RNA sequencing (RNA-seq) analysis of 35SARF18-OE-2 and Col.

The goals of this study are to find the target gene of ARF18, to explain why the ARF18 overexpression line has smaller leaves. Library construction and sequencing were performed as described elsewhere, based on RNA extracted with the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from 7-day-old seedlings of 35S-ARF18-OE-2 and Col. The RNA was purified by electrophoresis through a TBE-urea polyacrylamide gel. The gel regions harboring 18-30 nt fragments were excised and the RNA recovered. The resulting population of sRNAs was 5' and 3' RNA adaptor-ligated using T4 RNA ligase and amplified for 18 PCR cycles. The amplicons were purified and sequenced using an Illumina/Solexa Genome Analyzer (Beijing Genomics Institute, Shenzhen, China). Raw reads were trimmed by removing low-quality and adaptor sequences, and then they were aligned to the Arabidopsis genome using SOAP2 software. Transcript frequency was calculated using the reads per kilobase per million reads method. Differentially transcribed genes were identified using a method modified from Audic and Claverie (1997). Significance was based on the p value and the false discovery rate (FDR) (Audic and Claverie, 1997). The chosen thresholds were FDR = 0.001 and |log2| = 1. We identified 113 differentially expressed genes, of which 49 genes are up-regulated, and 94 genes are down-regulated. Overall design: The RNA was extracted from 7-day-old seedlings of Col and 35SARF18-OE-2 for deep sequencing.

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