Conformation/Identification of dPPRrcbL RNA targets using RIPSeq

Stroma extracts were isolated from 2-week-old transgenic dPPRrbcL or WT plants and incubated with HA-specific antibodies. IgGs were captured with Protein A Dynabeads (Thermo Fisher scientific) and recovered RNA was used for generation of libraries with the NEBNext® Ultra™ II RNA Library Prep Kit. Primary reads were aligned to the Arabidopsis chloroplast genome (accession number NC_000932.1) using CLC Genomics Workbench. Reads of the two replicates aligned in CLC Genomics Workbench were extracted as coverage (reads per nucleotide). Graphs were created with excel using the extracted coverage values RNA Immunoprecipitation with HA-specific antibody using WT and dPPRrbcL stroma extracts. As dPPRrbcL is expected to be a soluble chloroplast-localized protein, stroma extracts were used for RNA-Immunoprecipitation to confirm association with the rbcL mRNA and to identify potential off-targets.