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| Status |
Public on Nov 03, 2023 |
| Title |
Soil sample 1 |
| Sample type |
SRA |
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| Source name |
NA
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| Organism |
unidentified soil bacteria |
| Characteristics |
soil type: Cologne CAS soil
cell type: NA
ecotype: NA
treatment: Microbial community inoculated
time: Day 28
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| Treatment protocol |
The FlowPot experiment was set up following the previously described protocol in Duran et al., 2018 and Kremer et al., 2018 with slight modifications. Briefly, the 84 bacterial strains were cultivated from glycerol stocks first on TSB + agar (Tryptic Soy Broth, Sigma) for a week and then transferred to deep well 96-well- plates containing 800 μl of 50% TSB. 96-well-plates were left for six days at 25°C and subsequently pooled (in equal quantities, i.e. 600μl). This bacterial pool was centrifuged at 4,000 x g for 10 min and re-suspended in 10 mM MgCl2. OD600nm was adjusted to 0.05 (107 cells/mL) before inoculation. Fungal strains were cultivated from glycerol stocks on PGA (Potato Glucose Agar, Sigma-Aldrich) for two weeks. Pieces of mycelium were harvested using sterile tips into 1 mL of MgCl2 containing one stainless steel bead (3.2 mm) and subsequently crushed with a paint shaker (SK450, Fast & Fluid Management, Sassenheim, Netherlands) for 20 min and pooled in equal ratios (i.g. 800μl). Microbial mixtures were adjusted to a biomass ratio of 4:1 (eukaryotes:prokaryotes, as assessed by Joergensen and Emmerling, 2006), using 200μL bacterial inoculum and 200μL of fungal inocula in 50 mL 1/2 MS (Murashige + Skoog Medium including Vitamins, Duchefa) supplemented with MES buffer (2-(N-morpholino)ethanesulfonic acid), which were then inoculated into the FlowPot using a 50 mL syringe.
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| Growth protocol |
Before inoculation, A. thaliana Col-0 seeds were sterilized using ethanol and bleach and stratified for four days in the dark at 4°C. Between 4 and 5 hours after microbial inoculation, ten seeds were sown per pot and the closed boxes incubated at 21°C, for 10 hours with light (intensity 4) at 19°C and 14 hours in the dark for four weeks. After two weeks, boxes were opened under a sterile hood and excess seedlings were removed to keep 3 seedlings per FlowPot.
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| Extracted molecule |
total RNA |
| Extraction protocol |
After 4 weeks of FlowPot incubation, roots were harvested from planted pots and peat matrix was sampled from unplanted pots. The roots of all plants from three FlowPots were combined for a total of 9 plants per sample. Roots were thoroughly washed in water, dried on sterilized Whatman glass microfiber filters (GE Healthcare Life Sciences), transferred into eppendorf tubes and frozen in liquid nitrogen and stored at −80°C. Roots and peat matrix samples were homogenized in 2mL eppendorf screw cap tubes using the Precellys 24 tissue lyzer (Bertin Technologies, Montigny-le-Bretonneux, France) after deep freezing twice at 6,200 rpm for 30 s. DNA and RNA were co-extracted from both peat matrix and roots using the RNeasy PowerSoil Total RNA Kit (Qiagen, Hilden, Germany) and its extension for co-extraction of DNA. The RNA precipitation step temperature was adjusted to -20°C for roots samples.
The quality and concentration of RNA samples were quantified using a Bioanalyzer (Agilent, Waldbronn, Germany). Plant, bacterial and fungal ribosomal RNA were depleted from the roots samples using a combination of Illumina Ribo-Zero rRNA Removal Kit (Bacteria), Ribo-Zero rRNA Removal Kit (Yeast) and Ribo-Zero rRNA Removal Kit (Plant Seed/Root)(Illumina, San Diego, USA) following manufacturer’s protocol while only bacterial and fungal ribosomal RNA were depleted the for matrix samples. The quality and quantity were then quantified again using a Bioanalyzer (Agilent, Waldbronn, Germany). Library prep was done using the SMARTer Stranded RNA-Seq Kit (Takara Bio IncSaint-Germain-en-Laye, France) following manufacturer’s protocol. The quality and quantity were then quantified again using a Bioanalyzer (Agilent, Waldbronn, Germany). Illumina sequencing was then performed in-house using the HiSeq sequencer and custom sequencing primers.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
RNAseq fastq files were first processed using Trimmomatic v0.38, in order to filter and trim low-quality sequences out from raw Illumina reads. A pseudo-mapping approach relying on Salmon v0.14.1 (Patro et al., 2017) was then used to associate each read to a predicted gene in reference genomes. To do so, an index containing all CDS sequences from the microbial and host genomes was built. Resulting read counts associated with each microbial gene were then used to perform differential expression analyses, in roots vs soil matrix samples. To perform one differential expression analysis per strain, inter-strain identical genes identified by Salmon were excluded. We could then identify differentially expressed genes in each strain independently, if the number of reads was sufficient. To compare microbial transcriptomes, for each orthogroup defined by OrthoFinder (see subsection Reference genomes), read counts mapped on paralog genes were summed. This allowed us to perform a differential expression analysis at the orthogroup scale, for each microbe independently. Orthogroup differential expression was then compared between strains. Finally, read counts associated to a given orthogroup across all species were also summed, allowing differential expression analyses at the kingdom level. All differential expression analyses were performed with DESeq2 (Love et al. 2014), using shrinkage algorithm apeglm (Zhu et al., 2018) to correct LogFoldChange values.
Assembly: reference.fasta.gz (we used as a reference for our mapping, a simulated metagenome prepared by combining predicted transcript sequences from 107 genomes published in diverse databases (i.e. at-sphere.com, mycocosm.jgi.doe.gov, arabidopsis.org))
Supplementary files format and content: tar archives containing the Salmon mapping outputs
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| Submission date |
May 06, 2023 |
| Last update date |
Nov 03, 2023 |
| Contact name |
Fantin Mesny |
| E-mail(s) |
mesny@mpipz.mpg.de
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| Organization name |
Max Planck Institute for Plant Breeding Research
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| Department |
Plant-Microbes interaction
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| Lab |
Hacquard group
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| Street address |
Carl-von-Linné-Weg 10
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| City |
Cologne |
| ZIP/Postal code |
50829 |
| Country |
Germany |
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| Platform ID |
GPL33384 |
| Series (1) |
| GSE231841 |
Genome-resolved metatranscriptomics reveals conserved root colonization determinants in a synthetic microbiota |
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| Relations |
| BioSample |
SAMN34893822 |
| SRA |
SRX20245152 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7304760_quant_A.tar.gz |
5.8 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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