from each treat ment group, 10 plants were collected and mixed, to minimize the effect of transcriptome unevenness http://www.selleckchem.com/products/chir-99021-ct99021-hcl.html among plants. mRNA sequencing Total RNA was extracted by using an RNeasy Plant kit. RNA quality was calculated with a Bioanalyzer 2100 algorithm, high quality RNA was used. Total RNA samples were subjected to cDNA con struction for Illumina sequencing, in accordance with the protocol for the mRNA Seq sample preparation kit. Oligo magnetic beads were used to isolate poly RNA from the total RNA samples. The mRNA was fragmented by heating at 94 C for 5 min. First strand cDNA was synthesized using random hexamer primers for 10 min at 25 C, 50 min at 42 C, and 15 min at 70 C. After the first strand had been synthesized, dNTPs, RNaseH, and DNA polymerase I were added to synthesize second strand DNA for 2.

5 h at 16 C. The ends of double stranded cDNA were repaired by using T4 DNA polymerase and Klenow DNA polymerase and phosphorylated by using T4 polynucleotide kinase. A single A base was added to the cDNA molecules by using Klenow exo nuclease, and the fragments were ligated to the PE adapters. cDNAs with 200 25 bp fragments were collected. The purified cDNA was amplified by 15 cycles of PCR for 10 s at 98 C, 30 s at 65 C, and 30 s at 72 C using PE1. 0 and PE2. 0 primers. Mapping of short reads, detection of bridging sequences, and prediction of transcripts For each sample, cDNA was sequenced by an Illumina Genome Analyzer II. Data on nine technical replicates were accumulated for Fig ure 1. Data on four technical replicates were summed for Table 1.

In our preliminary experiment, two independent sequen cing runs using the same cDNA were highly correlated. The default Illumina pipeline quality filter, which uses a threshold of CHASTITY 0. 6, was used to identify clusters with low signal to noise ratios. CHASTITY is defined as the ratio of the highest of the four intensities to the sum of the highest Anacetrapib two. Passed filter reads were mapped onto both the Nipponbare reference genome and the spliced exon junction sequences by SOAP ver. 1. 11, allowing up to 2 bp of mismatch or up to 3 bp of indels. SEJ sequences were constructed by conca tenating the 40 bases at the 3 end of the upstream exon to the 40 bases at the 5 end of the downstream exon for all RAP2 transcripts at a locus. To calculate the cumulative coverage of the genome or annotated regions, reads were mapped by BWA with the default option.

To predict transcripts, a series of Tipifarnib cancer programs Bowtie, TopHat, and Cufflinks was used. Briefly, mRNA Seq reads were mapped against the whole reference genome by using Bowtie software. An initial consensus of exon sequences was extracted from the mapped reads. The reads that did not align to the genome but that were mapped to these potential junctions by TopHat were considered to bridge splice junctions. Cufflinks constructs gene models on the basis of the exons and brid ging sequences predicted by Bowtie and TopHat. ORFs were predicted by BLASTX search