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Sample GSM2337172

Query DataSets for GSM2337172

Status

Public on Feb 22, 2018

Title

WZY4WKS2

Sample type

SRA

Source name

petiole

Organism

Vitis vinifera

Characteristics

inoculation: X. fastidiosa wzy
time point: 4 weeks
tissue position: systemic
biorep: 1

Treatment protocol

Vitis vinifera ‘Cabernet Sauvignon’ vines were needle-inoculated with either X. fastidiosa wild type, wzy mutant, or 1X PBS buffer control. Cells of X. fastidiosa strains were harvested from plates of PD3 media in 1X PBS buffer, and suspensions were adjusted to OD600 0.25. Each vine was inoculated twice (with a 20μL drop) on the main stem. Petioles were harvested at each time point and then immediately placed into liquid nitrogen.

Growth protocol

We used wild type X. fastidiosa strain Temecula1 and a wzy mutant strain. X. fastidiosa wild type and wzy mutant strains were grown for 7 days at 28°C on solid PD3 medium without or with kanamycin at 5μg/mL, respectively.

Extracted molecule

polyA RNA

Extraction protocol

Total RNA was extracted from 0.1 g of ground tissue per sample. The frozen tissue was homogenized by inversion in 500 µl of extraction buffer (PureLink Plant RNA Reagent, Ambion cat# 12322-012) for 5 min at room temperature. After homogenization, 100µl of Plant RNA isolation Aid (Ambion cat# AM9690) was added to the suspension and mixed by inversion for 5 min at room temperature. Samples were centrifuged at top speed for 5 min at room temperature to pellet insoluble debris and other contaminants. The supernatant was transferred to a new tube and then 100µl of 5M NaCl and 300µl chloroform were added and mixed for 5 min by inversion. Samples were centrifuged at 12,000 xg for 10 min at 4oC to separate the phases, and the upper aqueous phase was transferred to a new tube. Samples were treated with DNase RNase-free (5 U) and incubated for 20 min at 37oC. After DNase treatment, 500µl of chloroform was added and mixed for 5 min as described above. To separate the phases, the samples were centrifuged again at 12,000 xg for 10 min at 4oC, and the upper aqueous phase was transferred to a new tube. An equal volume of isopropyl alcohol was added, after the samples were mixed by inversion and incubated at room temperature for 10 min. Precipitated RNA was centrifuged at 12,000 xg for 10 min at 4oC, and the supernatant was discarded. The RNA pellets were washed with 1mL of 80 % ethanol and centrifuged at 12,000 xg for 2 min at room temperature. Then the pellets were air-dried at room temperature and re-suspended in 30µl RNase-free water.
Illumina TruSeq RNA Sample Prep Kit v2 protocol

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 3000

Description

xf_raw_counts-late.txt

Data processing

Quality trimming of raw reads was done with Sickle v.1.210 with a threshold of 20 (Q>20).
Quality filtering was done with Scythe v.0.991 with a prior of 0.4
Bowtie2 v.2.1.0 was used to map the processed reads to the reference with the following options: -q --end-to-end --sensitive --no-unal -p20.
Read counts were extracted from the bowtie2 alignments using the script sam2counts.py v.0.91
Genome_build: Vitis vinifera cv. PN40024 (http://www.genoscope.cns.fr/externe/Download/Projets/Projet_ML/data/12X/annotation/)
Supplementary_files_format_and_content: xf_raw_counts-early.txt and : tab delimited text file, raw read counts determined by mapping the sequencing reads
Supplementary_files_format_and_content: xf_raw_counts-late.txt: tab delimited text file, raw read counts determined by mapping the sequencing reads

Submission date

Oct 05, 2016

Last update date

May 15, 2019

Contact name

Abraham Morales-Cruz

E-mail(s)

[email protected]

Organization name

Joint Genome Institute

Street address

1 Cyclotron Road