|
| Status |
Public on Jun 01, 2015 |
| Title |
A17_root nodule_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
wild type (A17)
|
| Organisms |
Sinorhizobium meliloti; Medicago truncatula |
| Characteristics |
tissue: Root nodule
|
| Treatment protocol |
Root nodules from dnf mutants and wild type cultivars Jemalong and A17 were placed in 30 µl of RLT buffer (RNeasy Mini Kit, Qiagen) and frozen in liquid nitrogen.
|
| Growth protocol |
Medicago truncatula [Gaertn.] cv Jemalong, A-17 and dnf plant seeds were lightly scratched with sandpaper and sterilized in undiluted bleach for 5 min. Seeds were rinsed with sterile water and imbibed for 8 h at room temperature and 48 h at 4°C, germinated overnight in inverted petri dishes in the dark, and planted into pre-sterilized vermiculite in cone-tainer tubes (Stuewe & Sons). Plants were watered every third day with 5 ml 0.1x BNM. Five day old seedlings were inoculated with 5 ml of a suspension (OD600 = 0.1) of CL150 in 0.1x Buffered Nodulation Medium (BNM) (Ehrhardt et al. 1992)(Barnett et al. 2004 Proc Natl Acad Sci USA 101:16636-16641). Cultures of S. meliloti CL150 were prepared for plant inoculation as described previously (Mitra and Long 2004, Plant Physiol.134: 595-604).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The nodules were ground with a microcentrifuge tube pestle as the buffer thawed. The lysate was brought to a volume of 450 µl with RLT buffer and processed through a Qiashredder column (Qiagen) and an RNeasy Mini spin column (Qiagen). The RNA was DNase-treated (DNase I, Thermo Fisher Scientific) in the presence of RNase inhibitor (SuperaseIN, Ambion) (Barnett et al. 2004, PNAS, 101:16636-16641). The DNase-treated RNA was re-purified using RNeasy MinElute columns (RNeasy MinElute Kit, Qiagen).
|
| Label |
biotin
|
| Label protocol |
One microgram template RNA was amplified and biotin-labeled using the MessageAmp Bacteria kit (Ambion). Amplified RNA (20 µg) was fragmented in 40 µl 200 mM Tris-Acetate, pH 8.1, 500 mM potassium acetate, 150 mM magnesium acetate at 94 degree centigrade for 35 min. The reaction was brought to 78.5 µl with water and hybridized to Affymetrix GeneChips as described previously (Barnett et al. 2004, PNAS, 101:16636-16641)
|
| |
|
| Hybridization protocol |
Twenty micrograms of biotinylated aRNA was hybridized to each chip at 48 degrees celsius for 16 hours as described by Barnett et al. 2004. GeneChips were washed and stained in the Affymetrix Fluidics Station
|
| Scan protocol |
Chips were scanned at the Stanford PAN facility using an Affymetrix scanner 3000 7G
|
| Description |
Root nodule harvested from Medicago truncatula A-17 21 days after inoculation with Sinorhizobium meliloti CL150
|
| Data processing |
Affymetrix CEL files were analyzed using R and BIOCONDUCTOR software packages (Gentleman et al. 2004, Genome Biol 5:R80). Probe summarization and normalization were carried out with the AFFY package and the RMA algorithm (Irizarry et al. 2003, Biostatistics 4: 249-264). Since the proportion of plant to bacterial RNA varied between different root nodule zones and between nodules of different plant mutants. To account for varying plant to bacterial RNA ratios in different samples, normalization was done separately for M. truncatula and S. meliloti probe sets.
|
| |
|
| Submission date |
Dec 30, 2013 |
| Last update date |
Sep 09, 2020 |
| Contact name |
Melanie J Barnett |
| Organization name |
Stanford University
|
| Department |
Biology
|
| Lab |
Sharon R. Long
|
| Street address |
371 Jane Stanford Way
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL9757 |
| Series (1) |
| GSE53705 |
Gene expression analysis in developmentally arrested root nodules formed by Sinorhizobium meliloti and Medicago truncatula |
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