Bioplatforms Australia Datasets Initiative
IlluminaHiSeq 2000 Sample submission form
ResearcherContact Details
Email:
Phone:
diana.garnica@anu.edu.au
02 61256963
Address:
Room 3.077, building 134, RSBS, ANU campus, Acton 0200
Name: John Rathjen-Diana Garnica
Institution / Organisation:
Australian National University
Sample Information
Organism / Species
Sample type
DNA/RNA
Part of organism RNA/RNA extracted from
Extraction method
Comments / special considerations
Puccinia spp.
RNA
Purified haustoria
Trizol, DNAse I treatment and clean up protocol from RNeasy Plant Mini Kit (Qiagen)
RNA quality was verified by Nanodrop and Bioanalyser RNA pico 600 ship. Further QC was performed via cDNA synthesis and RTPCR assessment of predicted effector genes.
Growth protocol of fungus and/or plant (medium, soil, water regimen, light/day, fertilisers etc):
Seedlings of the Pst-susceptible wheat cultivar Morocco were grown in the greenhouse under 70% relative humidity at 21°C and 16:8 light:dark cycle.
Seven-day old seedlings were inoculated with fresh urediniospores of P. striiformisf.sp. tritici strain 104E137A-, and incubated for 48h in 100% humidity
at 9°C in the dark. Subsequently plants were transferred to a growth chamber at 17°C with a 16:8 light cycle. Infected tissue of was collected 9 days after
infection (just prior to sporulation) and immediately processed for haustoria isolation. If the infection was for spore collection, infected plants were left
for ~ 19 days till spores were produced.
BPA Project: Ramaciotti Sequencing submission form
1
Treatment protocol (i.e. route of administration of pathogen):
Explained in previous section.
Further Information on experimental design (i.e. timepoints and biological replicates):
Haustoria were purified by a Percoll gradient method. Twenty g of infected wheat leaves were treated as above, but after passing through the 20 µm
mesh, thefiltratewas centrifuged at 1080 g for 15 min at 4°C and the resulting pellet was resuspended in 20 ml of 1x isolation buffer [0.2 M sucrose, 20mM
MOPS pH 7.2] containing 30% Percoll. The suspension was divided into four tubes and centrifuged at 25,000 g for 30 min at 4°C. The first 10 ml of each
tube was recovered, diluted 10 times with 1x isolation buffer and centrifuged at 1080 g for 15 min at 4°C. The pellets were resuspended in 20 ml of 1x
isolation buffer containing 25% Percoll and centrifuged at 25,000 g for 30 min at 4°C. The first 10 ml of each tube was recovered, diluted 10 times with 1x
isolation buffer and centrifuged at 1080 g for 15 min at 4°C. The final pellet was frozen in liquid nitrogen and stored at -80°C prior to RNA isolation. Three
biological replicates were sent for sequencing.
Germinated urediniospores were obtained by germination of about 80 mg of fresh spores harvested from infected leaves 17 daion sterile distilled waterat
9°C for 15 h in the dark.Germinated urediniospores were collected by filtrationwith an 11 μm nylon mesh, and frozen in liquid nitrogen and stored at -80
prior to extraction of total RNA.Three biological replicates were sent for sequencing
BPA Project: Ramaciotti Sequencing submission form
2
Sample Name
Volume
(ul)
RIN
(RNA)
OD
260/280
OD
260/230
Conc.(ng/ul)
Additional Information.
(method used)
Sample 1
Pst 104E137A- sample 1
30
>200
Sample 2
Pst 104E137A- sample 2
30
>200
Sample 3
Pst 104E137A- sample 3
30
>200
Sent 4-30-12
Sample 4
Spores1
50
>200
Sent 4-30-12
Sample 5
Spores2
50
>200
Sent 4-30-12
Sample 6
Spores3
50
>200
Sent 4-30-12
Sample 6
Sample 7
Sample 8
Sample 9
Sample 10
Sample 11
Sample 12
Attach additional sheet if more samples
BPA Project: Ramaciotti Sequencing submission form
3
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
4