RNA sequencing of doxycycline-inducible TPRX3 overexpression in bovine fetal fibroblast cells
The PCR amplified TPRX3 fragment derived from Estonian Holstein 8-cell stage IVF embryos was inserted into TetON piggyBac plasmid backbone. 1 µg of pB-TetOn-bgi-TPRX3-IRES-GFP-PGK-Puro, 0.5 µg of pPB-CAG-hyPBase-IRES-Venus, and 0.5 µg of pPB-CAG-rtTA-M2-IN plasmids were used per electroporation using 1600 V 20 ms 1 pulse settings. The bovine fetal fibroblast cells were grown for 24 h and checked for Venus expression under GFP channel with emission at 486 nm. The antibiotic selection started 24 h post electroporation in Puromycin (0.2 - 0.4 µg/ml) for ~3 days and G418 (200 – 400 µg/ml) for ~7 days after electroporation by gradually increasing concentration depending on condition of cells. In the overexpression group, cells were treated with 1 µg/ml doxycycline for 24 h whereas in the control group, cells were left untreated. Treated cells were collected 24 h following treatment. Two replicates of doxycycline-treated and non-treated bovine fetal fibroblast cells were harvested and processed for RNA extraction using Direct-zol™ RNA Microprep kit (Zymo Research). cDNA libraries were prepared according to the kit SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara). cDNA concentrations were assessed by Qubit Fluorometer (ThermoFisher Scientific), and 47.9 ng of cDNA library from each sample were pooled. Paired end sequencing using the Illumina NovaSeq platform and demultiplexing were performed at Novogene.