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Sample GSM1167255

Query DataSets for GSM1167255

Status

Public on Oct 10, 2013

Title

B2-T1.CATG

Sample type

SRA

Source name

B2-T1.CATG

Organism

Solanum tuberosum

Characteristics

anchor: DpnII
infection: infected
tissue: leaf

Treatment protocol

Three compound leaves per plant (3rd, 4th and 5th from the top) were spray inoculated on the abaxial side each with 200-400µl sporangia suspension (20 000 sporangia/ml, 65 000-130 000 zoospores/ml) using a pump glass flask. The plants were then covered with clear plastic bags to insure high humidity for optimal growth of P. infestans. Inoculations and tissue sampling were performed between 8:00 and 11:30 A.M. Individual leaflets of similar size were collected from the 4th and 5th compound leaf just before inoculation (T0), one (T1) and two (T2) days post inoculation. Leaflets were immediately frozen in liquid nitrogen. The 3rd compound leaf served as infection control. Infection symptoms were observed between the fourth and seventh day post inoculation. For each time point, leaf tissue was harvested from a different plant to avoid wounding and priming effects. The infection experiment was repeated five times with new batches of plants and inoculum (biological replicates). The best three experiments were chosen for further analysis.

Growth protocol

29 genotypes were multiplied by shoot cuttings. Six to seven weeks old plants (three plants per genotype) were transferred to a growth chamber (Snijders Scientific B.V., Tilburg, Netherlands), and acclimated for few days to 16h light at 22°C and 8h dark at 20°C before infection with P. infestans. P. infestans was propagated on detached leaflets of 6-10 weeks old susceptible plants (cvs. Désireé or Grata) or on tuber slices of the susceptible variety Grata. Inoculum was prepared as described (Gyetvai et al. 2012).

Extracted molecule

total RNA

Extraction protocol

For each time point (T0, T1 and T2) one leaflet each of 14, 6 and 9 genotypes in genotypic groups A1, A2 and B2, respectively, were pooled. Frozen pooled leaf tissue was powdered in a CryoMill (Retsch, Haan, Germany). Total RNA was extracted from powdered tissue using the Plant RNA Isolation Kit of Invitrogen (Karlsruhe, Germany) following the manufacturer’s protocol. RNA purification, removal of genomic DNA contamination, concentration measurements and quality tests were performed as described (Draffehn et al. 2010). RNA integrity was additionally assessed by electrophoresis using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA).
SuperSAGE libraries were generated at GenXPro GmbH (Frankfurt, Germany) essentially as described (Matsumura et al. 2010). To prevent amplification biases the TrueQuant technology was applied as described by B. Rotter (Patent application Nr. WO2009152928). Besides NlaIII (recognition site: 5’-CATG-3’), DpnII (recognition site: 5’-GATC-3’) was used as second anchoring enzyme, to capture transcripts without a NlaIII site. Therefore, the 26 bp tags carry either CATG or GATC at their 5’ end. The libraries were pooled and sequenced by Solexa/Illumina technology (Illumina, Inc., USA).

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

B2-T1.CATG

Data processing

Library strategy: SuperSAGE
RTA SCS.2.6, GERALD.1.6
GenXPro SuperSAGE-Tag analysis pipeline „GenXProgram v1.2“
Annotation to S.tuberosum and P. infestans cDNA
Genome_build: STGI.042210 for potato transcripts ( http://compbio.dfci.harvard.edu/tgi/) and /NCBI/ENTREZ/NCBI_P.infestans_mRNA+REFSEQAUG10/NCBI_P.infestans_mRNA+REFSEQ.Fasta, BROAD_Ins/phytophthora_infestans_t30-4_1_genes.fasta, and BROAD_Ins/phytophthora_infestans_t30-4_1_transcripts.fasta
Supplementary_files_format_and_content: Excel files that contain raw data tag counts, the tag counts related/normalized to 1 million, the pairwise comparison of different conditions and information about differentially expressed tags (including p-value and fold change).

Submission date

Jun 18, 2013

Last update date

May 15, 2019

Contact name

Christiane Gebhardt

E-mail(s)

[email protected]

Organization name

Max Planck Institute for Plant Breeding Research

Street address

Carl-von-Linné-Weg 10