NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM5954947 Query DataSets for GSM5954947
Status Public on Mar 22, 2022
Title bc4289_RNAseq_L2_rep2
Sample type SRA
 
Source name whole worms
Organism Caenorhabditis elegans
Characteristics strain: bc4289
developmental stage: L2-L3
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.
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina MiSeq
 
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
 
Submission date Mar 15, 2022
Last update date Mar 22, 2022
Contact name Sevinc Ercan
Organization name New York University
Department Biology
Lab Ercan
Street address 100 Washington Square East
City New York
State/province NY
ZIP/Postal code 10003
Country USA
 
Platform ID GPL15716
Series (1)
GSE198682 Chromosomal duplications increase gene dosage and mRNA level in C. elegans
Relations
BioSample SAMN26677021
SRA SRX14468279

Supplementary file Size Download File type/resource
GSM5954947_SEA146_BC4289_L2_rep2.fastq_genes.fpkm_TPM.tab.gz 450.1 Kb (ftp)(http) TAB
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap