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| Status |
Public on Jul 06, 2019 |
| Title |
Hv_mock rep3 |
| Sample type |
SRA |
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| Source name |
Hordeum vulgare roots
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| Organism |
Hordeum vulgare |
| Characteristics |
experiment: tripartite interaction in soil
age: 7 days old seedlings transferred to soil and sampled 6 days later
growth: 4 days old seedlings transferred into split-root chambers with natural soil (RT)
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| Growth protocol |
Natural ’Cologne soil’ (CS) was collected at the Max Planck Institute for Plant Breeding Research (50.958 North / 6.856 East, Cologne, Germany) in March and in September 2010 from experimental agricultural sites that were not exposed to agrochemicals for several years, stored and prepared for use as previously described (Bulgarelli et al. 2012. Nature 488(7409): 91-95. DOI: 10.1038/nature11336). Mixed mycelial confrontation in soil: Transgenic Serendipita vermifera (MAFF305830) expressing GFP and B. sorokiniana ND90Pr were pre-cultured in liquid MYP medium at 28°C, continuously shaking at 120 rpm for 6 and 4 days, respectively. For mixed inoculation of sterile Cologne soil, mycelia were mixed at a 1:1 (w/w) ratio and re-suspended in sterile distilled water to a final concentration of 250 mg mL-1. Sterile Petri dishes were filled with sterile soil and mycelial suspensions were added onto the soil surface. As controls, the same amount of mycelium of each fungus was re-suspended in sterile distilled water and spread over the soil surface. Following 2 days of incubation at 28°C in the dark, fungal mycelia were harvested from the soil surface and snap-frozen in liquid nitrogen for RNA extraction. Plant-fungi tripartite interaction in soil: Mycelial suspensions of GFP-expressing Serendipita vermifera (40 mg mL-1) and conidia of B. sorokiniana (3.4x104 spores/mL) were used to inoculate roots of 7-day-old barley seedlings (Hordeum vulgare cv. Golden Promise) either in combination or alone. For endophyte inoculations, roots were dipped into mycelial suspensions of Serendipita vermifera. Plantlets were placed into split-root chambers where both sides were filled with 250 mL of the 1:1 sterile Cologne soil:sand mix. For inoculations with B. sorokiniana, 30 mL conidia suspensions were distributed over the surface of the soil:sand mix covering the root. For mock controls, distilled water was used as treatment. Six plants were used for each treatment in the split-root experiment. Separate sides of the split-root chambers were considered independent treatments. Roots were harvested at 6 dpi, washed thoroughly and root segments were collected. Four cm root segments from soil-grown plants were removed 5 cm below the root-shoot junction. Plant material from the same treatment was pooled and snap-frozen in liquid nitrogen.
6 days after inoculation; 7 days old seedlings transferred into split-root chambers with natural soil (sterile) and cultivated in a growth chamber with a day/night cycle of 16/8 h at 22/18°C, 60% humidity and a light intensity of 108 µmol m-2 s-1
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| Extracted molecule |
total RNA |
| Extraction protocol |
Hyphae were flash frozen and RNA was harvested using Nucleospin RNA Plant kit (Macherey-Nagel, Germany). Roots were flash frozen and RNA extracted using TRIzol reagent. Illumina TruSeq RNA Sample Prep Kit was used with 1 - 3ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols; Illumina TruSeq RNA Sample Prep Kit was used with the total RNA upto 2µg from the “confrontation in soil” and upto 3µg from the “tripartite interaction in soil” experiments in order to construct the sequencing libraries.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
1A_III
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| Data processing |
Genome_build: Hordeum vulgare: 160404_barley_pseudomolecules_masked; Bipolaris sorokiniana: jgi.doe.gov Cocsa1; Serendipita vermifera: jgi.doe.gov Sebve1
Reference Genomes: Hordeum vulgare updated version of 2016: http://webblast.ipk-gatersleben.de/barley_ibsc/downloads/)
Reference Genomes: Bipolaris sorokiniana isolate ND90Pr (Ohm et al., 2012; Condon et al., 2013) released by JGI (https://www.ncbi.nlm.nih.gov/genome/3236, https://genome.jgi.doe.gov/portal/Cocsa1/Cocsa1.download.html)
Reference Genomes: Serendipita vermifera (MAFF305830) (Lahrmann et al., 2015). (https://www.ncbi.nlm.nih.gov/genome/18204, genome_assembly_id=218407, https://genome.jgi.doe.gov/Sebve1/Sebve1.home.html)
Processing of raw reads: Quality of raw reads was assessed using the FastQC tool v0.11.4. Paired-end and single-end reads were cleaned using the corresponding program in Trimmomatic v0.36 (Bolger et al., 2014) with the following parameters: LEADING:5 TRAILING:5 SLIDINGWINDOW:4:15 MINLEN:50. Bases with a quality lower than 5 were trimmed from the leading and trailing ends. Individual reads were scanned to cut along 4-mer sliding window if the average quality per base was found to be lower than 15. Only reads with a minimum length of 50 bp were selected for further analysis.
