GSM3579959: WT_SC_R2: WT rep2; Arabidopsis thaliana; RNA-Seq
Total RNA was extracted using NucleoSpin RNA II kit (Macherey-Nagel) Using 3-15 μg of RNA as starting material, a mRNA enrichment was performed with the MicroPoly(A) Purist kit (AMBION). To prepare the RNA-Seq library, the SOLID Total RNA-seq kit (Life Technologies) was used. After obtaining the library, an equimolar mixture of it was used to perform an emulsion PCR using the automatic system of EZ Beads (Life Technologies). Then, the bead enrichment was performed, followed by its deposition in the sequencing wells. The sequencing step was performed using a SOLID 5500XL equipment of 75 nucleotides using the Exact Call Chemistry. Single end reads in fastq format were mapped against the TAIR10 Arabidopsis transcriptome (www.arabidopsis.org) using kallisto (v0.43.0 with default parameters except l=200 and s=30), (Bray et al., 2016). Data was combined in R (v3.3.2) and analyzed. Principal component analysis was carried out with prcomp (scale=T). Differential expression was assessed with edgeR in classic mode (Robinson et al., 2010) followed by Benjamini Hochberg correction (Benjamini and Hochberg, 1995). Enrichment analysis were conducted using topGO (R package version 2.22.0) with Fisher's Exact Test (Fisher, 1922) followed by Benjamini Yekutieli correction (Benjamini and Yekutieli, 2001). The level of significance was fixed at 0.01 (1%) after correction.
GEO Accession
GSM3579959