Qubit assay

DNA extracts from blood and ear tissue samples from Wadi Sareen were assessed for DNA quality via agarose gel electrophoresis on a 2% gel, and 21 samples (TAH044-55, TAH057-59 and TAH060-64) with suitable non-degraded DNA selected for the library preparation stage. DNA was quantified using a Qubit Assay (Thermofisher Scientific) and normalised to 7 ng–1 µl.
A double digest RAD (ddRAD) library was produced according to a modified protocol of Peterson et al. (2012 (link)) (see also Bourgeois et al. (2018 (link)) for the modified protocol) using the restriction enzymes SphI and SbfI and a gel excision section of 320–590 bp following restriction of the DNA with the two enzymes. Individuals in the library were barcoded using a barcode on each end of the fragment (double barcode combination) and each individual was repeated twice with a different barcode across the library to improve evenness of coverage. The barcoding system allows DNA from multiple individuals to be “tagged” and pooled into a single lane of sequencing. An additional positive control was included to allow for quality control of the experimental process and for assessment of genotyping error-by-read depth. The resulting library was quantified using a Qubit Assay (Thermofisher Scientific) and then sequenced in both directions (Read 1 and Read 2) using a single lane of MiSeq Sequencing Technology (Illumina).

We extracted the total RNA from the six experimental individuals (three animals per group) by Trizol (Invitrogen, Carlsbad, CA, USA), the RNA was then treated by DNase digestion and purified by Qiagen RNeasy column (Invitrogen, Carlsbad, CA, USA), as previously described [35 (link)]. We checked the integrity and quality by a NanoDrop 1000 spectrophotometer and by visualization on a 1.5% agarose gel. The microRNA-Seq library was built utilizing NEBNext Multiplex Small RNA Library Prep Set for Illumina (Set 1) following the instructions provided by the manufacturer (NEB, E7300S/L, USA). The library construction was started with 800ng total RNA. Firstly, the 3’ adaptor and 5’ adaptor were sequentially ligated to the RNA. Then, we performed the reverse transcription to synthesize the first strand. Next, we executed the PCR amplification and added the 6-bp index to the DNA products; different libraries were assigned different indexes. Subsequently, we selected and recycled the DNA fragments from 140 to 150 bp using 6% polyacrylamide gel (6% Novex^® TBE PAGE gel, Life Technology, USA), which were corresponding to RNAs from 21 to 30 bp. After purification, the concentration was measured by the Qubit assay (Life Technology, Q32850). Finally, we sequenced the libraries identified by the 6-bp index through an Illumina HiSeq 2000 sequencer, as described previously [36 (link)].