Mapping to reference genomes: Processed and filtered reads from root samples were aligned against the genomes of barley, Serendipita vermifera and B. sorokiniana, whereas reads from the fungal confrontation samples were mapped onto the fungal genomes only. Mapping was performed using the spliced aligner TopHat v2.1.0 (Kim et al., 2013) with the following parameters: --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --minimum-anchor 8 --splice-mismatches 2 --minimum-intron-length 50 --maximum-intron-length 500,000 --maximum-insertion-length 12 --maximum-deletion-length 12 --maximum-multi hits 1. Non-spliced reads for which alignments had more than 2 mismatches were discarded. Spliced reads with a minimum 8 bases that aligned on each side were kept only if the spliced alignment in the anchor region did not exceed 2 mismatches. Reads with multiple alignments mapping to different locations/genes/genomic regions were discarded. Only uniquely mapped reads were retained in the aligned BAM files.
Estimation of gene expression: The number of reads mapped per gene was counted using HTSeq-Count in the ‘Intersection Non Empty’ mode (Anders et al., 2015). Given the BAM file of aligned reads and the gene feature format (GFF) file, HTSeq counts how many reads mapped per gene and retrieves locations at which reads were mapped on a reference genome. Resulting read counts were normalized against effect of gene length and differences in total mapped reads (library size) per sample using RPKM (Reads Per Kb per Million reads) to obtain a normalized expression level of each gene per sample.
Differential gene expression analysis: Genes with a minimum of 1 RPKM in all replicates of at least one sample were used for differential expression analysis with the Bioconductor software package edgeR (v3.15.1) (Robinson et al., 2010; McCarthy et al., 2012)
Supplementary_files_format_and_content: tab-delimited text files include raw read count, RPKM values, CPM, REI values per gene per sample (Condition)
Supplementary_files_format_and_content: Bipolaris_sorokiniana_blast2GOannotation_rawrc_rpkm_rei.txt: Bipolaris sorokiniana genes; blast2GO annotations; raw read count (reads mapped to Bipolaris sorokiniana genome) per gene (annotated genes of Bipolaris sorokiniana ); normalised read count - RPKM (Reads Per Kilobase Million); edge R normalised read count CPM (count per million); normalised Relative Expression Index REI (calculated on RPKM)
Supplementary_files_format_and_content: Hordeum_vulgare_blast2GOannotation_rawrc_rpkm_rei.txt: Hordeum vulgare genes; blast2GO annotations; raw read count (reads mapped to Hordeum vulgare genome) per gene (annotated genes of Hordeum vulgare ); normalised read count - RPKM (Reads Per Kilobase Million); edge R normalised read count CPM (count per million); normalised Relative Expression Index REI (calculated on RPKM)
Supplementary_files_format_and_content: Serendipita_vermifera_blast2GOannotation_rawrc_rpkm_rei.txt: Serendipita vermifera genes; blast2GO annotations; raw read count (reads mapped to Serendipita vermifera genome) per gene (annotated genes of Serendipita vermifera ); normalised read count - RPKM (Reads Per Kilobase Million); edge R normalised read count CPM (count per million); normalised Relative Expression Index REI (calculated on RPKM)
Supplementary_files_format_and_content: Serendipita_vermifera_JGI_geneID.txt: Serendipita vermifera transformed gene IDs (used in the study); corresponding JGI gene names; JGI transcript ID; JGI protein ID
Supplementary_files_format_and_content: Bipolaris_sorokiniana_JGI_geneID.txt: Bipolaris sorokiniana transformed gene IDs (used in the study); corresponding JGI gene names; JGI transcript ID; JGI protein ID
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| Submission date |
Apr 30, 2019 |
| Last update date |
Jul 06, 2019 |
| Contact name |
Alga Zuccaro |
| E-mail(s) |
[email protected]
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| Phone |
+492214707170
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| Organization name |
University of Cologne
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| Department |
Botanical Institute
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| Lab |
AG Zuccaro
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| Street address |
Zuelpicher Str. 47b
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| City |
Cologne |
| State/province |
NRW |
| ZIP/Postal code |
50674 |
| Country |
Germany |
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| Platform ID |
GPL22077 |
| Series (1) |
| GSE130517 |
RNA-Seq data used to explore transcriptional dynamics in multipartite fungus-plant interactions: Hordeum vulgare, Serendipita vermifera, Bipolaris sorokiniana |
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| Relations |
| BioSample |
SAMN11539417 |
| SRA |
SRX5771600 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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