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Sample GSM399598 Query DataSets for GSM399598
Status Public on Jun 16, 2009
Title WT_endosperm_BS_seq
Sample type SRA
Source name Wild-type endosperm
Organism Arabidopsis thaliana
Characteristics ecotype: Columbia-0
genotype: wild-type
tissue: endosperm
Growth protocol Standard long day growth conditions
Extracted molecule genomic DNA
Extraction protocol Seeds at the mid-torpedo stage to early-maturation stage (7-9 days after pollination, ecotype Col-0) were dissected in 0.3 M sorbitol, 5 mM MES (pH 5.7) on a slide under a dissecting microscope. We synthesized custom Illumina adapters in which cytosines were replaced by 5-methylcytosines, so that the adapters would survive bisulfite conversion. We chose to synthesize paired ends (PE) adapters, which allow each molecule to be sequenced from both ends, thus facilitating subsequent alignment to the genomic scaffold. We isolated 0.5-1 micrograms of genomic DNA from endosperm dissected from wild type and dme seeds, as well as control wild type embryos and aerial tissues. Specifically,plant tissues were ground in CTAB (cetyltrimethylammonium bromide) and genomic DNA was isolated as described by Murray and Thompson (Nucleic Acids Research. 8: 4321-4325, 1980, PMID: 7433111). DNA was sheared by sonication to fragments of 100-500 bp, and the adapters were ligated following the Illumina protocol. The library was then treated twice with sodium bisulfite (which converts unmethylated Cs to Us) using the Qiagen EpiTect kit, and amplified by 18 cycles of PCR using PfuTurboCx DNA polymerase (Stratagene), a proofreading enzyme that tolerates uracil in the template strand. Bands around 300 bp were gel-purified and cloned for validation.
Library strategy Bisulfite-Seq
Library source genomic
Library selection RANDOM
Instrument model Illumina Genome Analyzer II
Description DNA methylation analysis of Arabidopsis tissue by bisulfite sequencing
library_strategy: Other (BS-seq)
library_source: genomic
library_selection: random
Data processing Processed data files (.gff): alignment, single_C_fractional_methylation, and 50bp_window_fractional_methylation files, are linked below as supplementary files.
Alignment: We used Perl scripts to convert all the Cs in the ‘forward’ reads (and in the scaffold) to Ts, and all the Gs in the ‘reverse’ reads and scaffold to As, and initially aligned the converted reads to the converted TAIR8 scaffold using SeqMap as individual reads, allowing up to two mismatches per read. We subsequently used a Perl script to insure that paired reads mapped to opposite strands within 300 bp of each other.
Single_C: We used Perl scripts to recover the original sequence of each mapped read and, for each C (on either strand), count the number of times it was sequenced as a C or a T.
50bp_window: We used a Perl script to calculate fractional methylation (#C/(#C+#T)) within a 50 bp sliding window for each sequence context (CG, CHG, CHH).
Each processed data file has 9 tab-delimited columns (described in the README.txt file attached to the Series record).
Submission date May 01, 2009
Last update date May 15, 2019
Contact name Toshiro Nishimura
Phone 5106429550
Organization name University of California at Berkeley
Department Plant and Microbial Biology
Lab Daniel Zilberman
Street address 211 Koshland Hall
City Berkeley
State/province CA
ZIP/Postal code 94720
Country USA
Platform ID GPL9302
Series (1)
GSE15922 Genome-wide demethylation of Arabidopsis endosperm
SRA SRX005323
BioSample SAMN02197764

Supplementary file Size Download File type/resource
GSM399598_WT_endosperm_BS_seq_alignment_batch-1.gff.gz 470.1 Mb (ftp)(http) GFF
GSM399598_WT_endosperm_BS_seq_alignment_batch-2.gff.gz 443.4 Mb (ftp)(http) GFF
GSM399598_WT_endosperm_BS_seq_alignment_batch-3.gff.gz 529.0 Mb (ftp)(http) GFF
GSM399598_WT_endosperm_BS_seq_alignment_batch-4.gff.gz 403.0 Mb (ftp)(http) GFF
GSM399598_WT_endosperm_BS_seq_alignment_batch-5.gff.gz 450.7 Mb (ftp)(http) GFF
GSM399598_WT_endosperm_BS_seq_singleC.gff.gz 261.1 Mb (ftp)(http) GFF
GSM399598_WT_endosperm_BS_seq_w50.gff.gz 44.6 Mb (ftp)(http) GFF
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