Sherlock ax dna isolation kit

For invasive prenatal studies, DNA was extracted from amniocytes using the Sherlock AX DNA isolation kit (A&A Biotechnology, Gdansk, Poland), according to manufacturer’s instructions. Array comparative genomic hybridization (aCGH) in the fetus was performed using the 60K CytoSure Constitutional v3 microarray (Oxford Gene Technology, Oxford, UK).
For further genetic testing, DNA was extracted from the peripheral blood of the newborn and his parents using Gentra Purgene Blood Kit (Qiagen, Germantown, MD, USA). Parental and proband DNA samples were tested for the presence of the CNV deletion using junction-specific PCR with DreamTaq DNA Polymerase (Thermo Scientific, Waltham, MA, USA), followed by Sanger sequencing to map the deletion breakpoints. To determine the parental origin of the detected CNV deletion, trio-based genome sequencing (GS) was performed using NEBNext^® Ultra™ II FS DNA Library Prep Kit for Illumina (New England BioLabs, Inc. Ipswich, MA, USA) and paired-end sequenced (2 × 150 bp) on NovaSeq 6000 (Illumina, San Diego, CA, USA). The parental origin of the observed chromosomal abnormality was determined by analyzing the informative single-nucleotide polymorphisms (SNPs) within the deletion region.

DNA from sl-pHCs cell lines was extracted using the Sherlock AX DNA isolation kit (A&A Biotechnology, Gdansk, Poland), according to the manufacturer’s instructions. Array comparative genomic hybridization (aCGH) was performed using the 60K CytoSure Constitutional v3 microarray (Oxford Gene Technology, Oxford, UK), according to the manufacturer’s protocol. Quality control measures were monitored using CytoSure Interpret Software (Oxford Gene Technology). Data analysis was performed using CytoSure Interpret Software (Oxford Gene Technology, Oxford, UK) and a circular binary segmentation algorithm. The CNVs were classified using the CytoSure Interpret Software (Oxford Gene Technology, Oxford, UK). The microarray used in this analysis does not contain SNP probes nor detect polyploidy, balanced chromosomal rearrangements, and loss of heterozygosity.

All SNPs within SLC16A1 were detected based on RNA-seq data previously obtained from Arabian horses according to EquCab2.0 reference [22 (link), 29 (link)]. Then, the fast and less cost-effective PCR-RFLP method was designed to identify polymorphisms in exon 5 (ss#3021042926), and PCR-HRM was used to identify mutation in the 5’UTR (ss#3021042925). The details of the methods used are presented in Table 1. For 48 randomly selected samples, the both amplicons were sequenced using Sanger sequencing to confirm the results obtained. The sequencing was performed using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosysytems, Thermo Fisher Scientific), PCR products were purified using BigDye XTerminator Purification Kit (Applied Biosysytems) and next sequenced on 3500xl Genetic Analyzer (Applied Biosysytems). DNA samples from blood and hair follicles were isolated with the use of Sherlock AX DNA Isolation kit (A&A Biotechnology, Gdynia, Poland), according to protocol. Both polymorphisms were genotyped for all horses.