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Sample GSM5335131

Query DataSets for GSM5335131

Status

Public on May 05, 2022

Title

Vv_ONT_Mock_12h

Sample type

SRA

Source name

Embryogenic callus

Organism

Vitis vinifera

Characteristics

clone: Pinot noir UCD6
tissue: embryogenic callus
time-point: 12_hour
genotype: wild type
treatment: mock

Treatment protocol

For mock treatment, the embryogenic callus cultures were subjected to vigorous shaking in hormone-free C1P liquid medium to break the lumps of callus apart. For the yeast treatment, the embryogenic callus cultures were firstly subjected to mock treatment and then exposed to live H. uvarum cultures. For both mock and yeast treatments, samples were harvested at 1, 3, 6, and 12 hours. A common untreated 0 hour time point for mock and the biotic treatment was taken prior to any treatment.

Growth protocol

Embryogenic callus (EC) cultures were established from Vitis vinifera cv Pinor noir clone UCD5 and maintained in C1P medium which contains half strength MS medium, half strength MS micro elements, 0.1 % (w/v) casein hydrolysate, 3 % (w/v) sucrose, 0.5 % (w/v) gelrite, 250 μg myo-inositol, 5 μg nicotinic acid, 5 μg pyridoxine-HCl, 5 μg thiamine-HCl, 5 μg Ca-pantothenoate, 50 μg D-biotin, 1 μM BAP and 5 μM 2,4-D.

Extracted molecule

polyA RNA

Extraction protocol

For short-read RNA-seq, total RNA of harvested embryogenic callus were isolated according to the manufacturer’s instructions of Spectrum Plant Total RNA Kit (Sigma). Each purified RNA sample was treated with DNase I following the TURBO DNA-free kit protocol (Ambion) to remove contaminating genomic DNA. For long-read sequencing, Total RNA was extracted and separated using NORGEN Plant microRNA purification kit (Norgen Biotek) according to the manufacturer’s instruction. Genomic DNA contamination was removed with the standard protocol of the TURBO DNA-free kit (Thermo Fisher).
For short-read RNA-seq, RNA libraries were prepared using standard Illumina protocols. For long-read cDNA-seq, the libraries were prepared following the protocol of Oxford Nanopore cDNA-PCR kit (SQK-PCS109).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

MinION

Description

MinION
Vv_ONT_FlairQuantify_gene_tpm_ECstress12h.txt
Vv_ONT_bedtools_TE_counts_ECstress12h.txt

Data processing

For short-read sequencing:
Illumina Casava v1.8.2 used for basecalling.
Sequenced reads were trimmed for adaptor sequence following removal of low-quality sequence and tRNA and rRNA reads, then mapped to V. vinifera 12X PN40024 reference genome using HISAT2 v2.1.0 with parameters --rna-strandness RF --dta -k 100.
Raw read counts for genes were calculated using htseq-count v0.10.0 with parameters -f bam -t exon -i transcript_id -s reverse -m intersection-nonempty.
Gene FPKM was calculated by dividing gene's raw read counts per kb gene transcript length with per million mapped reads.
For TEFingerprint analysis, sequenced reads were trimmed for adaptor sequence following removal of low-quality sequence and tRNA and rRNA reads, then mapped to TE sequences extraced from all annotated TE loci using bwa mem (with default setting).
Read counts for TE loci were calculated by htseq-count v0.10.0 (-f bam -t exon -i transcript_id -s reverse -m intersection-nonempty), bedtools v2.27.0 (bedtools coverage), and TEFingerprint v0.1.4 (tef preprocess --exclude-tails and then tef compare -m 11 -e 600 -q 30 -t 8).
Average read depth of each TE locus was calculated by dividing the sum of read bases overlapping with a TE locus (obtained from bedtools intersect v2.27.0) with the read-covered bases of the TE locus (obtained from bedtools coverage v2.27.0).
Read counts for TE families were calculated by TEtranscripts with parameters --mode multi --stranded reverse.
TE family FPKM was calculated by dividing TE familiy's read counts per kb TE length with per million mapped reads.
For long-read sequencing:
Oxford Nanopore Technology Guppy v3.1.5 used for basecalling.
The resulting fastq files were processed by Pychopper2 (v2.0.1) to capture full-length reads and remove adapter sequences.
The full-length reads were mapped to the 12X PN40024 V. vinifera reference genome using minimap2 v2.17 with the option -ax splice for long-read splice alignment. For TE loci analysis, the minimap2 parameters -N 100 -ax splice were used instead. For TE family analysis, reads were mapped to V. vinifera canonical TE sequences (Repbase) with the option -ax splice.
Gene TPM was calculated using FLAIR pipeline (v1.4).
Read counts for TE loci and TE families were calculated by bedtools v2.27.0 (bedtools coverage).
TE family TPM was calculated by dividing read counts of TE families with per million mapped reads.
Genome_build: 12X PN40024
Supplementary_files_format_and_content: Tab-delimited table with raw counts for every gene and every Illumina RNA-seq library.
Supplementary_files_format_and_content: Tab-delimited table with FPKM values for every genes and every Illumina RNA-seq library.
Supplementary_files_format_and_content: Tab-delimited table with raw count values (htseq-count) for every TE loci and every Illumina RNA-seq library.
Supplementary_files_format_and_content: Tab-delimited table with raw count values (bedtools) for every TE loci and every Illumina RNA-seq library.
Supplementary_files_format_and_content: Tab-delimited table with TE's average read depth of mapped region for every TE loci and every Illumina RNA-seq library.
Supplementary_files_format_and_content: Tab-delimited table for raw count values (TEFingerprint) for every TE loci and every Illumina RNA-seq library.
Supplementary_files_format_and_content: Tab-delimited text files include gene TPM values for each ONT cDNA-seq library.
Supplementary_files_format_and_content: Tab-delimited text files include TE loci read count values (bedtools) for each ONT cDNA-seq library.

Submission date

May 24, 2021

Last update date

May 05, 2022

Contact name

Christopher Winefield

E-mail(s)

christopher.winefield@lincoln.ac.nz

Organization name

Lincoln University

Street address

Ellesmere Junction Road/Springs Road