Neoprep stranded mrna seq kit

For FWA expression in DMS3-ZF T2 lines (Figure S2C), RNA from pools of 12 day-old seedlings grown on 1/2 MS plates in long days period was extracted using Direct-zol kit (ZYMO research). For the rest of experiments, RNA from adult leaves grown on soil of one plant was extracted using Direct-zol kit (ZYMO research). 75ng of total RNA was used to prepare libraries using the Neoprep stranded mRNA-seq kit (Illumina). Alternatively, 1 μg of total RNA was used to prepare libraries using the TruSeq Stranded mRNA kit (Illumina).

RNA was extracted using Direct-zol RNA Miniprep kit (Zymo). For RNAseq involving ZF108–TET1cd and ZF108–YPet plants, 75 ng of total RNA was used to prepare libraries using the Neoprep stranded mRNA-seq kit (Illumina). For RNA-seq involving ZF1CACTA1–TET1cd, ZF2CACTA1–TET1cd, SunTagFWAg4-14aa, and SunTagFWAg4-22aa plants, 1 μg of total RNA was used to prepare libraries using the TruSeq Stranded mRNA-seq kit (Illumina). Reads were first aligned to the TAIR10 gene annotation using Tophat (46 (link)) by allowing up to two mismatches and only keeping reads that mapped to one location. When reads did not map to the annotated genes, the reads were mapped to the TAIR10 genome. Number of reads mapping to genes were calculated by HTseq (47 (link)) with default parameters. Expression levels were determined by RPKM (reads per kilobase of exons per million aligned reads) using customized R scripts.