GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM941768

Query DataSets for GSM941768

Status

Public on Sep 14, 2012

Title

Pericarp at 25DAP of P1-rr

Sample type

SRA

Source name

Pericarp at 25DAP of P1-rr

Organism

Zea mays

Characteristics

tissue: pericarp
Stage: 25DAP
isogenic line: P1-rr

Growth protocol

The P1-rr stock designated as 65-CFS-305 (Brink and Styles, 1966) was obtained from the National Seed Storage Laboratory, Ft. Collins CO. Near isogenic lines containing P1-rr and P1-ww were generated by six backcross generations of 65-CFS-305 with A619 (P1-ww) followed by 3 selfing generations to confirm homozygousity. The original 65-CFS-305 line has both P1-rr and the P2 gene in the 4 County 63 background while the inbred line A619 has a non-functional P1 locus (P1-ww) and lacks P2 (Szalma et al., 2005). The introgressed region from 65-CFS-305 in A619 was determined to extend on chromosome 1 from 35-66 Mb (AGPv2) by mapping with Illumina Maize SNP50 array (Ganal et al., 2011). The final NIL also contained a second, non-target introgressed region of chromosome 6 89-102 Mb. Pericarps were peeled from 14 and 25 days after pollination (DAP) maize ears avoiding any aleurone tissue

Extracted molecule

total RNA

Extraction protocol

The preparation of mRNA-Seq libraries was performed according to instructions from the Illumina mRNA-Seq Sample Preparation Kit (RS-100-0801 Illumina). Total RNA extracted from P1-rr and P1-ww pericarps harvested at 14 and 25 DAP and silks harvested 1-2 days post emergence were treated with DNaseI (Promega) twice to ensure no genomic DNA contamination. Two μg of total RNA was subjected to two rounds of hybridization to oligo(dT) beads (Illumina). The polyA-enriched mRNA was fragmented by heating at 94 °C for 5 minutes in Fragmentation Buffer supplied by Illumina, followed by ethanol precipitation. The fragmented mRNA was added into 1x First Strand Buffer (2.5mM dNTP, 10mM DTT, RNaseOUT, and random primers mix). After adding Superscript II, first strand cDNA synthesis was performed at 25°C for 10 minutes, 42°C for 50 minutes and 70°C for 15 minutes. The second strand cDNA synthesis was carried out in GEX Second Strand Buffer (25mM dNTP, 1μl RNase H and μl DNA Pol I for 2.5 hours at 16°C), and purified by PCR Purification Kit (Qiagen). After end repair and A-base addition, adapter ligation was performed using custom made adapters or Illumina multiplexing system. For custom-made adapters, reverse oligo-adapters consisting of SLX-R_P1 and SLX-R_P2 and forward oligo adapters consisting of SLX-N_P1 and SLX-N_P2, where underlined nucleotides (see Table S6) corresponds to a barcode sequence, which was either AATCG, AACAC, AACGT, or AACTG in P1-ww at 14 or 25 DAP and P1-rr 14 or 25 DAP, respectively. For using Illumina multiplexing system, the overall procedure was according to the manufacturer’s instructions (MultiplexPE_SamplePrep_1005361_RevB). We used Index 1 (ATCACG) or 2 (CGATGT) for P1-rr or P1-ww silk, respectively. For both procedures, ligated fragments were subjected to electrophoresis for selection of fragments of ~300 bp in length, followed by PCR amplification (20 cycles). PCR products were purified with Agencourt AMPure (Beckman Coulter). Library DNA was checked for concentration and size distribution in an Agilent Bioanalyzer before being subjected to Illuimina GA sequencing using the OSUCCC Nucleic Acid Shared Resource-Illumina GAII Core and the OARDC Molecular and Cellular Imaging Center (The Ohio State University, Ohio).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer II

Data processing

Basecalls performed using CASAVA version 1.4
RNA-seq reads were aligned to the maize genome assembly version 2 using TopHat version 1.2.0 -G option using the gff file for Filtered Gene Set provided by maizegenome.org
Alignment files were compared for differential gene expression using the Cuffdiff option of the Cufflink program (version 1.0.3)
Genome_build: Maize Golden Path B73 RefGen_v2
Supplementary_files_format_and_content: genes.fpkm_tracking and gene_exp.diff are tabulator delimiter files generated with Cuffdiff. genes.fpkm_tracking tracks the summed FPKM of transcripts sharing each gene_id and gene_exp.dif tracks the results of tests difference sin the summed FPKM of transcripts sharing each gene_id.

Submission date

Jun 01, 2012

Last update date

May 15, 2019

Contact name

Erich Grotewold

Organization name

Michigan State University

Lab

Grotewold Laboratory

Street address

603 Wilson Road