Neoprep library prep system

The current study uses both publicly available (Guo et al., 2016 (link), 2019 (link); Li et al., 2017 (link); Ulfah et al., 2016 (link); Wang et al., 2015 (link)) and newly generated whole‐genome sequencing data (Figure 1a,b). The new sequence data (n = 341 individuals from 12 breeds) were generated from libraries prepared using the NeoPrep Library Prep System (Illumina) and sequenced to, on average, 8× genome coverage by paired end (2×150 bp) sequencing on the IIlumina Hiseq X Ten system at CLOUD HEALTH. Table S1 provides detailed information on all the 43 populations used. The generated sequence dataset has been uploaded to NCBI with BioProject numbers PRJNA597842, PRJNA547951, and PRJNA552722.

Total RNA was extracted using the same procedure for all samples: homogenized in Qiazol with a pestle. Total RNA was extracted from the homogenate using the Qiagen RNeasy Universal Plus Mini kit (Qiagen, Hilden, Germany) with DNase treatment to remove traces of genomic DNA. Libraries were prepared on the Neoprep Library Prep System (Illumina, San Diego, USA) starting from 100 ng total RNA and following the manufacturer’s recommended protocol for the TruSeq stranded mRNA Library Prep Kit for Neoprep. Neoprep runs were performed using software version 1.1.0.8 and protocol version 1.1.7.6 with default settings for 15 PCR cycles and an insert size of 200 bp. Libraries were arranged in randomized order on library cards. To avoid batch effects, we used library cards with the same lot number for all samples for which direct comparisons of expression levels were planned (lot no. 20123465: CGE at generation 103, males, whole body, all ancestral and hot evolved samples; lot no. 20173962: CGE at generation 103, females, whole body, all ancestral and hot evolved samples; lot no. 20182049: CGE at generation 140, females and males, whole body and gonadal tissue). 50 bp single-end reads were sequenced on an Illumina HiSeq 2500.