Computational protocol: Caenorhabditis elegans BUB-3 and SAN-1/MAD3 Spindle Assembly Checkpoint Components Are Required for Genome Stability in Response to Treatment with Ionizing Radiation

Similar protocols

Protocol publication

[…] Mutagenesis and screening procedures were performed as described in . Single nucleotide polymorphism (SNP) mapping was performed according to the protocol described in . For whole-genome sequencing, genomic DNA was extracted and purified using a ChargeSwitch gDNA mini tissue kit (Invitrogen) and sent to GenePool (http://genepool.bio.ed.ac.uk/) for Illumina (Solexa) sequencing. Paired-end sequencing was set to achieve 24× coverage (100 bp paired-end reads for a total of 24,000,000 reads). Quality of the reads was checked using FastQC. Reads were then aligned to the C. elegans reference genome (WBcel235.74) using BWA-MEM. Variants in the strains TG1813 and TG2435 were called using the software SAMtools and Bfctools. Heterozygous variants and variants that were not unique to the mutant strain were filtered out. We then extracted homozygous variants within the 900 kb region determined by SNP mapping. Homozygous unique variants were then ranked based on the severity of the predicted effect on the genome. The gt2000 mutation was supported by 18 sequence reads including reads from both directions, confirmed visually using the Integrative Genomics Viewer software. [...] For time-lapse imaging of live embryos, we followed an imaging procedure as described in . Young adults were irradiated as described in the embryonic lethality assay. One-cell embryos were dissected in M9 buffer 24 hr post irradiation and immediately mounted on 2% agarose pads. Images were acquired every 10 sec using a spinning-disk confocal microscope (IX81; Olympus) with spinning-disk head (CSU-X1; Yokogawa Electric Corporation) and MetaMorph software (Molecular Devices). For image processing, we used the ImageJ software. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Caenorhabditis elegans
Chemicals Cisplatin, Methyl Methanesulfonate