title: Whole genome sequencing of EMS-induced suppressors of tost-1 maternal-effect lethality in Caenorhabditis elegans
design: Whole genome sequencing of EMS-induced suppressors of tost-1 maternal-effect lethality in Caenorhabditis elegans
library_construction_protocol: After confirmation of suppressor phenotypes and backcrossing to the parental tost-1(xf196 ts) strain, suppressor strains were expanded for genomic DNA extraction. Mixed-stage worm populations were cultured on 90 mm NGM plates seeded with E. coli OP50 for one generation, then transferred to 150 mm plates for an additional generation to increase biomass. Worms were harvested by washing plates with M9 buffer, collected by centrifugation, and washed multiple times to remove bacteria. Worm pellets were processed immediately for DNA extraction using the Gentra Puregene Tissue Kit (Qiagen) following the manufacturer's protocol for animal tissues. DNA concentration and purity were assessed by spectrophotometry before proceeding to library preparation. Genomic DNA was extracted using Gentra Puregene Tissue Kit (Qiagen). DNA (1.5 μg) was diluted to 55 μl in TE buffer and fragmented using Covaris S2 sonicator (Intensity: 5; Duty Cycle: 10%; Cycles/burst: 200; Time: 120 s; 2 cycles). Fragmented DNA was analysed on TapeStation with High Sensitivity D1000 ScreenTape.  Libraries were prepared using NEBNext Ultra II DNA Library Prep Kit starting with 10 ng fragmented DNA and 9 PCR cycles. Libraries were profiled on 2100 Bioanalyzer (Agilent) and quantified using Qubit 1× dsDNA HS Assay Kit. All 22 samples were pooled equimolarly and sequenced on NextSeq2000 P2 FC (2×100 cycles plus 2×8 index cycles).
identifiers.namespace: European Bioinformatics Institute
identifiers.id: E-MTAB-15751:Strain2_32a_p