Bioplatforms Australia Datasets Initiative
Illumina HiSeq 2000 Sample submission form
Researcher Contact Details
Name: Jonathan Powell
Email: jonathan.powell@csiro.au
Institution / Organisation: CSIRO Plant Industry
Address: 306 Carmody Rd, St Lucia
Sample Information
Organism / Species
Sample type
DNA/RNA
Part of organism RNA/RNA extracted from
Extraction method
Comments / special considerations
BPA Project: Ramaciotti Sequencing submission form
Phone: 61733272340
Brachypodium distachyon ecotype Bd21-3 mock inoculated/inoculated with Fusarium pseudograminearum
RNA
~ 1.5 cm sections from base of 33 seedlings per sample
Rneasy Plant Mini Kit
RINs have been obtained for all samples and confirmed >8. Concentrations and spectrophotometric properties have been
estimated using Nanodrop. Further QC has been performed via cDNA synthesis and qPCR assessment of key defence gene
upregulation in inoculated versus mock samples, which has been confirmed. Additional QC including RINs and accurate
quantification of concentrations will be performed by the Ramaciotti Centre upon sample receipt, prior to sequencing.
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Growth protocol of fungus and/or plant (medium, soil, water regimen, light/day, fertilisers etc):
Seedlings were inoculated at 3 days post germination and wrapped in paper towel as per Yang et al. 2010 European Journal of Plant Pathology 128; 495502, then grown for 3 days under constant light in the laboratory. CS3427 fungus was cultured from a V8 agar plug in ¼ V8 media in flasks at room
temperature.
Treatment protocol (i.e. route of administration of pathogen):
For each sample, 3 days post-germination seedlings were placed into Falcon tubes. 3 mls Fusarium pseudograminearum CS3427 spores at a concentration
of 5 x 10^5 (in sterile water), or sterile water for mock samples, was added. Tubes were rotated on a tube roller at medium speed setting for 5 minutes.
Inoculum was drained and seedlings were placed onto a fresh piece of paper towel to remove excess inoculum. For each sample, 11 seedlings were rolled
into each of three paper towel rolls and each roll was placed into an individual Falcon tube and 35 mls sterile water added.
Further Information on experimental design (i.e. time points and biological replicates):
Treatment types: Two treatments, 3 days post inoculation and 3 days post mock inoculation
Biological replication: Tissue from thirty-three individual seedlings pooled for each sample
Sample number: Four samples per treatment type
BPA Project: Ramaciotti Sequencing submission form
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Sample Name
Volume
(ul)
RIN
(RNA)
OD
260/280
OD
260/230
Conc.(ng/ul)
Additional Information.
(method used)
Sample 1
Bd21-3 Mock 1
20
9.2
783 ng/μl
Further QC to be performed by
Ramaciotti Centre upon receipt
Sample 2
Bd21-3 Mock 2
20
9.2
768 ng/μl
Further QC to be performed by
Ramaciotti Centre upon receipt
Sample 3
Bd21-3 Mock 3
20
9.1
693 ng/μl
Further QC to be performed by
Ramaciotti Centre upon receipt
Sample 4
Bd21-3 Mock 4
20
9.1
627 ng/μl
Further QC to be performed by
Ramaciotti Centre upon receipt
Sample 5
Bd21-3 Fp 1
20
9
717 ng/μl
Further QC to be performed by
Ramaciotti Centre upon receipt
Sample 6
Bd21-3 Fp 2
20
8.9
474 ng/μl
Further QC to be performed by
Ramaciotti Centre upon receipt *Low
260/230
Sample 7
Bd21-3 Fp 3
20
8.7
585 ng/μl
Further QC to be performed by
Ramaciotti Centre upon receipt
Sample 8
Bd21-3 Fp 4
20
8.9
648 ng/μl
Further QC to be performed by
Ramaciotti Centre upon receipt
Attach additional sheet if more samples.
BPA Project: Ramaciotti Sequencing submission form
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Sample Requirements
RNA
•
•
•
Samples should be intact and not degraded as assessed by a Bioanalyzer. The RNA Integrity Number (RIN) value should be greater than 8.
OD 260/280 ratio of 2, and a 260/230 of 1.8-2.
5 μg of total RNA at min concentration of 200ng/ul (optimal 500ng/ul). The concentration should be measured using the Ribogreen
fluorescent assay.
Samples should be resuspended in nuclease-free water or elution buffer.
RNA that has been extracted using Trizol or any phenol based method must undergo an additional column purification
•
•
Sample shipment details

Samples to be shipped on dry ice.
Facility
The Ramaciotti Centre
Address
Lowy Cancer Research Centre
C25
via Gate 11 Botany Street
University of New South Wales
Randwick, NSW 2052
Contact person
Tonia Russell
Phone: (02) 93851658
Email: illumina@unsw.edu.au
Please email a copy of the completed form to Anna Fitzgerald (afitzgerald@bioplatforms.com) at the time of sample submission and complete Google Docs metadata form.
BPA Project: Ramaciotti Sequencing submission form
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