Bioanalyser nanochip

Cardamine insueta, C. amara, and C. rivularis plants used in this study were collected from Urnerboden. All plants were grown together in a plant cultivation room with 16 h light and 8 h dark cycle. The plants were planted in single pots, placed on trays, and watered from below.
Submergence treatment was started in the morning at 07:00. Two mature leaves were detached and submerged in water. We isolated RNA from the floating leaflets of the three species at nine time points after the start of submergence treatment (0, 2, 4, 8, 12, 24, 48, 72, and 96 h) using Qiagen RNeasy kit (Qiagen, Maryland, United States). RNA quality was assessed by Bioanalyser Nanochip (Agilent, Santa Clara, United States) and libraries quantified by Qubit (Thermo Fisher, Waltham, MA, United States). In total 27 libraries (3 species × 9 time points) were prepared according to NEBNext Ultra^textTM Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, United States) followed by paired end sequencing (100 bp × 2) on a HiSeq2000 with a HiSeq Paired-End Cluster Generation Kit and HiSeq Sequencing Kit (Illumina, San Diego, CA, United States). Trimmomatic (ver. 0.36) (Bolger et al., 2014 (link)) was used for discarding the low-quality reads with parameters of “PE -threads 4 -phred33 ILLUMINACLIP:adapters.fa:2:30:10 LEADING:20 TRAILING:20 SLIDINGWINDOW:4:20 MINLEN:50”.

Transmural needle biopsies (1–2 mg tissue) from the cardiac anterior left ventricle were obtained as described previously^{21 (link)} from 11 patients with aortic valve stenosis (AS). Patients undergoing coronary artery bypass graft (CABG) surgery (n = 6) served as controls. Clinical data of the patients are shown in supplemental table S1. RNA was isolated using the RNeasy kit (Qiagen) and quality of RNA was determined using Bioanalyser Nanochip (Agilent Technologies). RNA purity and concentration was measured with the NanoDrop Spectrophotometer (Nanodrop Technologies). The human total RNA was hybridized to Illumina Human miRNAv2 Expression Panel arrays by ServiceXS (Leiden, The Netherlands). The lumi 2 package from Bioconductor was used to annotate and quantile normalize the expression data. To assess differential expression, the limma 3 package was used and genes with an adjusted p-value of <0.05 were considered significantly differentially regulated. With the genes of which the expression correlated strongly (r2 > 0.5) with CILP1, a statistical overrepresentation test was performed using the PANTHER classification system^{22 (link)}.

An Agilent Bioanalyser Nanochip (Agilent, Melbourne, VIC, Australia) was used for quality control of total RNA. cDNA libraries were synthesized and prepared using the Illumina Truseq Stranded mRNA Kit (Illumina), according to the manufacturer’s instructions. cDNA libraries were then sequenced using the HiSeq 2000 sequencing system (Illumina) at AGRF. The primary sequence data were generated using the Illumina CASAVA 1.8.2 pipeline. The sequence reads were aligned against the Homo sapiens genome (Build version HG19) and mapped using the Tophat aligner (v1.3.1; http://ccb.jhu.edu/software/tophat/manual.shtml). The transcripts were assembled and tested for differential expression using the Cufflinks tool (v2.2.1; http://cole-trapnell-lab.github.io/cufflinks/).