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| Status |
Public on Nov 29, 2012 |
| Title |
Roots phosphorous deficient rep3 PdR3 |
| Sample type |
SRA |
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| Source name |
Roots -P rep3
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| Organism |
Lupinus albus |
| Characteristics |
tissue type: Proteoid roots
treatment: phosphorous deficient soil
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| Treatment protocol |
White lupin (Lupinus albus L. var. Ultra) plants were grown in a growth chamber at 20/15 °C under a 16 h photoperiod (300 µmol photons m-2 s-1) as described by Gilbert et al. (2000). The Pi-sufficient [1 mM Ca(H2PO4)2] and Pi-deficient [1 mM CaSO4] solutions were provided to white lupin plants with three biological replications per condition. Roots and leaves were harvested at 14 days after emergence (DAE).
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| Growth protocol |
White lupin (Lupinus albus L. var. Ultra) plants were grown in a growth chamber at 20/15 °C under a 16 h photoperiod (300 µmol photons m-2 s-1) as described by Gilbert et al. (2000). The Pi-sufficient [1 mM Ca(H2PO4)2] and Pi-deficient [1 mM CaSO4] solutions were provided to white lupin plants with three biological replications per condition. Roots and leaves were harvested at 14 days after emergence (DAE).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified from 12 samples [(+P & -P) x (roots & leaves) x (three biological replicates)] using RNeasy KIT (Qiagen, Valencia, CA, USA). Contaminating genomic DNA was removed from each RNA sample using DNase I. RNA samples were quantified using Quant-iT⢠RiboGreen® RNA Reagent (www.invitrogen.com) and the RNA integrity was checked with RNA6000 Nano Assay using the Agilent 2100 BioanalyzerTM (Agilent Technologies, Palo Alto, CA). cDNA library preparation and sequencing reactions were conducted in the Biomedical Genomics Center, University of Minnesota. Illumina library prep, clustering and sequencing reagents were used throughout the process following the manufacturerâs recommendations (www.illumina.com). Briefly, mRNAs were purified using poly-T oligo-attached magnetic beads and then fragmented. The first and the second strand cDNAs were synthesized and end repaired. Adaptors were ligated after adenylation at the 3â-ends. After gel purification, cDNA templates were enriched by PCR. cDNA libraries were validated using a High Sensitivity Chip on the Agilent2100 BioanalyzerTM (Agilent Technologies, Palo Alto, CA). The cDNA library was quantified using PicoGreen Assay and by qPCR. The samples were clustered on a flow cell using the cBOT. After clustering, the samples were loaded on the Illumina GA-II machine. The rep1 cDNA libraries were run on one lane per library. For the rep2 and rep3 libraries, barcode was given to each library during the cDNA library construction then two libraries were run together per lane. The samples were sequenced using a single read with 76 cycles. Initial base calling and quality filtering of the Illumina GA-IIx image data were performed using the default parameters of the Illumina GA Pipeline GERALD stage (www.illumina.com). Additional filtering for homopolymers and read size (<76bp) was performed using custom written code.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
The initial base calling and quality filtering of the Illumina GA-IIx image data was performed using the default parameters of the Illumina GA Pipeline GERALD stage (www.illumina.com). Further filtering based on the read size (size>75bp) and homopolymers was performed using custom written code. The Oases program (Sammeth, Foissac, & Guigo 2008) together with the velvet algorithm (Zerbino and Birney, 2008) with k-mer 29 was used for de novo assembly of the Solexa GA-IIx EST reads. Next, 8,441 Sanger ESTs downloaded from the public database (http://www.ncbi.nlm.nih.gov) were combined with de novo assembled Illumina sequences using CAP3 program using the default parameter (Huang X. & Madan A. 1999). Finally, the redundant sequences were collapsed into one using CD-HIT-EST algorithm (Li W. & Godzik A 2006) producing a total of 125,821 Lupin Gene Index (LAGI 1.0) sequences with average length of 1,155 bp. For digital gene expression analysis, the raw digital gene expression counts were measured by quantifying the number of Illumina GA-IIx reads that were mapped to the reference sequences (LAGI1.0) using bowtie program (Langmead et al., 2009) using the default parameter. The raw digital gene expression counts were normalized using the RPKM (reads/Kb/Million) method previously described (Mortazavi et al., 2008; Nagalakshmi et al., 2008). To identify differentially expressed genes, an expression profile matrix was built representing the digital gene expression count for each gene in each library, then imported into the Genedata Expressionist Analyst module (http://www.genedata.com/). A t-test was performed to identify differentially expressed genes in 4 pair-wise comparison (PdR vs. PsR, PdL vs. PsL, PdR vs. PdL, PsR vs. PsL) (p<0.01, â¥2-fold difference).
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| Submission date |
Aug 02, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Sam Yang |
| E-mail(s) |
yangsh38@hotmail.com
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| Phone |
612-626-6582
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| Organization name |
USDA
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| Department |
ARS
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| Lab |
Carroll Vance
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| Street address |
411 Upper Buford Circle
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| City |
St Paul |
| State/province |
MN |
| ZIP/Postal code |
55108 |
| Country |
USA |
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| Platform ID |
GPL14013 |
| Series (1) |
| GSE31132 |
An RNA-Seq Transcriptome Analysis of Orthophosphate-Deficient White Lupin Reveals Novel Insights into Phosphorus Acclimation in Plants |
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| Relations |
| SRA |
SRX087930 |
| BioSample |
SAMN00691236 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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