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| Status |
Public on Dec 31, 2020 |
| Title |
lmn_WT_a |
| Sample type |
SRA |
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| Source name |
L1 larval stage sorted for non green (no balancer)
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
genotype: wild-type, Bristol
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| Growth protocol |
Worms were expanded on 15cm peptone rich plates with OP50. Synchronized L1 larvae were obtained by hyopchlorite treatment of gravid adults to recover eggs. Eggs were left to hatch 16h at room temperature in M9 buffer and then re-feed with OP50 for 2.5 hours. Before RNA extraction, worms were washed 3x in M9 and re-suspended in 100μl of M9, 400μl of Trizol® (Ambion) and snap-frozen in liquid nitrogen.
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| Extracted molecule |
total RNA |
| Extraction protocol |
After 3x in M9, L1 worms were re-suspended in 100μl of M9, 400μl of Trizol® (Ambion) and snap-frozen in liquid nitrogen. Extraction of RNA used 4 freeze-thaw cycles from liquid nitrogen to a 42 °C heat bath, followed by the addition of 200 μl of Trizol® to each sample. Vigorous vortexing at RT in 5 cycles (30 sec vortex, 30 sec on ice), was followed by 5 min at RT. RNA extraction was with 140 μl chloroform, vigorous shaking for 15 sec, and 2 min. The samples were centrifuged at 12000 rcf at 4 °C, and the aqueous phases were transferred to fresh tubes. An equal volume of 70 % EtOH was added slowly and the homogeneous mixture was transferred to a Qiagen RNeasy spin column (RNeasy kit, QIAGEN 74104).
Library preparation for the L1 samples was performed with the TrueSeq Total RNA preparation kit, stranded (Illumina). The quality of the resulting libraries was assessed with an Agilent Bioanalyzer and concentrations were measured with a Qubit fluorometer prior to pooling. 50 bp single-end sequencing was done on an Illumina HiSeq 2500.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
For each genotype the RNA-seq data from two biological replicate samples from the L1 stage were mapped to the C. elegans genome (ce10) using the R package QuasR v1.22.0 (www.bioconductor.org/packages/2.12/bioc/html/QuasR.html). QuasR includes the short read aligner bowtie using the parameters "-m 1 --best --strata --phred33-quals" to consider only uniquely mapping reads (command: "proj <-qAlign("samples.txt","BSgenome. Celegans.UCSC.ce10")") and subsequently extract count tables of reads mapping within exon gene annotation from WormBase (WS220).
To normalize for sequencing depth, each sample was divided by the total number of reads and multiplied by the average library size. Transformation into log2 space was performed after the addition of a pseudocount of 8 in order to minimize large changes in expression caused by low count numbers. The various count tables used throughout this study were normalized separately.
The EdgeR package v 3.24.0 was used to determine fold changes (Fc) and false discovery rates (FDR) of differential transcript abundances.
Genome_build: ce10
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| Submission date |
Aug 28, 2019 |
| Last update date |
Dec 31, 2020 |
| Contact name |
Susan Gasser |
| E-mail(s) |
[email protected]
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| Organization name |
Friedrich Miescher Institute
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| Street address |
Maulbeerstrasse 66
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| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
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| Platform ID |
GPL18245 |
| Series (2) |
| GSE136576 |
RNA-seq: Loss of a heterochromatin anchor rescues altered genome organization and EDMD muscle defects triggered by a laminopathy mutation |
| GSE136577 |
Loss of a heterochromatin anchor rescues altered genome organization and EDMD muscle defects triggered by a laminopathy mutation |
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| Relations |
| BioSample |
SAMN12648093 |
| SRA |
SRX6767241 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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