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Status |
Public on Jul 01, 2020 |
Title |
WT_RNA_Rep1 |
Sample type |
SRA |
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Source name |
Saccharomyces cerevisiae
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Organism |
Saccharomyces cerevisiae |
Characteristics |
strain: BY4741 genotype: WT lans-set2 status: N/A time after light change: N/A chip antibody: N/A genome build: sacCer3
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Treatment protocol |
For ChIP-seq: WT, set2∆, and rph1∆ cells were collected when the OD600 reached 0.8−1.0. Cells expressing LANS-Set2 (set2∆ and rph1∆) were maintained throughout culturing in 500 μW/cm2 blue light (set2∆ and rph1∆) or in the dark (set2∆) until the OD600 reached 0.8−1.0 when an initial time point was collected then samples were shifted from the dark to light or the light to dark. For RNA-seq: WT and rph1∆ cells were collected when the OD600 reached 0.8−1.0. set2∆ cells expressing LANS-Set2 were maintained throughout culturing in 500 μW/cm2 blue light or in the dark until the OD600 reached 0.8−1.0 when an initial time point was collected then samples were shifted from the dark to light or the light to dark.
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Growth protocol |
For ChIP-seq: Cells (either WT, set2∆, set2∆ expressing LANS-Set2, rph1∆, or rph1∆ expressing LANS-Set2) were resuspended in YPD (WT, set2∆, and rph1∆) or SC−LEU (set2∆ expressing LANS-Set2 and rph1∆ expressing LANS-Set2) and grown overnight at 30 °C. In the morning cell density was measured at OD600 and cultures were diluted in appropriate media to a final volume of 70 mL and final OD600 of 0.35. Cultures were maintained at 30 °C until the OD600 reached 0.8−1.0. For RNA-seq: Cells (either WT, rph1∆, or set2∆ expressing LANS-Set2) were resuspended in YPD (WT and rph1∆) or SC−LEU (set2∆ expressing LANS-Set2) and grown overnight at 30 °C. In the morning cell density was measured at OD600 and cultures were diluted in appropriate media to a final volume of 70 mL and final OD600 of 0.3. Cultures were maintained at 30 °C until the OD600 reached 0.8−1.0.
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Extracted molecule |
total RNA |
Extraction protocol |
For ChIP-seq: Cells were fixed with a 1% final concentration of formaldehyde, the fixation was quenched, cells were washed, and pellets were frozen at −80 °C. After the completion of each time course, cells were lysed using 140 mM FA lysis buffer (50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, pH 7.5, containing protease inhibitor cocktail). Lysed cells were sonicated (Diagenode Bioruptor UCD-200) on high intensity 5 times for 5 minutes each (cycles of 30 seconds “ON” and 30 seconds “OFF”) and clarified by centrifuging at full speed for 15 minutes. Overnight immunoprecipitations of S. cerevisiae chromatin with appropriate antibodies were prepared with S. pombe chromatin spike-in control corresponding to 15% of S. cerevisiae chromatin as estimated by Bradford assay (Bio-Rad). 50 μL of Protein G Dynabeads were added to each 500 μL immunoprecipitation reaction and reactions were incubated for 2 hours. Washes were performed with 1 mL of 140 mM FA-lysis buffer, 500 mM FA-lysis buffer (50 mM HEPES, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, pH 7.5, containing protease inhibitor cocktail), LiCl solution (250 mM LiCl, 10 mM Tris, 0.5% each of NP-40 and sodium doxycholate and 1 mM EDTA) and TE pH 8.0. Elution buffer (1% SDS, 0.1 M NaHCO3) was used to elute the DNA (15 minutes shaking at 65 °C followed by centrifugation at 2,000 RPM for 2 minutes). 10 μL of 5 M NaCl was added to the eluates and 10% inputs and samples were incubated at 65 °C overnight to carry out de-crosslinking. Samples were treated with RNase for 1 hour and proteinase K for 1hour before ChIP DNA Clean & Concentrator (Zymo Research) to extract the DNA. For RNA-seq: Cells (10 mL of log-phase cultures) were collected by centrifugation and frozen at −80 °C. , RNA was isolated by acid phenol extraction. RNA (10 μg) was treated with DNase (Promega) and purified (RNeasy column, QIAGEN). For ChIP-seq: Bar-coded sequencing libraries were prepared as recommended by the manufacturer (KAPA Hyper Prep Kit), pooled and sequenced (Hi-Seq 2500, Illumina). Briefly, extracted DNA was end-repaired and A-tailed and adapters were ligated. Bead-based size selection was performed, libraries were amplified, and bead-based size selection was repeated. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq 2500 following the manufacturer's protocols. For RNA-seq: RNA (2.5 μg) was processed using rRNA depletion beads specific to yeast (Illumina). Bar-coded sequencing libraries were prepared as recommended by the manufacturer (TruSeq Stranded Total RNA Library Preparation Kit, Illumina), pooled and sequenced (Hi-Seq 4000, Illumina). Briefly, libraries were constructed by bead-based rRNA depletion, cDNA synthesis, and PCR. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina Hi-Seq 4000 following the manufacturer's protocols.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 4000 |
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Data processing |
