Comparative testis transcriptome identified genes associated with male infertility of cattleyak
Total RNA was extracted from the six frozen testis samples using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. Magnetic beads with Oligo (dT) are used to isolate mRNA from the total RNA. Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then cDNA is synthesized using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments are connected with adapters. At last, the library could be sequenced using Illumina HiSeqTM 2000. Raw reads produced by Illumina HiSeqTM 2000 were filtered into clean reads and aligned to Bos_taurus_ncbi database containing 63379 non-redundant sequences (http://www.ncbi.nlm.nih.gov /protein? term=txid9913[Organism]) with SOAPaligner/SOAP2. The alignment data was utilized to calculate distribution of reads on reference genes and perform coverage analysis and other downstream analysis.