GSM8997454: replum-ovule, mutant, rep1; Erythranthe nudata; RNA-Seq
Three biological replicates of homozygous Wt and mutant plants were selected for harvesting 6 gynoeciums/plant from opened flowers, and then the valves were removed to collect the ovules attached to the septum. Ovules/septum were immediately collected in cold TRIzol LS (Ambion) for further total RNA isolation. We used the RNeasy Micro Kit (Qiagen) with the following modifications: ovules/septum in cold TRIzol were ground gently with a pellet pestle in a 1.7 mL microcentrifuge tube for 2 min then mixed with 220 uL of chloroform. The solution was then shaken vigorously by hand for 15 s, then incubated for 2 min at room temperature followed by a centrifugation at 12,000g for 15 min at 4 °C to separate phases. Total RNA was isolated according to the manufacturer's instructions, which includes in column Dnase-I treatment for removal of contaminated DNA. We generated and sequenced a total of 6 RNA sequencing libraries. The RNA integrity number for all samples >7.3, as determined by an Agilent 2100 Bioanalyzer. Six strand-specific complementary DNA (cDNA) libraries were prepared from approximately 100 ng of total RNA by the North Carolina State University Genomic Science Laboratory (GSL) using a NEB Ultra Directional Library Prep Kit for Illumina then sequenced on an Illumina NovaSeq 6000.