|
| Status |
Public on Feb 26, 2024 |
| Title |
wild type mixed embryos experiment 3rep1 |
| Sample type |
SRA |
| |
|
| Source name |
whole animal
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
tissue: sKM361
strain: N2
dev stage: mixed embryos
genotype: wild type
alias in_text_and_figures: wild type
Sex: hermaphrodite
tissue: whole animal
|
| Growth protocol |
Nematode culture was performed on NGM plates seeded with OP50. Worms were maintained at 20°C except where otherwise noted. Embryo samples were harvested by hypochlorite treatment. For L4 and young adult samples, L1s were hatched overnight in M9 supplemented with cholesterol, then grown on NGM with OP50 for 48h at 20°C (for L4s) or for 44h at 25°C (for young adults).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were snap frozen, then resuspended in Trizol and vortexed for 15 minutes at room temperature. Thereafter, total RNA was purified according to the Trizol manufacturer’s instructions (ThermoFisher).
Library preparation was performed using the NEBNext Small RNA Library Prep Set for Illumina with modifications. Size selection was performed only after reverse transcription, using 8% urea gels to purify ~65-75nt RT products. Prior to loading on the gel, each RT reaction was treated with 5000units of RNAse H (New England Biolabs) for 30 minutes at 37C.
|
| |
|
| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
Library preparation was performed using the NEBNext Small RNA Library Prep Set for Illumina with modifications. Size selection was performed only after reverse transcription, using 8% urea gels to purify ~65-75nt RT products. Prior to loading on the gel, each RT reaction was treated with 5000units of RNAse H (New England Biolabs) for 30 minutes at 37C.
|
| Data processing |
Small RNA libraries were trimmed using Cutadapt 3.4 (122), then mapped using bowtie2 2.4.4 with settings “--no-unal --end-to-end –sensitive” (123). BAM files were sorted and indexed using samtools 1.13, then assigned to miRNAs using htseq 0.13.5 (124, 125). For genome mapping and miRNA assignment, custom reference files were used which included the spiked in RNA oligos. Statistical analysis was performed using DESeq2 and GraphPad Prism (126). For spike-in normalization (Figure S3), size factors for DESeq2 analysis were calculated by dividing spike-in reads in each sample by the median number of spike-in reads per sample. For all other analyses, default DESeq2 normalization was performed using all endogenous miRNA-mapping reads (spike-in reads were excluded from analysis).
Assembly: genome_c_elegans.PRJNA13758.WS280.genomic_with_spike_mut_mir35.fa
Assembly: cel_mirGeneDB_spikeins_010622.gff
Supplementary files format and content: xlsx file containing counts of reads mapped to miRNA and spike in RNA annotations
|
| |
|
| Submission date |
Feb 23, 2024 |
| Last update date |
Feb 26, 2024 |
| Contact name |
Katherine McJunkin |
| E-mail(s) |
mcjunkin@nih.gov
|
| Phone |
3048811944
|
| Organization name |
National Institutes of Health
|
| Street address |
50 South Drive, Building 50 Room 3148
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL27998 |
| Series (1) |
| GSE256489 |
Catalytic residues of microRNA Argonautes play a modest role in microRNA star strand destabilization in C. elegans [miRNA] |
|
| Relations |
| BioSample |
SAMN32677699 |
| SRA |
SRX19003426 |
