Identifying Modifier Candidates via ENU-Induced Mutagenesis

For ENU-induced mutagenesis, the NMF291^−/+ males were i.p. injected with ENU (80–110 mg/kg Body Weight (B. W.)) for three consecutive weeks, as previously reported (Salinger and Justice, 2008 ; Chen et al., 2020 (link)). After an infertility test, the ENU-treated males were crossed to untreated NMF291^−/+ females for G₁. Of these offsprings, the NMF291^−/− mice were used for behavioral tests, and the mice with less ataxia phenotype and improved lifespan were selected for family pedigree determination.
For identification of the modifier candidates, the ENU family members with or without phenotypic improvement were applied for exome sequencing. For exome capture and library construction, the instructions of NimbleGen SeqCap EZ Exome Library SR Platform (Roche) were followed. Briefly, genomic DNA was fragmented to 200–300 bp with ultrasonic shearing. End-repair, A-tailing, adapter ligation, and pre-capture ligation were performed by using a KAPA LTP Library Preparation Kit (Roche). After exome capture, the resulting samples were amplified by KAPA HiFi HotStart Ready Mix using Pre-LM-PCR Oligos 1 and 2. Libraries passing QC were sequenced (Illumina HiSeq 2500 platform) and data processing and variant discovery were performed by using GATK platform (McKenna et al., 2010 (link)). Variant annotation and classification were achieved using ANNOVAR (Wang et al., 2010 (link)).