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| Status |
Public on Mar 23, 2023 |
| Title |
N2 strain; POS1 treatment; rep 2 |
| Sample type |
SRA |
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| Source name |
whole animal
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| Organism |
Caenorhabditis elegans |
| Characteristics |
tissue: whole animal
strain: N2
treatment: POS1
age: day 1 adult
Sex: hermaphrodite
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| Treatment protocol |
RNAi was induced by feeding and experiments were carried out on plates, at 20°C, based on methods previously described (Ahringer, 2006; Kamath et al., 2001). To target endogenous germline-expressed genes, we fed worms HT115 E. coli bacteria that had been transformed with the pL4440-derived par-1 (H39E23.1) or pos-1 (F52E1.1). The par-1 and pos-1 vectors were obtained from the Ahringer feeding library (Kamath & Ahringer, 2003). We used E. coli carrying the empty pL4440 vector as a negative control. Bacteria were streaked from frozen stocks onto LB agar plates with carbenicillin (25 ug/mL) and tetracycline (12.5 mg/mL); liquid cultures were inoculated with 5-10 colonies from <1 week old plates, into LB broth with carbenicillin (50 ug/mL) and tetracycline (12.5 mg/mL) and incubated for 16-18hrs shaking at 37°C, then amplified in a 1:200 dilution with carbenicillin (50 ug/mL) for 6hrs. Seeded plates were incubated in the dark at room temperature and used no earlier than 44hrs and no later than 78hrs. Experimental worms were bleach synchronized, allowed to grow for three days, then bleach synchronized on non-RNAi plates. The following day L1 larvae were transferred to RNAi plates and exposed to RNAi for two days, at which point day 1 adult hermaphrodites were harvested and stored in Trizol at -80°C after 2 minutes vortexing until RNA extraction.
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| Growth protocol |
Worms were cultured following standard protocol (Stiernagle, 2006 and described here), though we added 1.25% agarose to plates used to maintain non-N2 wild isolates, to avoid burrowing. Worms were maintained at 20°C without starving for at least three generations before initiating an experiment, with the exception of QX1211, which was maintained at 18°C to avoid induction of the mortal germline phenotype. Worms used for RNA-seq were grown during RNAi or control treatment as described in ‘Treatment protocol’.
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| Extracted molecule |
total RNA |
| Extraction protocol |
All samples were collected and processed simultaneously and in triplicate, starting with replicate plates of worms. The following protocol follows (He, 2011). Worms stored at -80°C in TRIzol (Invitrogen #15596026) were thawed and then freeze-thawed 6 times by freezing via submersion in liquid nitrogen, heat at 37°C on a heating block, then vortexing for 10 seconds and placed on ice. 200 uL Trizol was added and samples were incubated for 5 minutes at room temperature. 140 uL chloroform was added and samples were shaken at 1450 rpm for 1 minute, followed by a 2 minute incubation at room temperature. These samples were spun at 12,000g for 15 minutes at 4°C. The top aqueous phase was removed to a new tube and used downstream. One volume of 70% ethanol was added to this aqueous phase and then samples were vortex mixed and quickly spun down. 500 uL of this mixture was transferred to RNeasy columns (Qiagen #74104) and RNA extracted following Qiagen’s instructions. RNA was eluted in 50uL RNAse-free water.
Libraries were prepared with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #7760) following the manufacturer’s instructions. 500ng of fresh RNA was used as input for cDNA generation with this kit with 10 cycles of PCR. Libraries quality checked using an Agilent 2100 Bioanalyzer. BluePippin (Sage Science) was used to size-select fragments size 210-650bp in the pooled library using a 2% agarose gel.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
library fragment length information provided to Salmon - fldMean: 334.4183668; fldSD: 80.30734131
AP01-16
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| Data processing |
Basecalling, fastq generation, and de-multiplexing were performed in Illumina BaseSpace v5.3.7, September 2019, via bcl2fastq v2.2.0
Illumina TruSeq adapters were trimmed from RNA-seq reads using Trimmomatic v0.3.9, parameters: ILLUMINACLIP:TruSeq3-SE.fa:1:30:12
Strain-specific transcriptomes were generated by patching strains’ SNPs and INDELs from CeNDR’s (elegansvariation.org) 20210121 release onto the N2 reference genome release ws276 using g2gtools v0.1.31 (installed via conda v4.7.12, Python v2.7.16) followed by transcriptome extraction. CeNDR’s isotype-representative strain was used for any strain that was not an isotype representative (here, EG4349’s genome was used for strain EG4348). For each non-reference strain, first, INDELs were chained onto the reference genome using g2gtools vcf2chain and SNPs were patched onto the reference genome FASTA using g2gtools patch. Next, INDELs were chained onto the SNP-patched genome using g2gtools transform and strain-specific GTFs were created from this updated genome FASTA via g2gtools convert. Strain-specific transcriptomes were generated from these strain-specific genome FASTAs and GTFs using gffread v0.12.7
Strain-specific transcriptome indexes were generated with Salmon v1.4.0 command salmon index, parameters: -k 31 --keepDuplicates (all others default; no decoys used)
RNA quantification was performed via pseudo-alignment to strain-specific transcriptomes with Salmon v1.3.0 command salmon quant with parameters -l SR --dumpEq --rangeFactorizationBins 4 --seqBias, and library-specific --fldMean and --fldSD
Transcript-level abundances were converted to gene-level abundances with tximport v1.20.0 (in R v4.1.0) tximport function, parameter type = "salmon" after including 0 abundance for any transcripts not included in a wild isolate but included in the ws276 reference GTF
Gene-level abundances were normalized using DESeq2 v1.32.0 (in R v4.1.0) deseq() function with default parameters prior to differential expression analysis. This normalization and downstream differential expression analysis were only performed for genes with >10 reads summed across samples after tximport
Genome_build: ws276 (wormbase)
Supplementary_files_format_and_content: Tab-delimited text files with abundances for each transcript as output by salmon, one per sample (*_quant.sf.gz). One row per transcript. Columns (see Salmon documentation for even further detail): Name (transcript ID), Length (transcript length), Effective Length (sample-specific effective length of transcript computed by salmon), TPM (transcripts per kilobase million abundance estimate using sample-specific effective length), NumReads (estimated number of reads assigned to this transcript).
Supplementary_files_format_and_content: Matrix file (tab-delimited text) with normalized gene abundances (deseq2_normalized_counts.txt.gz) for each sample (column) and gene included in differential expression analysis (row; gene names are row names). Derived using the counts() function in DESeq2 v1.32.0 with parameter normalized = T. All genes with >10 reads (summed across samples, counts from tximport, see above) prior to this normalization are included, n = 18589. These are the counts used by DESeq2 for differential expression analysis.
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| Submission date |
Dec 13, 2021 |
| Last update date |
Mar 23, 2023 |
| Contact name |
Avery Davis Bell |
| Organization name |
Georgia Institute of Technology
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| Department |
School of Biological Sciences
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| Lab |
Paaby Lab
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| Street address |
North Avenue
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| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30332 |
| Country |
USA |
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| Platform ID |
GPL19757 |
| Series (1) |
| GSE190803 |
RNA-seq profiles of 5 differentially RNAi sensitive wild C. elegans strains in control and embryonic RNAi conditions |
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| Relations |
| BioSample |
SAMN24009573 |
| SRA |
SRX13397898 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5732263_AP01-16_quant.sf.gz |
619.7 Kb |
(ftp)(http) |
SF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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