prewarmed YPG for second induction and harvested at 0min, 15min, 30min and 60 min.
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| Growth protocol |
S. cerevisiae grown in YPD at 30°C
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
RNA was extracted with MasterPure RNA extraction kit from Lucigene The DNA was purified with phenol, precipitated with ethanol and sodium acetate at -20 °C, washed with cold 70% ethanol, and resuspended in 30 µl TE buffer.
RNA-Seq: First rRNA was depleted from total RNA with Illumina ribozero Gold kit. Then stranded RNA library was generated with NEBnext Ultra Directional RNA Library Prep kit for illumina. MNase and ChIPSeq-Seq: 100 ml culture per S. cerevisiae sample and 30 ml of S. pombe culture was crosslinked using 1% formaldehyde for 15 minutes at room temperature. Formaldehyde was quenched by 0.125 M glycine for 5 minutes. Then cells were washed three times with cold TBS, flash-frozen in liquid nitrogen and stored at -80 °C. Frozen cell pellets were resuspended in zymolyase solution (1 M sorbitol, 50 mM Tris-HCl pH 7.5, 1% beta-mercaptoethanol, 0.1 U/µl zymolyase). The zymolyase digestion proceeded at +37 °C for 30 min (S. cerevisiae) or 90 min (S. pombe). Spheroplasts were isolated by centrifugation at 6000xg for 10 minutes at +4 °C. Spheroplasts were resuspended in NP buffer (10 mM Tris-HCl pH 7.5, 1 M sorbitol, 50 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 0.075% NP-40 (Tergitol), 1% beta-mercaptoethanol, 0.5 mM spermidine, 1% yeast protease inhibitor cocktail). The suspensions were pre-warmed and then 0.5 U/µl of MNase for S. cerevisiae and 2 U/µl for S. pombe was added per sample. MNase digestion proceeded at 37 °C for 40 min (S. cerevisiae) or 30 min (S. pombe) and stopped by addition of EGTA. The supernatants containing chromatin fragments were collected and diluted with 1 ml RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 0.1% SDS, 140 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate) with 1% yeast protease inhibitor cocktail. Equal volumes of S. pombe chromatin were spiked in to S. cerevisiae chromatin samples at this point, corresponding to around 3% of S. cerevisiae DNA. Each sample was immunoprecipitated with anti-H3K4me3 (Abcam ab8580) and anti-H3K4me2 (Abcam ab7766) antibodies. Protein A/G beads (Pierce) were coupled to antibodies and resuspended in diluted chromatin and rotated o/n at 4 °C. The next day, beads were washed with RIPA buffer, RIPA-500 buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 0.1% SDS, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), LiCl wash buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 250 mM LiCl, 0.5% v/v NP-40, 0.5% w/v sodium deoxycholate), and TE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA pH 8). Volume of each wash was 150 µl. Chromatin was eluted from beads in 2x10 µl ChIP elution buffer (Tris-HCl pH 8 50 mM, 1% SDS, 10 mM EDTA pH 8). The immunoprecipitated chromatin as well as a 20 µl sample of input chromatin were decrosslinked by TE, RNase cocktail, Proteinase K and 6 µl SDS (10% w/v), incubating overnight at 65 °C. DNA was purified by ethanol precipitation. Illumina sequencing libraries were prepared from the DNA using the NEBNext Ultra II kit without dual size selection and using 1.4X volume of AMPure XP beads for the purification steps. The libraries were sequenced on Illumina’s NextSeq 500, paired-end, 39 bases from each end SLAMSeq: Metabolic labeling of newly synthesized RNA molecules was done as previously described (Voichek et al., 2016). Briefly, 4-thiouracil (4tU) (Sigma) was dissolved in NaOH(83mM), Newly synthesized RNA was labeled for indicated time spans (10 min) at a final concentration of 5mM 4-thiouridine (4sU, Carbosynth). MES buffer with a final concentration 10mM PH 5.9 was added to media to avoid pH change as a result of NaOH addition. At each time points before harvesting, 4tU was added to media for ten minutes ahead of desired time point. cells were snap frozen in liquid nitrogen immediately at the time point required and RNA purification was done with MasterPure Yeast RNA Purification Kit. Total RNA was subjected to thiol(SH)-linked alkylation by iodoacetamide (Sigma, 10 mM) at 50C for 15 minutes (Herzog et al., 2017), the reaction was stopped by 20 mM DTT and RNA was re-purified by ethanol precipitation. After rRNA depletion with RiboPool Depletion Kit, library was prepared by Ultra™ II Directional RNA Library Prep Kit for Illumina® and sequenced single end with read length 150bp on Nextseq 500 Illumina sequencer.
