|
Status |
Public on Jan 21, 2015 |
Title |
wt_rep1_ear |
Sample type |
SRA |
|
|
Source name |
1mm ear primordia
|
Organism |
Zea mays |
Characteristics |
tissue: 1mm ear primordia experiment: RNAseq genotype: fea4/+ heterozygous
|
Treatment protocol |
Triplicate pools of more than ten 1mm ears were harvested from homozygous fea4 mutants and heterozygous wild-type sibling plants. Freshly dissected ear tissue was fixed in ice-cold acetone, followed by vacuum infiltration for 20 minutes, and three acetone changes of one hour each.
|
Growth protocol |
fea4-ref was isolated from an M2 screen of EMS-mutagenized A619 inbred maize, and was deposited in the Maize Genetics Co-Op Stock Center as fea179. The mutation was introgressed 4-5 times into various inbred lines for phenotypic analysis in segregating families. fea4-rel*09-5171 and fea4-rel*07-167 were found in a screen for enhancers of ramosa2 in the A619 inbred background. Ear primordia from segregating populations were collected from field grown plants at the CSHL uplands farm facility.
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted from pools of ear tissue using the PicoPure RNA Isolation kit (Life Technologies), according to manufacturer instructions. Messenger RNA was enriched by two successive purifications with oligo-dT coupled dynabeads (Invitrogen). Approximately 50ng of mRNA was used as input for the ScriptSeq v2 RNAseq system (Epicentre). This kit allowed the addition of barcoded adapters (Index #4,5,6,7) to enable multiplexed sequencing in a single lane of an Illumina HiSeq2000. Prior to sequencing, the average size distribution of the libraries was verified on a high sensitivity bioanalyzer chip. The libraries were diluted to 10nM and concentration was verified by qPCR using standards (KAPA Biosystems).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
|
|
Description |
all_genes_expression_values_supp_table.txt
|
Data processing |
Libraries were quantified on an Agilent bioanalyzer (Agilent) using a DNA 1000 chip, and sequenced using the Illumina HiSeq2000 platform at the CSHL Genome Center. All image processing and base calling was done with the Illumina Real Time Analysis software on the HiSeq as the run progressed. Binary basecall files were then transferred to a shared Linux server for further processing and archival. The latest version of the Illumina processing software CASAVA was used to generate fastq files. Tophat (version 2.0.9) was used to align reads to the maize reference genome (AGPv2) based on an a priori set of 110,028 predicted maize gene models (Working Gene Set v5b.60; maizesequence.org). We then quantified read counts per gene and normalized Fragments Per Kilobase exon per Million reads mapped (FPKM) values using Cufflinks (version 2.0.2) and a high-confidence subset of 39,656 maize gene models, the Filtered Gene Set (FGS v5b.60; maizeseqeunce.org). Differential expression analysis was carried out using the R package DESeq (v.1.14.0;(Anders and Huber, 2010). An adjusted p-value of < 0.05 was used to call differentially expressed genes after multiple testing correction. Further details are provided in Supplemental Methods in associated Pautler et al. paper. Genome_build: Zea mays refgen_agpv2 Supplementary_files_format_and_content: Single processed data file for RNAseq analysis includes expression information for all Filtered Gene Set (FGS) genes based on analysis with Cufflinks v.2.0.2 and differential expression analysis with DESeq. Gene is called significant for differential expression if adjusted p-value < 0.05. Normalized read count averages among all samples (baseMean.Avg), wild-type biological replicates (baseMean.wt) and fea4 mutant biological replicates (baseMean.fea4) based on DESeq analysis. These values were used in determine fold changes. Raw read counts for each sample and normalized fragments per kilobase per million reads (FPKM) based on Cufflinks analysis.
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|
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Submission date |
Oct 01, 2014 |
Last update date |
May 15, 2019 |
Contact name |
Andrea L Eveland |
E-mail(s) |
aeveland@danforthcenter.org
|
Organization name |
Donald Danforth Plant Science Center
|
Street address |
975 N. Warson Road
|
City |
St. Louis |
State/province |
MO |
ZIP/Postal code |
63132 |
Country |
USA |
|
|
Platform ID |
GPL15463 |
Series (1) |
GSE61954 |
FASCIATED EAR4 encodes a bZIP transcription factor that controls shoot meristem size in maize |
|
Relations |
BioSample |
SAMN03085616 |
SRA |
SRX718184 |