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Sample GSM5273156 Query DataSets for GSM5273156
Status Public on Oct 11, 2022
Title 0 hr rep 5
Sample type SRA
Source name L1 larvae
Organism Caenorhabditis elegans
Characteristics developmental stage: L1 larvae
time point (hours): 0 hr
strain: N2
Growth protocol Well-fed N2 was maintained on OP50 at 20°C. For each biological replicate, 20 large (10 cm) NGM plates with gravid adults were hypochlorite treated to obtain over 200,000 embryos. Embryos were resuspended in S-basal at a density of 1 embryo/uL in an Erlenmeyer flask at 20°C in a shaker at 180 rpm. Time points used in the time series are 12 hours offset from hypochlorite treatment and signify when worms have begun hatching and are thus undergoing L1 arrest. L1 larvae hatch approximately 12 hours after hypochlorite treatment, and this is considered 0 hr. At least 10,000 L1s were collected per time point. For days 8 and 12, the number of L1s collected was doubled and quadrupled, respectively, to account for lethality at later time points. To collect samples, L1s in S-basal were first spun down at 3,000 rpm, and S-basal was aspirated off down to less than 100 uL. The pellet and residual S-basal was transferred to a 1.5 mL Eppendorf tube using a glass pipet. Samples were flash frozen in liquid nitrogen and stored at -80°C until RNA isolation.
Extracted molecule polyA RNA
Extraction protocol RNA was extracted using TRIzol Reagent (Thermo-Fisher Scientific) using the manufacturer’s protocol with some exceptions. 100 mL of sand (Sigma-Aldrich) was included to aid homogenization. Sand was first prepared by washing two times in 1 M HCl, washed eight times in RNAse-free water (to a neutral pH), and baked to dry.
Libraries were prepared for sequencing using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, E7530) with 100 ng of starting RNA per library and 14 cycles of PCR. Libraries were sequenced using Illumina HiSeq 4000.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 4000
Data processing Single-end 50 bp reads were mapped with with bowtie (Langmead 2010) using the following command: bowtie --best --chunkmbs 256 -k 1 -m 2 -S -p 2. HTseq was used to count reads mapping to genes (Anders et al. 2015) using version WS210 of the C. elegans genome obtained from Maxwell et al. (Maxwell et al. 2012).
Count tables were used to detect differential expression in R using edgeR (Robinson et al. 2010). Prior to differential expression analysis, count tables were filtered first to include only genes with counts per million (CPM) greater than one across four libraries. This resulted in 16,699 genes for further analysis. The edgeR glm functionality was used to fit a GLM, and a likelihood ratio test was performed for each gene; this ANOVA-like test was used to detect any differences across the twelve time points.
Genome_build: WS210
Supplementary_files_format_and_content: Count files for each library; these are the non-normalized input into edgeR for differential expression analysis. An excel final with edgeR output from differential expression analysis is also included.
Submission date Apr 30, 2021
Last update date Oct 11, 2022
Contact name Amy K Webster
Organization name University of Oregon
Street address Institute of Ecology and Evolution
City Eugene
State/province Oregon
ZIP/Postal code 97703
Country USA
Platform ID GPL22765
Series (2)
GSE173656 mRNA-seq time series of Caenorhabditis elegans L1 starvation
GSE173657 Alternative somatic and germline gene-regulatory strategies during starvation-induced developmental arrest
BioSample SAMN18935662
SRA SRX10722769

Supplementary file Size Download File type/resource
GSM5273156_0hr_rep5.counts.txt.gz 143.4 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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