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| Status |
Public on Jun 16, 2009 |
| Title |
Embryo_BS_seq |
| Sample type |
SRA |
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| Source name |
Embryo
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| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Columbia-0
genotype: wild-type
tissue: embryo
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| Growth protocol |
Standard long day growth conditions
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| 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.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
DNA methylation analysis of Arabidopsis tissue by bisulfite sequencing
library_strategy: Other (BS-seq)
library_source: genomic
library_selection: random
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| 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).
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| Submission date |
May 01, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Toshiro Nishimura |
| E-mail(s) |
tnish@berkeley.edu
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| Phone |
5106429550
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| Organization name |
University of California at Berkeley
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| Department |
Plant and Microbial Biology
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| Lab |
Daniel Zilberman
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| Street address |
211 Koshland Hall
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| City |
Berkeley |
| State/province |
CA |
| ZIP/Postal code |
94720 |
| Country |
USA |
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| Platform ID |
GPL9302 |
| Series (1) |
| GSE15922 |
Genome-wide demethylation of Arabidopsis endosperm |
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| Relations |
| SRA |
SRX005322 |
| BioSample |
SAMN02197763 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM399597_Embryo_BS_seq_alignment_batch-1.gff.gz |
514.2 Mb |
(ftp)(http) |
GFF |
| GSM399597_Embryo_BS_seq_alignment_batch-2.gff.gz |
600.8 Mb |
(ftp)(http) |
GFF |
| GSM399597_Embryo_BS_seq_alignment_batch-3.gff.gz |
610.5 Mb |
(ftp)(http) |
GFF |
| GSM399597_Embryo_BS_seq_alignment_batch-4.gff.gz |
526.2 Mb |
(ftp)(http) |
GFF |
| GSM399597_Embryo_BS_seq_alignment_batch-5.gff.gz |
541.6 Mb |
(ftp)(http) |
GFF |
| GSM399597_Embryo_BS_seq_single_C.gff.gz |
271.4 Mb |
(ftp)(http) |
GFF |
| GSM399597_Embryo_BS_seq_w50.gff.gz |
45.5 Mb |
(ftp)(http) |
GFF |
SRA Run Selector |
| Raw data are available in SRA |
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