Hiseq 3000 instrument

10 mL of yeast undergoing meiosis at 4h (OD600 1.9) was harvested at 1000g for 5 min at 4°C. Cells were washed with ice-cold TE buffer and mechanically lysed with glass beads. Samples were centrifuged for 2 min at full speed and transferred to a new microcentrifuge tube. 1 mL of 70% ethanol was added to the homogenized lysate. Total RNA was then extracted and purified using the RNeasy RNA isolation kit (Qiagen), including treatment with DNase. RNA integrity was assessed by urea RNA-PAGE and quantitated with Quantifluor (Promega). Enrichment of mRNA, library preparation and sequencing was performed by the Max Planck-Genome Centre (Cologne, Germany). Briefly, rRNA was depleted using the Ribo-zero rRNA removal kit (Epicentre). mRNA was enriched using oligo-dT beads (New England Biolabs) and the cDNA library was prepared using the TrueSeq RNA sample preparation kit (Illumina) according to the manufacturer’s instructions. Samples were then sequenced on an Illumina HiSeq-3000 instrument. The RNA-seq raw data employed in this study are deposited at the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/), under accession no. under accession no. GSE144835.

We isolated total RNA from skin biopsies of 3 dogs (DS032, DS042, LA1666 (PRJEB14110 and PRJEB14109)), hair follicle tissue from 1 dog (PRJEB14110), and a skin biopsy from 1 horse (UKH004) (PRJEB12979) using the RNeasy Fibrous Tissue Mini kit (Qiagen). Prior to RNA extraction the tissue was mechanically disrupted using the TissueRuptor device (Qiagen). The RNA samples were transformed into illumina TruSeq libraries and 2 x 150 bp sequencing reads were obtained on a HiSeq3000 instrument (illumina) at the Next Generation Sequencing Platform, University of Bern. The reads were filtered for low quality bases using a Phred quality score threshold of 15 for each base and reads longer than 50 bases were retained. The star aligner [27 (link)] was used to map the quality filtered reads to Ensembl CanFam3.1 reference using recommended parameters ‘—outFilterType BySJout’ and ‘—outFilterMultimapNmax 20’. We additionally used publicly available illumine skin RNA-seq datasets from the skin of two dogs, a Beagle and a dog of unspecified breed [22 (link)] as well as from the skin biopsy of one domestic horse showing Leopard complex spotting (EBI accessions PRJNA78827 and PRJEB3095). The reads of the downloaded RNA-seq data sets were also filtered and mapped to the reference genome in the same manner as described above. Exon level coverage was calculated using BedTools [28 ].

The selection of patients to be subjected to CES was based on three main criteria: (i) the lack of genetic diagnosis by PS; (ii) the availability of DNA from at least three relatives; and (iii) the quality of DNA samples. Thus, eleven index patients were selected (Figure 1) and captured using the SureSelect^XT Focused Exome design (Agilent Technologies, Santa Clara, CA, USA), consisting of exonic regions of ~4800 genes (12 Mb) that have been associated with diseases in databases as HGMD, OMIM and ClinVar. Libraries were prepared following the “SureSelect^XT Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library” protocol (Version B4, Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed in a Hiseq3000 instrument (Illumina, San Diego, CA, USA) using a HiSeq 3000/4000 SBS Kit (300 cycles) and a HiSeq 3000/4000 PE Cluster Kit.

The quantity and quality of the purified total RNA was assessed using a Thermo Fisher Scientific Qubit 3.0 fluorometer with the Qubit RNA BR Assay Kit (Cat # Q10211, Thermo Fisher Scientific) and an Advanced Analytical Fragment Analyzer System using a Fragment Analyzer RNA Kit (Cat # DNF-471, Agilent Technologies, Santa Clara, USA), respectively. Sequencing libraries were constructed using an Illumina TruSeq Stranded mRNA Library Prep kit (20020595, Illumina, San Diego, USA) in combination with TruSeq RNA UD Indexes (Cat # 20022371, Illumina) according to Illumina’s guidelines. The cDNA libraries were evaluated using a Thermo Fisher Scientific Qubit 3.0 fluorometer with the Qubit dsDNA HS Assay Kit (Cat # Q32854, Thermo Fisher Scientific) and an Agilent Fragment Analyzer (Agilent) with an HS NGS Fragment Kit (Cat # DNF-474, Agilent), respectively. Pooled cDNA libraries were sequenced 100 bp single-end on one lane of an Illumina HiSeq 3000 instrument (Illumina). All base call files were demultiplexed and converted into FASTQ files using Illumina bcl2fastq conversion software. The quality control assessments, generation of libraries, and sequencing were conducted by the Next Generation Sequencing Platform, University of Bern.

For library preparation, RNA quality and concentration was measured using the Fragment Analyzer from AATI (now Agilent) with the total RNA Standard Sensitivity protocol. The Sequencing library was prepared from 200 ng total RNA with the TruSeq® Stranded mRNA LT-Set B (RS-122-2102, Illumina Inc., San Diego, CA) producing a 275 bp fragment including adapters in average size. In the final step before sequencing, seven individual libraries were normalized and pooled together using the adapter indices supplied by the manufacturer. Pooled libraries have been clustered on the cBot Instrument from Illumina using the HiSeq® 3000 GD-410-1001 3000/4000 SR Cluster Kit (Illumina Inc., San Diego, CA). Sequencing was performed as 85 bp single-end reads and 7 bases index read on an Illumina HiSeq. 3000 instrument at a sequencing depth of approximately 60 million reads per sample using FC-410-1001, HiSeq® 3000/4000 SBS Kit (50 cycles, Illumina Inc., San Diego, CA).