2.4 RNA extraction and RNAseq library preparation
Budget limitations precluded analyzing the full set of samples, so we measured gene expression on a subset of 12 samples including control and Cu-treated plants from one population from the center of the mine (Sc3) and one from the edges of the mine (Sc4) (n = 3 replicates per population and treatment). Total RNA was isolated with the RNeasy Plant Mini Kit (74904, Qiagen) following the manufacturer instructions with small modifications (detailed protocol available in the SMs).
The cDNA libraries were prepared with the NEBNext® Ultra™ II Directional RNA Library Prep with Sample Purification Beads (E7765S, NEB) following the manufacturer’s protocol with 1 µg of total RNA as input material (see detailed protocol in SMs).