Megabace1000 genotyping system

Genomic DNA (gDNA) was purified from mouse ear biopsies with High Pure PCR template preparation kit (11796828001; Roche Holding AG, Basel, Switzerland) according to the manufacturer’s protocol. Purified gDNA was used for genotyping by high-resolution melting (HRM) SNP genotyping for rs33583463 (5:118596773 bp; mouse genome assembly GRCm38) and rs29824716 (5:120043597 bp; mouse genome assembly GRCm38), and by PCR-agarose gel electrophoresis method for the homemade microsatellite marker D5tbxhm17 (5:119660373 bp; mouse genome assembly GRCm38). HRM analysis was carried out on the Roche LightCycler 480 using High Resolution Melting Master (04909631001, Roche Holding AG, Basel, Switzerland) as described elsewhere [26 (link)]. Microsatellite marker genotyping was determined by analyzing PCR products on a MegaBACE1000 genotyping system (Amersham Biosciences, Little Chalfont, UK) or on a 3% agarose gel as previously described [9 (link)]. Primer sequences are provided in the Additional file 1: Table S1. Based on genotype, mice were allocated into homozygous congenic, sub-congenic or wild type control groups for various experiments.

Genomic DNA (gDNA) was purified from mouse ear or tail biopsies with a High Pure PCR template preparation kit (11796828001; Roche Holding AG, Basel, Switzerland) according to the manufacturer’s recommendation. Purified gDNA was used for genotyping by high resolution melting (HRM) single nucleotide polymorphism (SNP) genotyping for rs33583463 (5:118,596,773 bp, mouse genome assembly GRCm38) and by PCR-agarose gel electrophoresis method for homemade microsatellite marker D5tbxhm17 (5:119,660,373 bp, mouse genome assembly GRCm38). HRM analysis was carried out on Roche LightCycler 480 using High Resolution Melting Master (04909631001; Roche Holding AG, Basel, Switzerland) as described elsewhere (Thomsen et al. 2012 (link)). Microsatellite marker genotyping was determined by analyzing PCR products on a MegaBACE1000 genotyping system (Amersham Biosciences, Little Chalfont, UK), as previously described (Karlsson et al. 2003 (link)), or on a 3% agarose gel.
For DNA sequencing, all coding exons (and surrounding regions) of the Med13L gene for BR.RIIIS/J-Eae39r1 congenic and B10.RIII control mice were amplified with specific primer pairs and sequenced by Sanger technology (GATC biotech, Germany). Primer sequences used for genotyping and sequencing are available upon request.