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| Status |
Public on Mar 22, 2022 |
| Title |
sp117_RNAseq_embryo_rep1 |
| Sample type |
SRA |
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| Source name |
whole worms
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: sp117
developmental stage: embryos
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| Growth protocol |
Mixed developmental stage embryos were obtained by bleaching gravid adults. To isolate synchronized L2/L3 worms, gravid adults were bleached and embryos were hatched overnight in M9 buffer. The resulting starved L1s were grown for 24 hours at 22°C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA-seq: Total RNA was purified following Trizol manufacturer’s instructions after freeze-cracking samples five times. RNA was cleaned up using Qiagen RNeasy MinElute Cleanup kit. mRNAs were purified using Sera-Mag Oligo (dT) beads (Thermo Scientific) from 10 µg of total RNA. cDNA preparation was done in the presence of dUTP to prepare stranded RNA-seq libraries as in PMID:19620212, cDNA synthesis was performed in the presence of dUTP in order to prepare stranded mRNA-seq libraries. DNA-seq: Embryos were washed and dounced in FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1 % sodium deoxycholate; 150 mM NaCl). 0.1 sarkosyl was added before sonicating to obtain chromatin fragments of majority between 200-800 bp.
cDNA was ligated to Illumina adapters and amplified by PCR. Library DNA between 250-500 bp in size was gel purified.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Data processing |
RNA-seq:We aligned reads to the WS220 genome version using HISAT2 version 2.2.1 (Kim et al., 2019) using the parameter --rna-strandness R. Count data was calculated using HTSeq version 0.13.5 (Anders et al., 2015). The raw counts were normalized FPKM using cufflinks version 2.2.1 (Roberts et al., 2011), and then FPKM was converted to TPM. The raw counts were used for the R package DESeq2 version 1.30.0 (Love et al., 2014).
DNA-seq: aligned to genome version WS220 (ce10) using Bowtie2 (version 2.3.2) with default settings (Langmead and Salzberg 2012). All replicate and read number information is provided in Supplemental File 1. Samtools version 1.6 (Li 2011) was used to merge replicates before running bamCompare tool from Deeptools version 3.3.1 (Ramirez et al. 2016), using the following options: --binSize 500, --scaleFactorsMethod None, --normalizeUsing CPM, --operation log2, --minMappingQuality 30, --outFileFormat bedgraph, --ignoreDuplicates. Copy number analysis was performed with Root-cern version 6.08.06 and CNVnator version 0.3.3 (Abyzov et al. 2011) comparing data from mutant strains to reference genome version WS220 (ce10) using bin_size = 1000. Output files listing deletions and duplications are provided in Supplemental File 2. Overlap of CNVs with genes were determined by Galaxy (https://usegalaxy.org/) tools “Operate on Genomic Intervals” (Afgan et al. 2018).
Assembly: WS220
Supplementary files format and content: DNA-seq: bedgraphs from bamCoverage of aligned reads. RNA-seq: normalized counts (TPM and FPKM) for individual replicates. Log2fold change values generated using DEseq2
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| Submission date |
Mar 15, 2022 |
| Last update date |
Mar 22, 2022 |
| Contact name |
Sevinc Ercan |
| Organization name |
New York University
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| Department |
Biology
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| Lab |
Ercan
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| Street address |
100 Washington Square East
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10003 |
| Country |
USA |
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| Platform ID |
GPL15716 |
| Series (1) |
| GSE198682 |
Chromosomal duplications increase gene dosage and mRNA level in C. elegans |
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| Relations |
| BioSample |
SAMN26677017 |
| SRA |
SRX14468290 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5954951_SEA71_SP117_emb_rep1.fastq_genes.fpkm_TPM.tab.gz |
467.6 Kb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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