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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 |
For RNASEQ. The genome assembly and annotation for the RNA-Seq data analysis was downloaded from SGD database (version 64-1-1) (Xu et al., 2009). The quality of the RNA-Seq data was assessed with FastQC (Andrews, 2010). The reads were aligned to the genome with STAR and parameters --outFilterMismatchNmax 4 --alignIntronMin 13 --alignIntronMax 2482. Read counts were then summarised using featurecounts with parameter -s 2 -C . chimeric fragments was excluded from fragment counting. MultiQC was used to assess the performance of the preprocessing steps (Ewels et al., 2016). Differential gene expression analysis was perfomed with DESEQ2 package in R. The counts were normalized by coding transcriptome. For ChIPseq and MNase-seq Illumina adptor was detected and trimmed by TrimGalore. Trimmed reads were then alignment with BWA to Saccharomyces_cerevisiae genome assembly R64-1-1. High repetitive Ribosome DNA region was provided as blacklist and removed from alignment. PCR duplicates were marked with Picard MarkDuplicates and then removed for subsequent analysis. Deduplicted reads from biological replicates were then merged together. Bigwig files were then generated from merged bam files by deeptools bamcoverage command. Parameter --binSize 1 --MNase --minFragmentLength 100 --maxFragmentLength 200 --normalizeUsing CPM were used to take only mononucleosome, deconvolute and take only the center dyad genomic coordinate of each nucleosome. Bed files contains gene groups gerenerated from RNAseq data were provided to deepltools computatematrix command to extract the nucleosome occupancy and histone modification level of each gene within particular group. Deeptools plotProfile tool were then used to summarise above matrix into metagene plot with parameter –perGroup. SLAMseq: SLAMseq data was anaysed with slamdunk provided by nextflow pipeline(v 1.0.0). https://nf-co.re/slamseq . As stranded library was prepared with dUTP method and fastq files were first converted to reverse complementary reads to feed into slamdunk nf core pipeline. Adapter contamination and low quality region was trimmed with TrimGalore, trim length 30bp was used instead of default value. When aligned to genome, transcript annotation downloaded from SGD database (version 64-1-1) (Xu et al., 2009) were first converted into bed file and then used as input for parameter utrbed. At least 2 conversions cooccur in one reads is regarded as a confident call for nascent RNA. SNP masking was employed to uncouple Single Nucletide Polymorphism from converted nucleotides.
Assembly: V64-1-1
Supplementary files format and content: bigwig for ChIP-seq, txt files of normalised RNASEQdata and slamseq data
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| Submission date |
Apr 19, 2022 |
| Last update date |
Jan 09, 2023 |
| Contact name |
Vicent Pelechano |
| E-mail(s) |
vicente.pelechano.garcia@ki.se
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| Organization name |
ScilifeLab - Karolinska Institutet
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| Department |
MTC
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| Street address |
Nobels väg 16
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| City |
Solna |
| ZIP/Postal code |
SE-17177 |
| Country |
Sweden |
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| Platform ID |
GPL19756 |
| Series (1) |
| GSE201036 |
Differential regulation of mRNA stability modulates transcriptional memory and facilitates environmental adaptation |
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| Relations |
| BioSample |
SAMN27631006 |
| SRA |
SRX14909877 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6049407_H3_WT_t2_R2.mLb.clN.sorted.bw |
10.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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