For ChIP: Reads from the sequencer were demultiplexed using bcl2fastq (v2.20.0). Sequencing adapters on reads were trimmed using cutadapt (v1.12) using options -a GATCGGAAGAGC -A GATCGGAAGAGC and --minimum-length 36 in paired mode. After trimming, reads were filtered for quality using the fastq_quality_filter in FASTX-Toolkit (v0.0.12), with options -Q 33, -p 90, and -q 20. In-house scripts were used to limit potential PCR duplicates by limiting reads with the same sequence to a maximum of 5 copies and discarding the copies beyond that limit. Since the previous filtering steps may remove one end of a read pair but not the other, both read-pair fastq files were synced using in-house scripts to ensure proper order for alignment. As the ChIP experiments contained S. pombe spike-in, a chimeric S. cerevisae-S. pombe genome (sacCer3-ASM294v2) was generated using the genomeGenerate tool in STAR (v2.5.2b). This chimeric genome contains the full sequence of both species, allowing reads to align to their best overall fit between the two species. Once generated, read alignment was done using STAR (v2.5.2b) and options --outFilterMismatchNmax 2, --chimSegmentMin 15, --chimJunctionOverhangMin 15, --outSAMtype BAM Unsorted, --outFilterType BySJout, --outFilterScoreMin 1, and --outFilterMultimapNmax 1. Post-alignment, Samtools (v1.31) and bedtools (2.26) were used to generate bigWig files for downstream analyses. To account for S. pombe spike-in, the bigwig signal was normalized by the total number of S. pombe reads per million, using the -scale option within the bedtools genomecov tool. Normalized H3K36me3 signal was obtained by dividing H3K36me3 spike-in normalized signal by H3 spike-in normalized signal for each base pair for each replicate. Some regions were devoid of any H3 signal, and these regions were flagged and excluded from further analyses regardless of H3K36me3 signal. Deeptools (v2.5.4) was used to generate metagene plots using the normalized H3K36me3 signal. Deeptools was also used to obtain base pair by base pair signal over regions of interest such as genes, introns, and exons. This data was used to calculate the average signal per gene, excluding those regions flagged for lacking H3 signal. We set out to understand H3K36me3 signals over time, however differing levels of normalized H3K36me3 signal within genes made analysis difficult. To account for these differences, we scaled the average signal per gene throughout the time course between 0 (no signal) and 1 (the maximum signal of a gene over the time course). This yielded a relative (proportional) scale for each gene as a fraction of its maximum signal and allowed for easier comparisons of patterns within and across treatments. To yield a comprehensive gene value for plotting, H3K36me3 signal (for both mean or proportional) was the mean of all the replicates for the gene. For RNA: Reads from the sequencer were demultiplexed using bcl2fastq (v2.20.0). Reads were trimmed using cutadapt (v1.12) using options -a GATCGGAAGAGC -A GATCGGAAGAGC and --minimum-length 36 to remove any sequencing adapters. After trimming, reads were filtered for quality using the fastq_quality_filter in FASTX-Toolkit (v0.0.12), with options -Q 33, -p 90, and -q 20. Reads were aligned to the sacCer3 genome using STAR (v2.5.4b) with options --quantMode TranscriptomeSAM, --outFilterMismatchNmax 2, --alignIntronMax 1000000, --alignIntronMin 20, --chimSegmentMin 15, --chimJunctionOverhangMin 15, --outSAMtype BAM Unsorted, --outFilterType BySJout, and --outFilterMultimapNmax 1. To calculate the RNA abundance values, Salmon (v0.8.1) tool quant was used with options -l SR, --incompatPrior 0.0 to account for read strandedness. Samtools (v1.3.1), bedtools (v2.26), and R (v3.3.1) were used to interconvert files for downstream analyses. DESeq2 (v1.14.1) was used to determine which genes had differential expression. Venn diagrams were made using R package Vennerable (v3.0). Genome_build: sacCer3-ASM294v2 Genome_build: sacCer3 Supplementary_files_format_and_content: bigwig files for ChIP contain spike-in (S. pombe) scaled ChIP-seq signal for H3 or H3K36me3. Those ending with "normalizedByH3" contain H3K36me3 ChIP-seq signal that is normalized by matched H3 signal (spike-in scaled H3K36me3 / spike-in scaled H3). Bigwigs for RNA contain read depth normalized, non-stranded RNA-seq signal. Supplementary_files_format_and_content: sf files contain the gene expression data (TPM) as determined by sailfish for all replicates in either Light to Dark (LtD) or Dark to Light (DtL) treatments. Supplementary_files_format_and_content: Differential abundance files contain the DESeq2 differential genes between 0min and all other timepoints across Light to Dark (LtD) or Dark to Light (DtL) treatments. Supplementary_files_format_and_content: Model value matrices contain the GLMM model prediction values, errors, and credible intervals for each gene across the time course. High confidence genes are those where credible interval does not cross zero, and are labeled as having a non-zero trend in the table. Supplementary_files_format_and_content: Histone value matrices contain the average, spike-in scaled, H3 normalized H3K36me3 ChIP-seq signal for each gene and replicate.
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Submission date |
Jun 30, 2020 |
Last update date |
Jul 01, 2020 |
Contact name |
Austin J Hepperla |
E-mail(s) |
hepperla@unc.edu
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Organization name |
University of North Carolina at Chapel Hill
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Department |
Genetics
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Street address |
7018B Mary Ellen Jones Building
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City |
Chapel Hill |
State/province |
NC |
ZIP/Postal code |
27599 |
Country |
USA |
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Platform ID |
GPL21656 |
Series (1) |
GSE153539 |
An optogenetic switch for the Set2 methyltransferase provides evidence for transcription-dependent and independent dynamics of H3K36 methylation |
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Relations |
BioSample |
SAMN15403484 |
SRA |
SRX8639506 |
Supplementary file |
Size |
Download |
File type/resource |
GSM4646804_WT_RNA_Rep1.sf.txt.gz |
79.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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