Transgenic Murine Testis RNA Extraction

Total RNAs were separately isolated from the decapsulated testes of four transgenic adult mice. Following homogenization in 0.5 ml RLT buffer (Qiagen) supplemented with 1.5% β-mercaptoethanol (Amresco) with a PRO Scientific 200 homogenizer (PROScientific Inc., Oxford, CT), RNAs were extracted as described62 (link)63 (link). Total RNAs were DNase treated (Turbo DNase, Ambion) and resolved using the 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Prepared mouse total testes RNAs64 (link) were used to construct individual RNA-seq libraries according to established protocols63 (link). Briefly, pre-amplified cDNA libraries were generated from 5 ng of total testis RNA using the Seq-plex system (Sigma) and used to construct sequencing libraries (DNA Ultra-Low, NEB). RNA-seq libraries were subjected to paired-end sequencing on the Illumina Hi-Seq 2500 platform, as above.