GSM7946892: HFpEF + Veh_2307; Mus musculus; RNA-Seq
Approximately 106 cells or 20-30 mg of mouse heart tissue were lysed with TRI Reagent (Zymo Research) and frozen at -80 °C to ensure complete cell lysis. Total RNA was extracted and washed from lysed cells using the Direct-zol-96 RNA Kit (Zymo Research) according to the manufacturer's instructions. Samples were treated with DNase I for 15 min at room temperature. cDNA was reverse transcribed from approximately 1 μg of RNA through random hexamers using the SuperScript III First-Strand Synthesis System according to the manufacturer's instructions (Thermo Fisher Scientific). Real-time qPCR reactions were performed using the TaqMan universal PCR master mix (Thermo Fisher Scientific) with the TaqMan probes listed. RT-qPCR reactions were performed using the QuantStudio7 Flex Real-Time PCR systems (Thermo Fisher Scientific). Each reaction was performed in triplicate for a given RT-qPCR run, and each condition had four experimental replicates. Relative expression of the gene of interest was normalized to GAPDH as the housekeeping control using the 2–ΔΔCT method. P values were calculated with Student's t-tests. From each replicate, 100 ng total RNA was extracted via the polyA-tail-specific protocol according to Illumina, Inc.. The RNA libraries were prepared using a TruSeq Stranded mRNA kit (Illumina), which also removes ribosomal RNA. The libraries were sequenced as 100 base pair single-end reads using Illumina NovaSeq SP with an average of 29.8 million reads per sample.