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Sample GSM5745891

Query DataSets for GSM5745891

Status

Public on Dec 24, 2021

Title

Roots, 24hpt, Az39, B

Sample type

SRA

Source name

Whole roots

Organism

Arabidopsis thaliana

Characteristics

tissue: Whole roots
treatment: Azospirillum brasilense Az39
time: 24hpt (hours post treatment )

Treatment protocol

Lysogeny broth agar plates were inoculated with WT A. brasilense strain 39 and A. brasilense ipdc. Plates were incubated at 37oC for 48 hours. Isolated colonies were used to inoculate lysogeny broth and incubated for 24 hours in a rotary shaker at 37oC and 250 rpm. The pellets were washed and resuspended in 0.9% sterile saline and adjusted to a final OD595 = 1. Arabidopsis seedlings were treated four days after initial planting with 2 uL (OD595 = 1) Azospirillum suspension or 2 uL of 0.9% saline.

Growth protocol

Arabidopsis thaliana (WT Col.) seed were surface sterilized in ethanol and aseptically transferred onto MS-nutrient agar plates (1.1 g/L MS, 1% sucrose, 0.8% agar). Plates were orientated in a vertical manner and incubated in growth chambers with the following settings: 22oC, 16 hours light (150 µE/m2/s) and 8 hours of dark.

Extracted molecule

total RNA

Extraction protocol

Root systems were collected at 24 hours and 7 days post treatment and flash frozen in liquid nitrogen. Two biological replicates were collected for each time point with each replicate consisting of at least 50 root systems. Root tissue was homogenized with Invitrogen PureLink Plant RNA Reagent (Thermo Fisher Scientific, Waltham, MA, USA) to isolate nucleic acids, and DNA contamination was removed by treating the samples with Ambion TURBO-DNA-free ( Thermo Fisher Scientific, Waltham, MA, USA) per the manufacturer’s protocol. RNA quantity was determined with a Nanodrop spectrophotometer and RNA quality was determined with an RNA nanochip on the Agilent 2100 bioanalyzer system
Reverse transcription, fragmentation, end repair, adapter ligation, and PCR enrichment were conducted with the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, Whitby, ON, Canada) per the manufacturer’s protocols. Library size distribution was determined with a high sensitivity DNA chip on the Agilent 2100 bioanalyzer system. Library concentration and molarity was determined with the NEBNext Library Quant Kit (New England Biolabs, Whitby, ON, Canada) in conjunction with PerfeCTa qPCR ToughMix (QuantaBio, Beverly, MA, USA) and PicoGreen fluorospectrophotometry. Libraries were sent to Genome Québec (Montréal, QC, Canada) for 150 bp single-end sequencing on the Illumnia HiSeq 4000 platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Adaptors and low-quality bases removed with Trimmomtatic v. 0.36
Reads aligned to Arabidopsis thaliana TAIR10 reference genome with HISAT2 v. 2.2.1
Genes quantified with featureCounts
Genome_build: Arabidopsis thaliana TAIR10
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

Submission date

Dec 21, 2021

Last update date

Dec 24, 2021

Contact name

Olivia Wilkins

E-mail(s)

olivia.wilkins@umanitoba.ca

Organization name

University of Manitoba

Department

Biological Sciences