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Sample GSM876147

Query DataSets for GSM876147

Status

Public on Jan 10, 2013

Title

Meloidogyne_infected_rice_root_tip-time_point_1-replicate_3

Sample type

SRA

Source name

Rice root tip

Organism

Oryza sativa Japonica Group

Characteristics

tissue: root tip
strain: cv. Nipponbare (GSOR-100)
growth conditions: hydroponic culture for 2 days
origin: Philippines
infection: Meloidogyne graminicola
time point: 3 days after inoculation

Treatment protocol

The root (tips) were excised with a scalpel from the remaining root system. In the case of root knot nematode infected plants, where galls were taken as infected tissue, the root tips of uninfected plants were sampled as control tissue (GSE30367). For migratory nematode infection, the whole root tissue, without root tips, was sampled in infected and uninfected plants. For each treatment, two or three biological replicates, with each replicate containing a pool of 6 different plants.

Growth protocol

Oryza sativa cv. ‘Nipponbare’ (GSOR-100, USDA) was germinated on wet filter paper for 6 days at 30°C and then transferred to SAP-substrate (Reversat et al., 1999) and further grown at 26°C under a 16h/8h light-regime. The nematodes , originally isolated in the Philippines (M. graminicola) and Myanmar (H. oryzae), were cultured and extracted as described in Kyndt et al. 2012. Five days after transplanting in SAP the plants were inoculated with 250 nematodes per plant for M. graminicola and 400 nematodes per plant for H. oryzae. Control plants were mock-inoculated. One day after inoculation the plants were transferred to a hydroponic culturing system with Hoagland solution (Reversat et al., 1999) to synchronize the infection process. Infected and control tissue was collected at 3 and 7 days after inoculation.

Extracted molecule

polyA RNA

Extraction protocol

Approximately two microgram of total RNA from infected and non-infected roots and root tips were used for mRNA-Seq library construction according to the manufacturer’s recommendations (Illumina). The mRNA was isolated from the total RNA using oligo dT-attached magnetic beads and subsequently fragmented using divalent cations under elevated temperature. First-strand cDNA was generated with reverse transcriptase and random primers. After second-strand cDNA synthesis and adaptor ligation, cDNA fragments of approximately 200 bp were isolated by gel electrophoresis. cDNA fragments were amplified by 18 cycles of PCR using the multiplexing primers from the ‘Multiplexing sample preparation kit’. The denatured library was diluted to a final concentration of 6 pM and loaded on a single read flow cell. Multiplexing was applied to minimize lane effects. After cluster generation, the multiplexed library was sequenced on an Illumina Genome Analyzer II (76 cycles).

Library strategy

RNA-Seq

Library source

non-genomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer II

Description

Time point 1 = 3 days after inoculation

Data processing

Reads were mapped to the Oryza sativa subsp. Japonica reference genome (build MSU6.0) in two phases using TopHat (Trapnell et al., 2009, version 1.3.1) and Cufflinks (Trapnell et al., 2010, version 1.0.3). This software requires Samtools (Li et al., 2009, version 0.1.12a) which was downloaded from “http://samtools.sourceforge.net/”. The Bowtie index was constructed with the ‘bowtie-build’ command (Langmead et al., 2009, version 0.12.7) from the chromosomal fasta files at “ftp://ftp.ensemblgenomes.org/pub/release-10/plants/fasta/oryza_sativa/dna/”. In the first phase the reads from all samples were mapped together using TopHat, maximising sensitivity, to create the input file for Cufflinks. The Ensembl reference annotation in GTF format (“ftp://ftp.ensemblgenomes.org/pub/release-10/plants/gtf/oryza_sativa/”) was used to provide established junctions via the ‘–G’ option. Further, standard parameters were used with exception of following options: --bowtie-n, --initial-read-mismatches 1, -g 10, -a 10, -m 1, -i 30, --solexa1.3-quals. The next step consisted of applying Cufflinks to construct a minimal set of transcripts using reference annotation based transcript (RABT) assembly (Roberts et al., 2011), again based on the Ensembl MSU6 GTF annotation file. Standard settings were used with the exception of following options: -u, -I 500000, --min-intron-length 30. In the second phase all samples were remapped separately using the same TopHat settings with the following two exceptions: the ‘transcripts.gtf’ output from Cufflinks was used to supply junctions and ‘--no-novel-juncs’ was added to prevent TopHat from introducing new sample specific junctions that were not detected in the first phase. All mapped reads were summed per MSU6 gene or nTAR. Processed data file does not include counts for probable nematode transcripts (see Kyndt et al. 2012).

Submission date

Feb 15, 2012

Last update date

May 15, 2019

Contact name

Simon Lieven Imanuel Johannes Denil

Organization name

VUB/ULB

Department

University Hospital Brussels

Lab

BRIGHTcore