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| Status |
Public on Apr 21, 2008 |
| Title |
rdd_methylC-seq |
| Sample type |
SRA |
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| Source name |
Immature floral tissue
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| Organism |
Arabidopsis thaliana |
| Characteristics |
ros1-3 dml2-1 dml3-1, unopened flower buds
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| Treatment protocol |
Immature (unopened) flower buds were collected and frozen in liquid nitrogen.
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| Growth protocol |
All plants were grown in potting soil (Metro Mix 250; Grace-Sierra, Boca Raton, FL) at 23?C under a 16-hour light/8-hour dark cycle.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
MethylC-seq library construction protocol: Genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA), and 5 µg of was fragmented by sonication to 50-500 bp with a Bioruptor (Diagenode Sparta, NJ), followed by end repair and ligation of methylated adapters provided by Illumina (Illumina, San Diego, CA) as per manufacturer?s instructions for gDNA library construction. 100-200 ng of adapter-ligated gDNA of 120-170 bp was isolated by agarose gel electrophoresis, and subjected to two successive treatments of sodium bisulfite conversion using the EpiTect Bisulfite kit (Qiagen, Valencia, CA), using the subsequent FFPE purification step, as outlined in the manufacturer?s instructions. The reaction was then purified once more using the PCR purification kit (Qiagen, Valencia, CA). Five ng of bisulfite-converted, adapter-ligated DNA molecules were enriched by 18 cycles of PCR with the following reaction composition: 2.5 U of uracil-insensitive PfuTurboCx Hotstart DNA polymerase (Stratagene), 5 µl 10X PfuTurbo reaction buffer, 25 µM dNTPs, 1 µl Primer 1.1, 1 µl Primer 2.1 (50 µl final). The thermocyling was as follows: 95?C 2 min, 98?C 30 sec, then 18 cycles of 98?C 10 sec, 65?C 30 sec and 72?C 30 sec, completed with one 72?C 5 min step. The enriched library was purified with the PCR purification kit (Qiagen, Valencia, CA)and quantity and quality examined by spectrophotometry, gel electrophoresis, and limited sequencing of cloned library molecules.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
Sequence information was extracted from the image files with the Illumina Firecrest and Bustard applications and mapped to the Arabidopsis (Col-0) reference genome sequence (TAIR 7) with the Illumina ELAND algorithm. ELAND aligns 32 bases or shorter reads, allowing up to two mismatches to the reference sequence. For reads longer than 32 bases, only the first 32 bases will be used for alignment, while the remaining sequence will be appended regardless of similarity to the reference sequence. A Perl script was used to truncate the appended sequence at the point where the next four bases contain two or more errors relative to the reference sequence.
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| Data processing |
When mapping reads generated from bisulfite converted genomic DNA, converted cytosines will score as a mismatch and will adversely affect the ELAND alignment ability. Therefore reads were mapped against computationally bisulfite converted and non-converted genome sequences. As bisulfite conversion of cytosine to thymidine results in non-complementarity of the two strands of a DNA duplex, reads were mapped against two converted genome sequences, one with cytosine changed to thymidine to represent a converted Watson strand, and a second with guanine changed to adenosine to represent the converted Crick strand. For reads that aligned to multiple positions in the reference genome at 32 bases we utilized version (1.080214) of the cross_match algorithm (P. Green personal communication) to map these non-unique reads to a reference sequence that was repeat-masked for 50 bp perfect repeat sequence. To reduce clonal bias, short read sequences that mapped to the same start position were collapsed into a single consensus read. Where a base call within the consensus was contentious, the base to be retained was randomly selected.
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| Submission date |
Mar 21, 2008 |
| Last update date |
May 15, 2019 |
| Contact name |
Joseph R Ecker |
| E-mail(s) |
ecker@salk.edu
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| Phone |
8584534100
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| Organization name |
HHMI-Salk-Institute
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| Department |
Genomic Analysis Laboratory
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| Lab |
Ecker lab
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| Street address |
10010 North Torrey Pines Road
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL9062 |
| Series (2) |
| GSE10877 |
Highly integrated single base resolution maps of the epigenome in Arabidopsis |
| GSE10966 |
Highly integrated epigenome maps in Arabidopsis - whole genome shotgun bisulfite sequencing |
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| Relations |
| SRA |
SRX002498 |
| BioSample |
SAMN02195401 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM276812.txt.gz |
398.6 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data not provided for this record |
| Raw data are available in SRA |
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