Identification of BSF RNA targets using RIPSeq

Stroma extracts were isolated from 2-week-old WT plants and incubated with either BSF-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. BAM files were extracted and sorted in Galaxy . Sorted BAM files were converted into RPKM-normalized bigwig files and displayed in IGB. The differential enrichment of BSF/control of the two replicates was displayed across the entire chloroplast genome to identify the RNA targets of BSF. Overall design: RNA Immunoprecipitation with BSF-specific antibody and the respective pre-immuneserum as control using WT stroma extract. As BSF represents a soluble chloroplast-localized protein, stroma extract was used for RNA-Immunoprecipitation to identify the plastid RNA targets of the BSF protein. BSF-specific antibodies were produced in Rabbit using recombinant BSF as antigene.