Computational protocol: Global characterization of the root transcriptome of a wild species of rice, Oryza longistaminata, by deep sequencing

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Protocol publication

[…] Synthesis of cDNA and normalization for pyrosequencing was carried out by MWG (Ebersberg, Germany) using RNA from roots of soil-grown plants without N-fertilizer. High quality polyA+ RNA was isolated from total RNA as template for first- and second-strand synthesis. By using a semirandom priming approach for both strands, an even shotgun-like distribution of cDNA fragments was achieved. The fragments were size-fractionated and normalised by denaturing and re-association. Approximately 10 μg of cDNAs were sheared by nebulisation and sequenced on a 454 GS-FLX pyrosequencing platform. A total of 337,830 raw reads were obtained. SeqClean software was applied to eliminate low quality sequences, poly A/T sequences, adaptor sequences. The cleaned sequences were subjected to the CAP3 program [] for clustering and assembly with default parameters. All the consensus sequences were compared with NR database (GenBank). GO accessions were obtained via assignment of Arabidopsis gene identifiers with the strongest BLASTx alignments to the corresponding O. longistaminata ESTs. Comparison of the distribution of cellular component, biological processes or molecular function obtained using GO annotation was done using the GOSlim program sequences are available at under the accession Xa21_454, and at GenBank (dbEST acc. No. HS317469 - HS388835). [...] To validate the presence of novel ESTs detected by pyrosequencing in O. longistaminata, randomly selected sequences were used for expression analysis by RT-PCR (root RNA) and Southern blot (leaf DNA) analyses. About 100 ng total RNA was use to synthesize the first-strand cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, Carlsbad, CA) with Oligo(dT)12-18 primers. Specific primer pairs for cDNA amplification were designed by Primer3 software [] according to the EST sequences. PCR was performed in a 50 μL reaction volume containing 1 μL cDNA, 1× PCR buffer [10 mM Tris-Hcl (pH 8.0), 1.5 mM MgCl2], 0.2 mM dNTPs, 0.2 μM of each primer, and 1.5 U Taq polymerase (MolTaq). The annealing temperature was 60°C for all primer pairs. After 5 min at 94°C, 35 cycles were carried out with 45 s at 94°C, 45 s at 60°C, 1 min at 72°C for extension and final step of 10 min at 72°C. The PCR products were purified and sequenced by the Sanger method (LGC Genomics, Germany). For Southern blot analysis, 5 μg of genomic DNA was used for restriction endonuclease digestion with HindIII and subjected to Southern blot analysis with digoxygenin-labeled probes according to the protocol described by Neuhaus-Url et al. []. […]

Pipeline specifications

Software tools SeqClean, CAP3, BLASTX, Primer3
Databases GabiPD dbEST
Applications Metagenomic sequencing analysis, qPCR
Organisms Oryza longistaminata, Oryza sativa
Chemicals Nitrogen