Tissueruptor device

The cytokines IL-10, IFN-γ, and TNF-α were quantified in serum and jejunum samples by using Simplex Luminex kits for each immune parameter and ProcartaPlex Basic Rat kits (eBioscience, Vienna, Austria) in a Luminex 100 IS™ (Luminex Corporation. Austin, TX, USA). The jejunum samples (100 mg) were first manually disaggregated with sterile scalpels, and the resulting tissue sections were homogenized in 0.5 mL RIPA buffer containing a cocktail of protease inhibitors (Complete, Mini tablets, Roche Life Science, Mannheim, Germany). Homogenization was completed by using a TissueRuptor® device (Qiagen, Hilden, Germany), and the samples were kept on ice during the procedure. Then, the samples were centrifuged (16,000× g for 10 min at 4 °C), and the protein concentration of the resulting supernatant was quantified and adjusted to 10 mg/mL for cytokine assessment as recommended by the manufacturer. These measurements were carried out in duplicate for each sample.

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 ].

Cell-seeded scaffolds after the 24- and 48-h growth periods were snap-frozen in 350 μL TRIzol^® (Sigma) and stored at −80°C until preparation. On thawing, the scaffolds were homogenized using a TissueRuptor™ device (Qiagen) and centrifuged at 12,000 rpm to obtain an aqueous layer and this was subjected to a chloroform extraction and 70% ethanol precipitation. The RNA was then prepared using an RNeasy^® kit (Qiagen) according to the manufacturer's protocol. The RNA (100 ng/μL) was used to prepare cDNA using ImProm-II™ Reverse Transcription System (Promega) according to the manufacturer's instructions.