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Sample GSM4978008

Query DataSets for GSM4978008

Status

Public on May 05, 2021

Title

StAgo1-GFP 88069 3

Sample type

SRA

Source name

Infected leaves

Organisms

Solanum tuberosum; Phytophthora infestans

Characteristics

potato cultivar: StAgo1a-GFP (Sarpo Mira)
p. infestans strain: NL-88069
dpi: 5
tissue: Infected leaves
ip antibody: GFP (Chromotek, gtm-100)

Treatment protocol

Sporangia were harvested from 14 d old agar cultures by flooding cultures with water, and rubbing the culture with a glass rod. Sporangial suspensions were decanted, counted on a haemocytometer, and adjusted to 5 × 10^4 sporangia ml^−1. Attached leaflets were inoculated with a 10 μl droplet (500 sporangia) on either side of the leaf midrib, 4 weeks post transfer to soil from in-vitro conditions.

Growth protocol

Potato plants were grown in-vitro at 22°C with a 16-h photoperiod on Murashige-Skoog (MS) medium (Duchefa Biochemie B.V., Amsterdam, Netherlands), supplemented with 2% (w/v) sucrose and 0.3% (w/v) gelrite (Duchefa). After 3 weeks plants were transferred to pots with soil in a growth chamber with the same light and temperature regime. P. infestans were maintained at 20 °C on rye agar medium supplemented with 2 % sucrose (Caten & Jinks 1968), rifampicin (10 μg ml−1), and pimaricin (10 μg ml−1). Transgenic lines of P. infestans isolate 88069 were maintained as above, but with the addition of the antibiotic geneticin (G418; 10 μg ml−1) to the medium.

Extracted molecule

total RNA

Extraction protocol

Inoculated leaves were harvested and flash frozen in liquid nitrogen. StAGO1a-GFP co-IP were performed with anti-GFP antibody (Chromotek, gtm-100).
Barcoded sequencing libraries were constructed using the Ion Total RNA-Seq Kit v2 and Ion Xpress barcode primers. Sequencing was performed on the Ion Proton platform using the Ion PI™ Hi-Q™ Sequencing 200 Kit (Thermo Fisher Scientific).

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Ion Torrent Proton

Data processing

smallRNA processing: The function SamToFastq, included in the Picard tool package, was used to convert the original BAM format to FASTQ format (for the raw data which was not already in FASTQ format), on which a quality control was performed, using FastQC
smallRNA processing: Cutadapt, quality trimming (20), read length filtration 18 - 38 nt
smallRNA processing: Bowtie2 in combination with SAMtools view were used for tRNA and rRNA trimming and potato and P. infestans sRNA filtering to exclude the reads from the other genome for the different datasets, using 0 mismatches allowed per seed against each Fasta filter
smallRNA processing: SamToFastq converted the files to fastq
smallRNA processing: Seqtk was applied to convert the FASTQ files to FASTA.
Degradome processing: Raw RNA reads from degradome libraries were adaptor- and quality trimmed with Cutadapt
Degradome processing: Seqtk was applied to convert the FASTQ files to FASTA.
The sRNA and degradome fasta files and were analyzed in PAREsnip2.
PAREsnip output data was imported in R where PAREsnip2 replicates were merged and duplicated targets were discarded.
Resulting datasets were analyzed in our machine learning script (https://github.com/kristianHoden/degradome). False positives were discarded
The datsets were sorted in list according to their Normalized Fragment Abundance (NFA) and targeting transcriptome
Solanom tuberosum genome v4.04, Phytophthora infestans genome (ASM14294v1)
fasta format of small RNA and degradome libraries
Genome_build: Solanom tuberosum genome v4.04, Phytophthora infestans genome (ASM14294v1)
Supplementary_files_format_and_content: pinfDown - contain the datasets with targets in P. infestans with decreased NFA of the targets upon infection
Supplementary_files_format_and_content: pinfUp - - contain the datasets with targets in P. infestans with increased NFA of the targets upon infection
Supplementary_files_format_and_content: potDown - contain the datasets with targets in potato with decreased NFA of the targets upon infection
Supplementary_files_format_and_content: potUp - - contain the datasets with targets in potato with increased NFA of the targets upon infection
Supplementary_files_format_and_content: rDown - contain the datasets with targets in resistance (R) genes in potato with decreased NFA of the targets upon infection
Supplementary_files_format_and_content: rUp - contain the datasets with targets in resistance (R) genes in potato with increased NFA of the targets upon infection

Submission date

Dec 16, 2020

Last update date

May 05, 2021

Contact name

Kristian Erik Persson Hodén

E-mail(s)

kristian.hoden@gmail.com

Organization name

Swedish University of Agricultural Sciences

Department

Plant Biology