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Sample GSM2702787

Query DataSets for GSM2702787

Status

Public on Sep 16, 2017

Title

Nodule_dctA-_maeP_1

Sample type

SRA

Source name

Plant nodule tissue infected with bacterial cells.

Organism

Pisum sativum

Characteristics

plant cultivar: Homesteader
agent: Rhizobium leguminosarum bv viciae 3841
plant genotype: Wild type
bacterial strain: Rl379
bacterial genotype: dctA- maeP+
plant growth condition: Nitrogen free

Treatment protocol

Plants were inoculated with either a wild type dctA+ Rhizobium leguminosarum bv. viciae 3841 bacterial strain, or a R. leguminosarum bv. viciae 3841 derivative that was dctA- and expressed the maeP gene of Streptococcus gallolyticus in trans.

Growth protocol

Sterile seeds were germinated on 1% water agar plates for 72 hours at room temperature in the dark. Three seedlings were planted per Leonard assembly (Magenta jars containing a 1:1 [w/w] misture of quartz sand / vermiculite, and 250 mL of Jensen’s medium [per litre: 1 g CaHPO4, 0.2 g K2HPO4, 0.2 g MgSO4x7H2O, 0.2 g NaCl, 0.1 g FeCl2, 1 mg H3BO3, 1 mg ZnSO4x7H2O, 0.5 mg CuSO4x5H2O, 0.5 mg MnCl2x4H2O, 1 mg Na2MoO4x2H2O, 10 mg Na2EDTA, 2 mg NaFeEDTA, 0.4 mg biotin]) and inoculated with ~ 1 x 108 colony forming units of the appropriate R. leguminosarum strain 48 hours later. Plants were grown in a Conviron growth chamber with a day (18 h, 218C) and night (6 h, 178C) cycle. Plants were routinely watered with sterile distilled water, and once with 200 mL of sterile Jensen’s media 21 days post-inoculation with R. leguminosarum. Nodules were collected 25 days post-inoculation with R. leguminosarum.

Extracted molecule

total RNA

Extraction protocol

Nodules were collected from pea plants 25 days post-inoculation with R. leguminosarum. Plants were removed from the vermiculite-sand mixture, placed on ice, and nodules were picked from the roots and immediately flash frozen in liquid nitrogen. Each biological replicate consisted of nodules from three plants from a single Leonard assembly, and enough nodules were collected to fill a 2 mL centrifuge tube to approximately the 500 µL mark. Three biological replicates were prepared per strain, and nodules were stored at -80°C until use. For RNA isolation, all nodules per sample were placed in an autoclaved mortar together with 800 µL of 1% β-mercatoethanol in nuclease-free water (Bioshop) and immediately crushed using an autoclaved pestle. Approximately 500 µL of crushed nodule solution was transferred to a 1.5 mL centrifuge tube on ice, and mixed with 500 µL of room temperature hot phenol solution (per 6 mL: 5 mL of hot phenol buffer [20 mM Tris-HCl pH 7.5, 400 mM NaCl, 40 mM EDTA, 1% SDS, in nuclease-free ddH2O] and 1 mL unbuffered phenol). The mixtures were vigorously vortexed for 20 seconds then immediately incubated in a water bath at 95°C for 1 minute. Samples were centrifuged at 16,000 g for 10 minutes at 4°C, and the supernatant was transferred to a fresh 1.5 mL nuclease-free centrifuge tube. The supernatants were treated with two rounds of unbuffered-phenol:chloroform (1:1) extraction, followed by one round of chloroform extraction. The nucleic acids were then precipitated by mixing the supernatant with one-tenth sample volume of 3 M sodium acetate and 2x sample volume of isopropanol, mixing, and the mixture was incubated on ice for 30 minutes. The nucleic acids were pelleted by centrifugation at 16,000 g for 10 minutes, the pellet washed with ice cold 70% ethanol, and air dried. The pellet was resuspended in 80 µL of nuclease-free ddH2O, mixed with 10 µL DNase I buffer and 10 µL DNase I (20 units; NEB catalog # M0303S), and incubated for 20 minutes at room temperature. The 100 µL sample was then transferred to a fresh 1.5 mL nuclease-free centrifuge tube, extracted once with unbuffered-phenol:chloroform (1:1) and once with chloroform, and the RNA precipitated as before. Each RNA pellet was then resuspended in 100 µL water. A second round of DNase I treatment, extraction, and RNA precipitation was performed as described above. The final RNA pellets were each resuspended in 100 µL of water, the integrity of the RNA confirmed with PCR and MOPS-formaldehyde gels (and later with BioAnalyzer), and the remainder of the samples stored at -80°C until further use.
RNA libraries were prepared for sequencing using standard Illumina protocols by the Farncombe Family Digestive Health Research Institute located at McMaster University (Hamilton, Ontario, Canada). Depletion of the plant and bacterial rRNA was performed using Illumina ribo-zero kits with a 50:50 mixture of plant and bacteria probes.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 1500

Data processing

Quality control: The absence of adapter sequences in the raw reads, the high quality at all nucleotide positions of the reads, and the absence of low quality reads were confirmed with FastQC.
Read mapping: Raw sequencing reads were mapped to a fasta file containing the reference sequences of both the plant and bacterial partners (the file is described in the genome build field) using Bowtie 2 with default settings, a read count summary table was prepared with HTSeq-count using the ‘-a 0’ option.
Differential expression: Differential expression was determined using DeSeq2 with default settings in R, considering the plant and bacterial features together.
Genome_build: The annotated P. sativum cv. Camphor transcriptome (Alves-Carvalho et al. 2015. Plant J. 84:1–19) was downloaded, and the high and low copy files were concatenated into a single fasta file. The nucleotide fasta file of the coding regions of the Genbank assembly (GCA_000009265.1_ASM926v1) of the R. leguminosarum bv. viciae 3841 genome (Young et al. 2006. Genome Biol. 7:R34) was also downloaded. All nucleotide sequences from both organisms were combined into a single contiguous nucleotide fasta file, and a gff file indicating the location of all plant and bacterial features was prepared. In total, 53,171 features were considered: 45,908 P. sativum transcripts and 7,263 R. leguminosarum coding regions.
Supplementary_files_format_and_content: Combined_processed_data_plant.xlsx: Contains gene information and the DESeq2 output for each unigene in the P. sativum transcriptome. Columns include: Gene (transcript unique identifier), MercatorBin (the functional annotation of the gene as determined with Mercator), eggnogAnnotation (the functional annotation of the gene as determined with eggnog-mapper), and several DESeq2 output values (baseMean, log2FoldChange, lfcSE, STAT, pvalue, padj)
Supplementary_files_format_and_content: Combined_processed_data_bacterial.xlsx: Contains gene information and the DESeq2 output for each protein coding gene in the R. leguminosarum bv. viciae genome. Columns include: GeneID (the GeneID of the gene in the gff file), ProteinAccession (the protein accession number), LocusTag (the gene's unique locus tag), GeneName (the name of the gene), Function (the annotated function of the gene), and several DESeq2 output values (baseMean, log2FoldChange, lfcSE, STAT, pvalue, padj)

Submission date

Jul 14, 2017

Last update date

May 15, 2019

Contact name

Turlough M Finan

E-mail(s)

finan@mcmaster.ca

Organization name

McMaster University

Department

Department of Biology