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| Status |
Public on Jun 30, 2020 |
| Title |
rnaL20nsun1 |
| Sample type |
SRA |
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| Source name |
Whole animal
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| Organism |
Caenorhabditis elegans |
| Characteristics |
datapipeline: Pipeline2
temperature: 20
genotype: noNSUN
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| Treatment protocol |
Empty vector, nsun-1 (W07E6.1), nsun-2 (Y48G8AL.5), nsun-4 (Y39G10AR.21), and nsun-5 (Y53F4B.4) bacterial feeding clones were kindly provided by Prof. Julie Ahringer’s lab (82). Single colonies were inoculated in LB-Ampicillin 100 μg/ml and cultured for 8 h at 37°C. Bacterial cultures were seeded onto 50 mm NGM agar plates containing 1 mM IPTG and 25 μg/ml Carbenicillin at a volume of 200 μl of bacterial culture per plate, and left to dry for 48 hours. 50 synchronized L1 larvae were placed onto RNAi plates and left to grow until adult stage.
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| Growth protocol |
wild type and noNSUN animals were cultured at 15°C and 25°C for three generations
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| Extracted molecule |
total RNA |
| Extraction protocol |
Input RNA was extracted from the supernatant of the same samples used for polysome profiling and ribosome footprinting. RNA was depleted of DNA with a TURBO DNA-free kit (Invitrogen), according to the manufacturer’s instructions. Libraries were prepared with 750 ng of starting material using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina, following rRNA depletion using a NEBNext rRNA Depletion Kit (Human/Mouse/Rat)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Pipeline 1 - Raw reads were assessed for quality using FastQC (Andrews, 2010) and Trimmed for low quality bases and adapter sequences using Trimmomatic (version 0.39, parameters - ILLUMINACLIP:2:30:10 SLIDINGWINDOW:4:20 MINLEN:20) (Bolger et al., 2014). SortMERNA (Kopylova et al., 2012) was used to remove any rRNA sequences. Remaining reads were uniquely aligned to the C. elegans (WBCel235) reference genome using HISAT2 (Kim et al., 2015) (version 2.1.0). The longest transcript was chosen for each gene from the WBCel235 reference genome and the CDS for these transcripts was extracted. Reads were length stratified and checked for periodicity, only read lengths showing periodicity over the 3 frames were retained for further analysis (26 bp -30 bp). Reads aligned to the genome were shifted 12 bp from the 5′-end towards the 3′-end (Ingolia et al., 2009). Any reads aligned to the first 10 codons of each gene were then removed and the remaining reads with a 5′ end aligning to a CDS were kept for further analysis (Lecanda et al., 2016). Ribosome occupancy for gene in a sample was calculated as the number of shifted in frame RPFs aligned to the CDS of the gene (not including the first 10 codons). These values were inputted into DESeq2 (Love et al., 2014).
Pipeline 2 - Raw reads were assessed for quality using FastQC (Andrews, 2010) and Trimmed for low quality bases and adapter sequences using Trimmomatic (version 0.39, parameters - ILLUMINACLIP:2:30:10 SLIDINGWINDOW:4:20 MINLEN:25) (Bolger et al., 2014). SortMERNA (Kopylova et al., 2012) was used to remove any reads matching rRNA sequences. Remaining reads were aligned to the C. elegans reference genome (WBCel235) using HISAT2 (Kim et al., 2015) (version 2.1.0, default parameters). Read alignments were then counted using HTSeq-count (Anders et al., 2015) and gene counts inputted into DESeq2 (Love et al., 2014).
Genome_build: WBCel235
Supplementary_files_format_and_content: rawCounts.txt
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| Submission date |
Mar 03, 2020 |
| Last update date |
Jul 01, 2020 |
| Contact name |
Jonathan Lewis Price |
| Organization name |
University of Cambridge
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| Department |
The Gurdon Inst.
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| Lab |
Miska Lab
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| Street address |
The Gurdon Inst. Tennis Court Road
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| City |
cambridge |
| ZIP/Postal code |
CB2 1QN |
| Country |
United Kingdom |
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| Platform ID |
GPL13657 |
| Series (1) |
| GSE146256 |
Translational adaptation to heat stress is mediated by 5-methylcytosine RNA modification in Caenorhabditis elegans |
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| Relations |
| BioSample |
SAMN14262102 |
| SRA |
SRX7830157 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4369043_rnaL20nsun1.txt.gz |
71.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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