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Status |
Public on Sep 07, 2024 |
Title |
N2 worms, day 7, untreated rep3 (small RNA-seq) |
Sample type |
SRA |
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Source name |
whole worms
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Organism |
Caenorhabditis elegans |
Characteristics |
tissue: whole worms genotype: WT treatment: untreated
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Treatment protocol |
To prepare mRNA-seq sample, approximately 1000 synchronized Day 7 N2 worms were treated with control, embryo lysates or day 6 worms lysates for 24h.
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Growth protocol |
Worms were grown and maintained at 20 ºC and fed with E. coli OP50 on Nematode Growth Medium (NGM) (0.25% Bacto peptone, 0.3% sodium chloride, 1.7% agar, 5 ug/mL cholesterol, 25 mM potassium phosphate pH 6.0, 1 mM magnesium sulfate, 1 mM calcium chloride).
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Extracted molecule |
total RNA |
Extraction protocol |
Synchronized worms were collected from the plates, washed at least three times with M9 buffer, and resuspended in TransZol Up reagent (TransGen Biotech, #ET111-01). Worm samples were frozen in liquid nitrogen and freeze-thawed for at least five times. Total RNA was isolated by chloroform extraction followed by isopropanol precipitation, washed with 75% (v/v) ethanol and dissolved in RNase-free water. Small RNA libraries for small RNA seq were prepared by BGI Tech using DNBSEQ small RNA seq protocols.
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Library strategy |
ncRNA-Seq |
Library source |
transcriptomic |
Library selection |
size fractionation |
Instrument model |
DNBSEQ-T7 |
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Description |
untreated.miRNA.count.txt; untreated.piRNA.count.txt; untreated.22GRNA.count.txt; untreated.26GRNA.count.txt
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Data processing |
The raw sequence reads were cleaned from adapter sequences or low-quality sequences by SOAPnuke (developed by BGI) with the parameters "-n 0.001 -l 13 -q 0.1 --highA 1 --minReadLen 15 -- maxReadLen 44 --ada_trim". Quality control were conducted on both the raw and processed data using the FastQC tool (v.0.11.9, http://www.bioinformatics.babraham.ac.uk/projects/fastqc). The cleaned reads were aligned to the Caenorhabditis elegans genome (Ensembl WBcel235/ce11 assembly) using Bowtie v.1.0.0 (http://bowtie-bio.sourceforge.net) with the parameters ‘-v 1 -M 1 --best --strata --tryhard --chunkmbs 512 -S’. The alignment outputs were then converted to sorted BAM files via Samtools v.1.3.1. Gene annotations in GTF format were downloaded from Ensembl (ftp://ftp.ensembl.org/pub/release-110/gtf/caenorhabditis_elegans/). Structural reads, defined as reads mapping in the sense orientation to annotated rRNAs, tRNAs, snRNAs, or snoRNAs, were removed from further analyses using Bedtools v.2.30.0 (http://bedtools.readthedocs.io). Annotations for miRNA genes were derived from miRbase. miRNA reads were defined as those ranging from 21 to 24 bases that map in the sense orientation to the annotated miRNA genes. 21U reads were defined as 21-base reads beginning with T, which map in the sense orientation to piRNA genes, and these reads must align uniquely. Targets of piRNAs were predicted using piRTarBase. Gene-derived 22G-RNA were defined as reads ranging from 21 to 23 bases and 26G-RNA were 26-base reads. These reads were starting with G and mapping in the antisense orientation to annotated genes. The read counts per gene were quantified using featureCounts/Subread v2.0.1. Reads were further extracted and filtered with Awk, Samtools and custom Python scripts. Filtered reads were then converted to BED files, and subsequently to BedGraph files using Bedtools v.2.30.0. For visualization, the BedGraph files were converted to BigWig files and displayed using the IGV tool (v.2.9.4). Reads for each gene were normalized to the total mapped reads of individual gene class as reads-per-million (RPM). Differential expression analysis was conducted with DEseq2, with an adjusted p-value <0.05 set as the threshold for statistically significant differential expression for each gene class. Assembly: Caenorhabditis elegans genome (Ensembl WBcel235/ce11 assembly) Supplementary files format and content: tab-delimited text file includes raw counts for each sample
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Submission date |
Apr 25, 2024 |
Last update date |
Sep 07, 2024 |
Contact name |
Ying Liu |
E-mail(s) |
ying.liu@pku.edu.cn
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Organization name |
Peking University
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Department |
College of Future Technology
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Lab |
Laboratory of Mitochondrial and Metabolism Research
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Street address |
5 Summer Palace Road, Haidian District, Beijing
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City |
Beijing |
ZIP/Postal code |
100871 |
Country |
China |
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Platform ID |
GPL33586 |
Series (1) |
GSE265889 |
Effect of embryo lysates treatment on small RNA expression |
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Relations |
BioSample |
SAMN41084059 |
SRA |
SRX24370768 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
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