High resolution melting master

For qPCR, RNA was isolated from tissue or cells using TRIzol (Invitrogen 15596–026) according to the manufacturer's protocol. Subsequently, 200 ng RNA was treated with DNAse I (Invitrogen 18068–015). cDNA was synthesised using Superscript II reverse transcriptase (Invitrogen 18064–071) and diluted 4 times with milliQ water. Quantitative real time PCR was performed on a Lightcycler 480 (Roche) using SYBR green (Roche 04887352001). For miRNA quantification cDNA was synthesized with the miScript reverse transcription kit (Qiagen 218061), diluted 8 times with milliQ and qPCR was performed with High Resolution Melting Master (Roche) on a Lightcycler 480 according to the manufacturers' protocols. To validate miRNA-30c expression levels we also performed qPCR with the Taqman primer assay according to the manufacturers' protocol. Primer sequences are listed in Table S3. Analysis of qPCR data was performed using LinRegPCR analysis software [25] (link), [26] (link).

LGR were confirmed with either gene dosage or directly by sequencing the breakpoint. The gene dosage real-time quantitative PCR (qPCR) approach used four sets of primers: two sets inside the deletion and one set on each border. The qPCR was performed on a LightCycler 480^® (Roche Diagnostics, Penzberg, Germany) with High-Resolution Melting Master^® containing ResoLight^® dye (Roche Diagnostics), as described previously [28 (link)], and the analysis of quantitative result used the ΔΔCp method [29 (link)].
Characterization of large rearrangements was performed by classical PCR and Sanger sequencing. As a first screening, samples were amplified with the primer described by Puget et al. (Lit1—37 kb) and Swenssen et al. (Lit6—14 kb) [5 (link),8 (link)]. The sequencing of the breakpoint used primers on each non-deleted border of the deletion. The classical PCR approach was used. PCR products were analyzed on agarose gel and sequenced in both directions by using the PCR primers with the BigDye Terminator Cycle Sequencing Reaction Kit and an ABI Prism 3130XL automated sequencer (Applied Biosystems Waltham MS, US). The main primers are reported in Table 1. In cases in which a shortage of material prevented PCR or sequencing, the signal of the CGH array was used to estimate the average size of the deletion.

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.