RNA-seq profiling of six fetal tissues at day 63 of gestation from a reciprocal backcross of German Landrace and Piétrain
RNA-seq profiling of six fetal tissues at day 63 of gestation from a reciprocal backcross of German Landrace and Piétrain
Briefly, twenty-two F₁ sows from (German Landrace [GL] × Piétrain [Pi]) were backcrossed to three GL and three Pi sires to produce F₁×GL and F₁×Pi conceptuses. A total of 270 BC fetuses were collected at day 63 of gestation. From these, 38 fetuses were selected for this study. Tissues (liver, brain, muscle, kidney, ovary, and testis) were carefully dissected, immediately snap-frozen in liquid nitrogen, and stored at −80°C until RNA extraction. For the four non-gonadal tissues (liver, brain, muscle, and kidney), a set of 24 matched samples was obtained from the same fetuses (n=12 per BC group for each tissue). Furthermore, 17 ovary samples (9 F₁ × GL, 8 F₁×Pi) and 18 testis samples (10 F₁ × GL, 8 F₁×Pi) were collected. Of these gonadal samples, 10 ovaries and 11 testes originated from the same 24 fetuses used for the non-gonadal tissues. The remaining 7 ovary and 7 testis samples were obtained from additional fetuses not included in the non-gonadal set.  Total RNA was isolated from tissue samples using QIAzol Lysis Reagent (Qiagen, Germany), while the ovary, testis, and muscle tissues underwent additional homogenization with ceramic beads using a Precellys 5000 homogenizer. These tissues were chosen for homogenization as they are denser and more fibrous, requiring additional mechanical disruption for effective RNA extraction. Following RNA extraction, DNase treatment was applied to remove genomic DNA contamination, and RNA was purified using the RNeasy Mini Kit (Qiagen, Germany). RNA concentration and quality were assessed using a NanoDrop ND-1000 spectrophotometer (Peqlab) and a Bioanalyzer 2100 (Agilent Technologies). To prepare the RNA library, 1 µg of total RNA with a RIN > 8 was used for library preparation using an Illumina Stranded mRNA Prep, Ligation kit with 11 PCR-cycles of amplification according to the manufacture's recommendation (Illumina). The libraries were quality checked for fragment length distribution on Agilent 2100 Bionalyzer and normalized to an equal concentration of 10nM prior to pooling. 