|
| Status |
Public on May 17, 2014 |
| Title |
Cotyledon-stage seed coat |
| Sample type |
SRA |
| |
|
| Source name |
Laser-captured seed coat from soybean cotyledon-stage seed
|
| Organism |
Glycine max |
| Characteristics |
cultivar: Williams 82
developmental stage: cotyledon-stage seed
tissue: seed coat
|
| Growth protocol |
Soybean plants (Williams 82) were grown under standard greenhouse conditions (Le et al., PNAS 2010). Seeds with length between 2.7 mm and 3.0 mm containing cotyledon (COT) stage embryos were harvested.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Harvested seeds were fixed in 3:1 (v/v) ethanol:acetic acid and embedded in paraffin (Kerk et al., Plant Physiol.132. 27-35 (2003)). Seed parts (seed coat, embryonic cotyledons, and embryonic axis) were captured from ten-micron longitudinal sections using a Leica LMD6000. Genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA).
Approximately 100, 250, and 500 nanogram of genomic DNA isolated from embryonic axis, embryonic cotyledons, and seed coat, respectively, were subjected to library preparation following the methods of Hsieh et al. (Hsieh T-F et al. 2009. Science 324:1451-1454) with modifications. We spiked-in ~ three nanogram of unmethylated lambda DNA (Promega) to serve as a control for complete bisulfite conversion. Adapter-ligated genomic DNA was subjected to two rounds of bisulfite (BS) treatment using the EpiTect kit (Qiagen, Valencia, CA). BS-treated DNA was purified using AMpure XP beads (Beckman) and PCR-amplified for 10 cycles using ExTaq (EpiCentre) DNA polymerase. PCR-amplified DNA fragments were size selected using the AMpure XP beads (Beckman). Phi-X174 DNA was spiked in to the library by the sequencing facility before cluster formation and sequencing.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
COT-SC
Whole seed coat was captured from ten micron paraffin sections using Leica LMD6000 system.
|
| Data processing |
Basecalls were performed using RTA version 1.12.4.2.
Original data files from the Illumina sequencing pipeline were in the QSEQ format.
We aligned the raw reads to a pre-processed reference genome using BS Seeker [Chen et al. BMC Bioinformatics (2010)] allowing for two mismatches. The pre-processed reference genome consisted of sequences from the soybean genome (Glyma version 1.0.1) [Schmutz et al. Nature (2010)] obtained from the Phytozome website (http://phytozome.net), soybean chloroplast genome (GenBank: DQ317523), lambda reference genome (GenBank: J02459), and Phi-X174 reference genome (GenBank: J02482).
Reads containing three consecutive methylation in the non-CG sites were removed, possibly representing non-converted cytosines (Cokus et al. Nature 2008). Clonal reads possibly arising during the PCR amplification step were collapsed into one read.
Methylation level of sampled cytosine was calculated as (methylated calls / (methylated calls + unmethylated calls)).
Genome_build: Glyma version 1.01
Supplementary_files_format_and_content: Tab-delimited text file including methylation level of each sampled cytosine. Column definitions are included in the supplementary README.txt file that is linked to the Series record.
|
| |
|
| Submission date |
May 16, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Bob Goldberg |
| E-mail(s) |
bobglab@mcdb.ucla.edu
|
| Phone |
310-825-3270
|
| Organization name |
University of California, Los Angeles
|
| Department |
Molecular, Cell and Developmental Biology
|
| Street address |
610 Charles E Young Drive East
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platform ID |
GPL15008 |
| Series (1) |
| GSE57762 |
Methylation Changes in Soybean Cotyledon Stage Seed Parts |
|
| Relations |
| BioSample |
SAMN02782718 |
| SRA |
SRX545985 |
