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Sample GSM1005508

Query DataSets for GSM1005508

Status

Public on Dec 03, 2012

Title

H-P2

Sample type

SRA

Source name

H_root_P2

Organism

Zea mays

Characteristics

line: Huangzao4
disease-resistance: susceptible
tissue: radicle roots
inoculated with: teliospores of Sporisorium reilianum f. sp. Zeae
infection status: post-inoculation stage2 (P2)
develpmental stage: 6 days post inoculation after seed germination

Treatment protocol

Mock-inoculated roots with water were set as the Control group (CK), After inoculation, there were two different phenotypes based on the PCR results: set negative PCR result as Post-inoculation stage 1 (P1) and positive PCR result as post-inoculation stage 2 (P2), which were considered as two different infection statuses in the root after inoculation

Growth protocol

the seedlings were incubated in a culture chamber for further infection by S. reilianum at 23°C: 20°C (light : dark, 14:10 h) and 20% relative humidity

Extracted molecule

total RNA

Extraction protocol

Radicle roots were selected based on PCR results, and corresponding RNAs were harvested using Trizol reagent.
Digital gene expression (DGE): 500 to 2,000 ng of DNaseI-treated total RNA was used in library construction:double-stranded cDNAs were synthesized using oligo(dT) beads (Invitrogen). The cDNAs were then digested with an anchoring restriction enzyme (NlaIII or DpnII) and ligated to an Illumina-specific adapter, Adapter A, containing a recognition site or the type IIS restriction enzyme MmeI (New England Biolabs). Following MmeI digestion and dephosphorylation with shrimp alkaline phosphatase (USB Corp.), cDNAs were purified and a second Illumina adapter, Adapter B, containing a 2-bp degenerate 3# overhang, was ligated. Tags flanked by both adapters were enriched by PCR using Phusion DNA polymerase (Finnzymes) and Gex PCR primers 1 and 2 (Illumina) following the manufacturer’s instructions.PCR products were visualized on a 12% PAGE gel. The 85-bp DNA band was excised and purified using a Spin-X filter column (Costar). DNA quantification and quality analysis was performed using an Agilent DNA 1000 series II assay, and the DNA sample was diluted to 10 nM. Cluster generation and sequencing were run on the Illumina cluster station and Genome Analyzer (Illumina). Raw sequences were extracted from the resulting image files using the open source Firecrest and Bustard applications (Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer

Description

Sample 3

Data processing

We used Vmatch (www.vmatch.de) large-scale sequence analysis software to map the collapsed reads to the maize reference genome.the unique DpnII and NlaIII tags were queried for complete matches to all possible 20- and 21-mer sequences, respectively, using the Vmatch algorithm.
Tags that mapped uniquely up to three places in the maize genome were used to extract gene information using the Ensembl Application Perl Interface (http://uswest.ensembl.org/info/docs/api).
A working gene set was used (gene build 4a.53; maizesequence.org), and tags that mapped uniquely up to three places in the maize genome were selected to extract gene information using the Ensembl Application Perl Interface (http://uswest.ensembl.org/info/docs/api). In order to maximize the capture of complete UTRs, each gene model and the gene space was then computationally extended by 300 nucleotides at both 5' - and 3'-ends
In this study, genes that were differentially expressed between CK and P1, CK and P2, P1 and P2 stage were identified using a free open-source “DEseq” package based on R software, which allowed analysis of experiments with no biological replicates (http://www-huber.embl.de/users/anders/DESeq/)
Genome_build: B73 RefGen_v1
Supplementary_files_format_and_content: tab-delimited text files include P-values and log2 fold changse for each Sample

Submission date

Sep 18, 2012

Last update date

May 15, 2019

Contact name

shaopeng zhang

Organization name

Huangzhong agricultural university

Street address

No.1,Shizishan Street · Hongshan District

City

wuhan