|
| Status |
Public on Nov 27, 2013 |
| Title |
RA1_input_ear_YFP |
| Sample type |
SRA |
| |
|
| Source name |
ear primordia
|
| Organism |
Zea mays |
| Characteristics |
transgene: RA1-YFP
chip antibody: input
tissue: ear primordia (pool)
developmental stage: 1-5mm
genotype: ra1/ra1 B73
|
| Treatment protocol |
We created two native translational fusion constructs to drive the expression of tagged RA1 proteins in the endogenous expression domain. We fused the YFP and HA-FLAG tags in frame with the RA1 coding sequence at the N-terminus. Constructs were transformed into the HiII genetic background at the Iowa State University Plant Transformation Facility (Ames, IA). T0 generation transformed plants were crossed to the ra1-R mutant. T1 plants were then backcrossed to create a T2 generation segregating 1:1 for the transgene and for ra1.
|
| Growth protocol |
Tassel primordia were harvested ~4 weeks after planting, and immature ears were harvested after 6 weeks. Analysis of plant phenotypes in F1BC2 families segregating for the transgene and ra1 mutants, showed transgenic constructs were capable of complementing the mutant.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP preparation were performed as previously described (Bolduc et al. 2012; Morohashi et al. 2012). Anti-GFP (ab290, Abcam) and anti-HA (H3663, Sigma) antibodies were used for immunoprecipitation of RA1-YFP and RA1-HA, respectively, and protein A agarose/salmon sperm DNA beads (16-157, Millipore) were used to precipitate antibody complexes.
ChIP-seq library preparation were performed as previously described (Bolduc et al. 2012; Morohashi et al. 2012). ChIP DNA was subjected to end repair and A-base addition, followed by ligation with Illumina adapters made by annealing Mltplx_short and Mltplx_long oligos. Library amplification was performed by PCR using Phusion Hot Start II High-Fidelity DNA Polymerase (F549L, Thermo Scientific) with MltplxPCR1.0 and PCR2.0_indx1 to 7, followed by gel-size fractionation to obtain 200-500bp fragments.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Illumina GAII and HiSeq 2000 sequencing with SR50 were performed by the OSUCCC Nucleic Acid Shared Resource (The Ohio State University)
All image processing and base calling was done with the Illumina Real Time Analysis software 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.
ChIP-seq reads were aligned to the maize reference genome (AGPv2) and peak calling was performed with MACS version 1.4.0rc2 using only uniquely mapped reads. Peaks were identified as significantly enriched (p<1e-05) in each of the ChIP-seq libraries compared to input DNA.
Genome_build: Zea mays refgen_agpv2
Supplementary_files_format_and_content: Processed data files are output of MACS.1.4.0. The peaks.bed file includes (from left to right columns) chromosome, peak start, peak stop, -10*log10(pval). The summits.bed file includes (from left to right columns) chromosome, summit start, summit stop, tag pileup at summit.
|
| |
|
| Submission date |
Sep 20, 2013 |
| 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 (2) |
| GSE51048 |
Regulatory Modules Controlling Maize Inflorescence Architecture: ChIP-seq data |
| GSE51050 |
Regulatory Modules Controlling Maize Inflorescence Architecture |
|
| Relations |
| BioSample |
SAMN02360531 |
| SRA |
SRX357140 |
