Puregene tissue kit

Bisulfite sequencing was used to determine the methylation status of the F/R-BDP region in NO and EOC human tissue samples (Clark et al., 1994 (link)). Genomic DNA was isolated using the Puregene Tissue Kit (QIAGEN) and was chemically converted using the EZ DNA Methylation Kit (Zymo Research Corp.). The F/R-BDP region containing a CGI was amplified from the bisulfite-converted DNA using methylation PCR-specific primers, designed using MethPrimer (Supplementary file 2; Li and Dahiya, 2002 (link)). Gradient PCR reactions were performed using a C1000 Touch Thermal Cycler (Bio-Rad) to optimize annealing temperatures. After gel purification using the QIAquick Gel Extraction Kit (QIAGEN), PCR products were cloned using the TOPO TA Cloning Kit (Invitrogen). Between 9 and 15 individual clones were sequenced for each sample, and Sanger sequencing was performed by the UNMC DNA Sequencing Core Facility. DNA sequence information was analyzed using the Lasergene SeqMan Pro program (DNASTAR).

DNA was extracted using a Qiagen Puregene Tissue Kit following the DNA Purification from Tissue protocol. PCR inhibitors were removed from DNA using a Qiagen DNeasy PowerClean Clean Up Kit. A Qubit 2.0 fluorometer was used to measure the initial concentration of each extracted DNA sample and then the DNA was precipitated out, dried down and sent to Arbor Biosciences (Ann Arbor, MI) for library preparation, hybrid enrichment and sequencing, following details in Quattrini et al. [38 (link)]. The targeted enrichment of ultraconserved elements (UCE) and exonic loci was carried out using the hexacoral-v2 probe design, which includes 25 514 baits targeting 2499 loci [36 (link)]. Bioinformatic post-sequencing analyses were conducted following the Phyluce online documentation (https://phyluce.readthedocs.io/en/latest/tutorial-one.html), including raw read trim and matching of loci to UCE and exon probes. SPAdes v3.12.0 was used outside of the phyluce pipeline to assemble trimmed raw reads using the main executable script spades.py and a coverage cutoff of 2. Individually aligned UCE/exon loci were filtered to include only those that were present in at least 50% of the samples. All code used in this study are detailed in electronic supplementary material, Dataset S1.

DNA extractions were done using the Puregene Tissue Kit from Qiagen® following the manufacturer's instructions. Then, 500 ng of DNA were sheared to a 150–700 bp range using the Covaris® E210 instrument (Covaris, Inc., USA). Sheared DNA was used for Illumina® library preparation by a semi-automatized protocol. Briefly, end repair, A tailing and Illumina® compatible adaptors (BiooScientific) ligation were performed using the SPRIWorks Library Preparation System and SPRI TE instrument (Beckmann Coulter), according to the manufacturer protocol. A 300–600 bp size selection was applied in order to recover the most of fragments. DNA fragments were amplified by 12 cycles PCR using Platinum Pfx Taq Polymerase Kit (Life® Technologies) and Illumina® adapter-specific primers. Libraries were purified with 0.8× AMPure XP beads (Beckmann Coulter). After library profile analysis by Agilent 2100 Bioanalyzer (Agilent® Technologies, USA) and qPCR quantification, the libraries were sequenced using 100 base-length read chemistry in paired-end flow cell on the Illumina HiSeq2000 (Illumina®, USA).

Genomic DNA was extracted from the cheek swab samples using Qiagen’s Puregene tissue kit or Qiagen’s DNEasy DNA extraction kit. The entire coding sequence (except exon 11), as well as flanking intronic boundaries, were Sanger sequenced in the Italian greyhound (Supplementary Table S1). For the pumis, mudis, Chihuahua and wolf-dog hybrids, an amplicon encompassing exon 7 of the MLPH gene, and extending into the flanking introns (including alleles d² and the 1 bp insertion reported here), was obtained (Supplementary Table S1). For the mudis and wolf-dog hybrids not previously genotyped for the d¹ allele, a 268 bp amplicon was obtained with d¹ PCR primers F and R (Supplementary Table S1). Genotyping was performed with direct Sanger sequencing of the amplicon at Eurofins Genomics (Louisville, KY, USA), and visualized with Sequencher software (Gene Codes Corporation, Ann Arbor, MI, USA), or sequenced by Laboklin and analyzed with SeqScanner Software, respectively.

Frozen tissues samples were mechanically lysed with a Precellys homogenizer (Bertin technologies, France) in TRIzol reagent (ThermoFischer #15,596,026, Massachusetts, US) and processed according to the manufacturer’s instructions to obtain total RNA. DNA were extracted using the Puregene Tissue Kit (Qiagen #158,063, Germany). Both total DNA and RNA concentration were determined by spectrophotometry (Nanodrop 2000, ThermoScientific, Massachusetts, US). cDNA was produced from RNA using SuperScript IV Reverse Transcriptase (ThermoFischer #18,090,010, Massachusetts, US).