GSM8086964: E19-T1-47-AI: Conceptus_Endometrium Co-culture_Rep 4; Bos taurus; RNA-Seq
Endometrial total RNA was extracted and analyzed using RNA-Sequencing (RNA-Seq). Endometrial total RNA extraction was achieved using 5 mm stainless steel beads (Qiagen) and a bead mill homogenizer (Bead Mill 4; ThermoFisher) and Trizol (Invitrogen) in combination with an on-column method (RNeasy Mini Kit; Qiagen) involving a DNase step as previously described by Mathew et al. 2019. The resulting RNA quality and quantity were assessed using the 2100 Bioanalyzer (Agilent Technologies) and a NanoDrop 1000 (NanoDrop Technologies), respectively. Endometrial RNA integrity numbers (RIN) ranged from 5.2 – 9.3 and total endometrial RNA was analyzed using RNA-Sequencing (RNA-Seq) for the following culture tissues: 1) Endo (n=13; mono-culture); and 2) Endo-Conceptus Endo (n=15; co-culture). Conceptus RNA was extracted using the AllPrep DNA/RNA Mini Kit (Qiagen) with the optional on-column DNase digestion step according to the manufacturer's recommendations. The RNA quantity and quality were assayed using a NanoDrop 1000 and RNA Screen Tape (Agilent Technologies). The conceptus RIN ranged from 7.5 – 10. Conceptus section total RNA was assayed using RNA-Seq for the following culture tissues: Conceptus (n=8; mono-culture) and Endo-Conceptus Conceptus (n=9; co-culture). Library preparation including poly (A) enrichment and RNA-Seq of the explant and conceptus total RNA was performed by the University of Missouri DNA Core facility (Columbia, MO). The cDNA libraries were prepared using the Stranded RNA Prep Ligation Kit (Illumina) as described by the manufacturer. The final amplified cDNA constructs were purified by addition of Axyprep Mag PCR Clean-up beads (Corning) and the final construct of each purified library was evaluated using a Fragment Analyzer automated electrophoresis system (Agilent), quantified using the Invitrogen Qubit HS dsDNA assay kit and Qubit fluorometer, and diluted according to Illumina's standard protocol for sequencing on the NovaSeq 6000 (Illumina).