title: RNA-Seq of Arabidopsis thaliana extracellular ATP response
design: With TruSeq RNA Sample Preparation Kit v2 (Illumina, Cat. # RS-122-2002), the poly(A) RNA was isolated from 2 g of the total RNA from each treatment sample using magnetic oligo (dT) beads. Following purification, the mRNA was fragmented by zinc treatment at 94 C for 5 min and reverse-transcribed to synthesize first strand cDNA using SuperScript II reverse transcriptase (Invitrogen) and random primers. Second-strand cDNA synthesis was performed, with the products then being subjected to end-repair and phosphorylation, and addition of an A base to the 3 end of the blunt phosphorylated DNA fragments. Illumina multiple indexing adapters were ligated to the fragments, as described by Illuminas TruSeq RNA Sample Preparation V2 Guide (Illumina). The cDNA fragments flanked by Illumina PE adapters were selected and purified by AMPure XP beads for downstream enrichment. The cDNA fragments were amplified by PCR Primers PE 1.0 and PE 2.0 (Illumina) that anneal to the ends of the adapters, using the PCR program of 30 s at 98 C followed by 15 cycles of 10 s at 98 C, 30 s at 60 C, 30 s at 72 C and a final elongation step of 5 min at 72 C. The products were purified using AMPure XP beads to create an Illumina paired-end library. Library quality control was performed with a Bioanalyzer DNA 1000 Chip Series II (Agilent). A qPCR method was employed for quantifying libraries in advance of generating clusters. The libraries were diluted to a final concentration of 10 nM. The paired-end libraries were applied for cluster generation at a concentration of 10 pM on a flowcell in a cBOT (Illumina). Sequencing was performed on an Illumina HiSeq2500 platform by Macrogen with one lane 12 pooled libraries to generate 100 bp single-end reads. The base-calling and quality value calculations were performed by the Illumina data processing pipeline CASAVA v1.8.2 and v1.7.0, respectively. Various quality controls including removal of reads containing primer/adaptor sequences, trimming of read length and filtering of high-quality reads based on the score value were performed using Illumina CASAVA v1.7.0.