Transcriptome profiles of paternal testis and their offspring in response to gut microbiota dysbiosis using RNA sequencing analysis
Transcriptome profiles of paternal testis and their offspring in response to gut microbiota dysbiosis using RNA sequencing analysis
At 5 weeks of age, specific pathogen free (SPF) male C57BL/6J inbred mice were transferred to a conventional colony facility. The male mice were divided into separate cages (one mouse/cage) and randomly assigned to two groups: (i) Control group, which received regular diet and sterilized water for six successive weeks; (ii) Treated group, which received regular diet and sterilized water supplemented with a cocktail of non-absorbable antibiotics (neomycin 2.5 mg/ml, bacitracin 2.5 mg/ml and pimaricin 1.25 ug/ml) for six successive weeks. The water bottle and antibiotics cocktail were replaced freshly every 5 days. At the end of six weeks treatment or 4-week after treatment withdrawal, male mice from both groups were mated with treatment-naive 6 weeks old C57BL/6J inbred female mice. At the end of six weeks treatment or 4-week after treatment withdrawal, male mice from both groups were mated with treatment-naive 6 weeks old C57BL/6J inbred female mice. Subsequent to mating, male mice were sacrificed and testes were harvested for comparative transcriptome analysis. To characterize F1 placental and fetal brain phenotypes, pregnant dams were sacrificed either at E13.5 or at E18.5. While to phenotype F1 offspring in the postnatal period, offspring whole brain and brown adipose tissue were harvested on postnatal day 17 (P17). Of note, all tissue samples were snap frozen in liquid nitrogen and stored at -80C until processing for transcriptome profiling. Frozen tissue samples stored in -80C were thawed in ice and total RNA was extracted from four different tissue types: sire testis, F1 placenta, F1 brain and F1 brown adipose tissue (BAT), using RNeasy Protect Mini Kit (Qiagen) following the manufacturerâ€™s instructions. The quantity and quality of total RNA was evaluated on a NanoDrop Spectrophotometer and Agilent TapeStation system (Agilent, CA, USA) to ensure RIN>8.5. For library preparation, mRNA was first enriched using the NEBNext Poly(A) mRNA magnetic isolation module with 1ug input RNA. Purified mRNA was subsequently prepped into stranded libraries using the NEBnext Ultra II directional RNA library prep kit, following all manufactures guidelines. Amplified libraries were multiplexed and sequenced on an Illumina NextSeq 500 (PE40).