Plant Science

Profiling bacterial transcriptome in planta is challenging due to the low abundance of bacterial RNA in infected plant tissues. Here, we describe a protocol to profile transcriptome of a foliar bacterial pathogen, Pseudomonas syringae pv. tomato DC3000, in the leaves of Arabidopsis thaliana at an early stage of infection using RNA sequencing (RNA-Seq). Bacterial cells are first physically isolated from infected leaves, followed by RNA extraction, plant rRNA depletion, cDNA library synthesis, and RNA-Seq. This protocol is likely applicable not only to the A. thaliana–P. syringae pathosystem but also to different plant-bacterial combinations.

Gene expression is dynamically regulated on many levels, including chromatin accessibility and transcription. In order to study these nuclear regulatory events, we describe our method to purify nuclei with Isolation of Nuclei in TAgged Cell Types (INTACT). As nuclear RNA is low in polyadenylated transcripts and conventional pulldown methods would not capture non-polyadenylated pre-mRNA, we also present our method to remove ribosomal RNA from the total nuclear RNA in preparation for nuclear RNA-Seq.

This protocol describes small RNA library preparation from Vigna mungo total RNA followed by deep sequencing and analysis for microRNA identification.​

RNA editing is a widespread post-transcriptional phenomenon through which primary RNA sequences are altered by nucleotide insertion/deletion or base conversion. It occurs in a variety of organisms and cooperates with alternative splicing in increasing both proteomic and transcriptomic complexity. We describe here a method allowing RNA editing events detection by performing direct sequencing of both genomic DNA and cDNA from the same source.

High-throughput sequencing is a powerful tool for exploring small RNA populations in plants. The ever-increasing output from an Illumina Sequencing System allows for multiplexing multiple samples while still obtaining sufficient data for small RNA discovery and characterization. Here we describe a protocol for generating multiplexed small RNA libraries for sequencing up to 12 samples in one lane of an Illumina HiSeq System single-end, 50 base pair run. RNA ligases are used to add the 3’ and 5’ adaptors to purified small RNAs; ligation products that lack a small RNA molecule (adaptor-adaptor products) are intentionally depleted. After cDNA synthesis, a linear PCR step amplifies the DNA fragments. The 3’ PCR primers used here include unique 6-nucleotide sequences to allow for multiplexing up to 12 samples.

Medicago truncatula serves as a model plant for legume genetics and genomics. We used RNA-Seq to characterize the transcriptome during the early organogenesis of the nodule and during its functioning. We generated approximately 135.5 million high-quality 36-bp reads, which were then aligned with the M. truncatula genome sequence (Mt3.0 version) and with sequences from a custom splice-junction database, for the detection of transcribed regions and splicing sites. Mapping and analysis of the reads conducted to the detection of 37,333 expressed transcription units (TUs), 1,670 had never been described before and were functionally annotated. We identified 7,595 new transcribed regions, mostly corresponding to 5’ and 3’ UTR extensions and new exons associated with 5,264 previously annotated genes. We also assessed the complexity in the nodulation transcriptome by performing a Cufflinks analysis to determine the frequency of the various alternatively spliced forms. Thus, we identified 23,164 different transcripts derived from 6,587 genes. Finally, we carried out a differential expression analysis, which provided a comprehensive view of transcriptional reprogramming during nodulation.

All Illumina sequence data have been deposited in the NCBI short-read archive, and Sanger-sequenced PCR products have been deposited in GenBank (SRA048731). Assembled contigs longer than 200 bp have been deposited at TSA (JR366937-JR375780). Coverage data are available at http://ddlab.sci.univr.it/cgi-bin/gbrowse/medicago/.

microRNAs (miRNAs) are ubiquitous regulators of gene expression in eukaryotic organisms, which guide Argonaute proteins (AGO) to cleave target mRNA or inhibit its translation based on sequence complementarity. In plants, miRNA directed cleavage occurs on the target mRNA at about 10 to 11 nucleotide (nt) up stream to the site where the 5’ end of miRNA binds. Sequencing of the miRNA directed cleavage products (degradome) is widely employed as a way to both verify bioinformatic predictions of miRNA mediated regulation and identify novel targets of known miRNAs. Here we describe a protocol for preparation of degradome RNA complementary DNA library for high-through-put sequencing (dRNA-seq) using Illumina GA II sequencing platform, which is currently most popular and cost-effective. Using this protocol we successfully generated three dRNA-seq libraries using three solanaceae plants, including tobacco, tomato and potato. Although this protocol was developed with single-plexed adapter, it should be able to generate multiplexed libraries by replacing the 3’ adapter with multiplexing compatible 3’ adapter and replacing the PCR primer with indexed primers.