|
| Status |
Public on Feb 12, 2020 |
| Title |
lsm-8 met-2 set-25 (1148_2,unused*) |
| Sample type |
SRA |
| |
|
| Source name |
L3 larval stage sorted for non green (no balancer)
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: GW1148 (lsm-8-/- ; met-2-/- set-25-/-)
genotype: met-2(n4256) set-25(n5021) III; lsm-8 (xe17 [myo2p::mcherry::unc54 3'UTR])IV/nT1[qIs51](IV;V)
developmental stage: L3
sorting efficiency and genotype validation with wiggle files: * poor
|
| Treatment protocol |
The worms have been sorted for L3-L4 stage and non-green pahrynx (balancer) in all strains in order to get only the L3-L4 homozygous for the deletion allele(s). The N2 and GW638 have been sorted in the same conditions to get similar treated worms, even though they do not have a green/GFP balancer to sort out. During the validation for the genotype done with the wiggle files, we found that the marked samples (*) had relatively a high number of reads on the deletion of interest compared to all other related samples indicating that the sorting process was probably not very efficient with some heterozygous worms or other worms that might have contaminated partially the sample.
|
| Growth protocol |
Worms from each genotype were grown on 2-6 petone rich plates (15 cm) seeded with OP50 bacteria.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For the RNA-seq experiment L3-L4 worms from the four following genotypes: WT, met-2 set-25, lsm-8-/- and met-2 set-25; lsm-8-/- worms were isolated using the COPAS BIOSORT instrument in four independent biological replicates. The sorted worms were harvested, washed three times in M9 and finally re-suspended in 100μl of M9, 400μl of Trizol® (Ambion) was added and the samples were snap-freezed in liquid nitrogen. Total RNA was treated additionally with the Turbo DNA free kit (Ambion, AM1907), depleted for rRNA using Ribo-Zero Gold kit from Epicentre and depletion validated through Agilent Bioanalyzer analysis.
Library preparation was performed with a ScriptSeq v2 RNA-Seq library preparation kit (Epicentre). The quality of the resulting libraries was assessed with an Agilent Bioanalyzer and concentrations were measured with a Qubit fluorometer prior to pooling.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
The RNA-seq samples were mapped to the c.elegans genome (ce6) with the R package QuasR (www.bioconductor.org/packages/2.12/bioc/html/QuasR.html) using the included aligner bowtie considering only uniquely mapping reads for mRNA as well as for repeat element quantification. The command used to perform the alignments was "proj <- qAlign("samples.txt","BSgenome.Celegans.UCSC.ce6")" which instructs bowtie to align using the parameters "-m 1 --best --strata --phred33-quals". For splice junction quantification we used the spliced alignment algorithm SpliceMap. The command used to perform the alignments was "proj <- qAlign("samples.txt","BSgenome.Celegans.UCSC.ce6",splicedAlignment=TRUE)". The command used to create the various count tables was qCount(proj,exons,orientation="same"). For gene quantification (ce_mRNA_lsm8_*.txt), gene annotation from WormBase was used (WS190). The repeat element quantification was based on UCSC (genome.uscsc.edu) repeat annotation (ce_repeats_*.txt). 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. To quantify potential changes in splicing in lsm8 as apposed to N2, we quantified the expression of all the exon-exon junctions from the spliced alignments using no annotation (ce_junctions_*.txt). The command used to create the exon-exon junction count table was qCount(proj2,NULL,reportLevel="junction"). These junction counts were then normalized for library size (as described above) and overlapped with gene annotation to assign them to their host gene.
Genome_build: ce6
Supplementary_files_format_and_content: text files with raw or normalized counts.
|
| |
|
| Submission date |
Dec 22, 2016 |
| Last update date |
Feb 12, 2020 |
| Contact name |
Dimos Gaidatzis |
| E-mail(s) |
d.gaidatzis@fmi.ch
|
| Organization name |
Friedrich Miescher Institute
|
| Street address |
Maulbeerstrasse 66
|
| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL18245 |
| Series (2) |
| GSE92850 |
LSM2-8 and XRN-2 contribute to the silencing of H3K27me3 marked genes through targeted RNA decay I |
| GSE92851 |
LSM2-8 and XRN-2 contribute to the silencing of H3K27me3 marked genes through targeted RNA decay |
|
| Relations |
| BioSample |
SAMN06176586 |
| SRA |
SRX2441373 |
