Identification of PUMPKIN RNA targets using RIPSeq

Stroma extracts were isolated from 2-week-old WT plants and incubated with either PUMPKIN-specific antibodies or with the pre-immune serum. IgGs were captured with SiMAG-Protein G beads (Chemicell) and recovered RNA was used for generation of libraries with the ScriptSeq v2 RNA-seq Library Preparation Kit (Epicentre). Primary reads were aligned to the Arabidopsis chloroplast genome (accession number NC_000932.1) using CLC Genomics Workbench, the mean RPKM values of the replicates as well as the ratio of PUMPKIN vs Pre-immuneserum were calculated. Five prominent RNA targets (trnG-UCC, trnV-UAC, petB, petD and ndhA) were identified and validated via Slot Blot analyses. RNA Immunoprecipitation with PUMPKIN-specific antibody and the respective pre-immuneserum as control using WT stroma extract. As PUMPKIN represents a soluble chloroplast-localized protein, stroma extract was used for RNA-Immunoprecipitation to identify the plastid RNA targets of the PUMPKIN protein.