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| Status |
Public on Sep 21, 2012 |
| Title |
at-sperm-col_dme2 |
| Sample type |
SRA |
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| Source name |
AT Sperm dme-2 Columbia
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| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue type: pollen
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| Treatment protocol |
Stage 12-13 flower buds were emasculated and pollinated 48 hours later. Reciprocal crosses were performed using wild-type Col-0 and Ler ecotypes. In addition, dme-2 (Col-gl) heterozygous flowers were pollinated with wild-type (Ler) pollen, and fie-1 (Ler) heterozygous flowers were pollinated with wild-type (Col-0) pollen. For wild-type crosses, seven to eight days after pollination, F1 seeds (torpedo-stage to early-bent-cotyledon stage) were immersed in 0.3 M sorbitol and 5 mM Mes (pH 5.7) on a slide under a dissecting microscope. Embryo and endosperm were dissected using a fine needle and forceps. The seed coat was discarded. Wild-type embryos were twice centrifuged and the pellet resuspended in 0.3 M sorbitol and 5 mM Mes (pH 5.7) to remove contaminating endosperm. For crosses with the dme-2 or fie-1 mutations, F1 aborting seeds were identified and mutant endosperm was isolated. Approximately 500 wild-type endosperm, 1000 dme-2 or fie-1 endosperm, and 300 wild-type embryos were collected. Pollen was isolated from wild-type (Col-0) and dme-2 heterozygous plants (Col-gl ecotype) as described previously. Vegetative cell and sperm nuclei were extracted from mature pollen and fractionated by fluorescence activated cell sorting as described previously.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
About 150 ng of genomic DNA was fragmented by sonication, end repaired and ligated to custom-synthesized methylated adapters (Eurofins MWG Operon) according to the manufacturer’s (Illumina) instructions for gDNA library construction. Adaptor-ligated libraries were subjected to two successive treatments of sodium bisulfite conversion using the EpiTect Bisulfite kit (Qiagen) as outlined in the manufacturer’s instructions. One quarter of the bisulfite-converted libraries was PCR amplified using the following conditions: 2.5 U of ExTaq DNA polymerase (Takara Bio), 5 μl of 10X Extaq reaction buffer, 25 μM dNTPs, 1 μl Primer 1.1, 1 μl Primer 2.1 (50 μl final). PCR reactions were carried out as follows: 95 ̊C 3 min, then 12-14 cycles of 95 ̊C 30 sec, 65 ̊C 30 sec and 72 ̊C 60 sec. The enriched libraries were purified twice with solid phase reversible immobilization (SPRI) method using AM-Pure beads (Beckman Coulter) prior to quantification with a Bioanalyzer (Agilent).
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Arabidopsis thaliana sperm of dme-2 Columbia-gl (female).
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| Data processing |
Alignment: We used Perl scripts to convert all the Cs in the reads (and in the scaffolds) to Ts, and aligned the converted reads to the converted Col and Ler scaffolds, allowing up to two mismatches per read. 76 base reads were divided into the first 45 and the last 31 bases. 80 base reads were divided into the first 40 and the last 40 bases. 100 base reads were divided into the first 50 and the last 50 bases. Each half of the read was aligned independently using bowtie, allowing up to two mismatches. The coordinates of the two halves were subsequently correlated; the second half was discarded if it did not match the first. Reads that aligned to either Col or Ler with fewer mismatches were assigned to that ecotype.
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).
Genome_build: TAIR8
Supplementary_files_format_and_content: All files are in GFF format. Files contain fractional methylation data either for individual cytosines (single-c) or in 50 bp windows.
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| Submission date |
Jun 26, 2012 |
| 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 |
GPL13222 |
| Series (1) |
| GSE38935 |
Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes |
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| Relations |
| SRA |
SRX156132 |
| BioSample |
SAMN01057768 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM952444_TFH49.all.cg.merged.gff.gz |
43.5 Mb |
(ftp)(http) |
GFF |
| GSM952444_TFH49.all.chg.merged.gff.gz |
47.9 Mb |
(ftp)(http) |
GFF |
| GSM952444_TFH49.all.chh.merged.gff.gz |
218.9 Mb |
(ftp)(http) |
GFF |
| GSM952444_TFH49.all.w50-CG.merged.gff.gz |
19.9 Mb |
(ftp)(http) |
GFF |
| GSM952444_TFH49.all.w50-CHG.merged.gff.gz |
20.5 Mb |
(ftp)(http) |
GFF |
| GSM952444_TFH49.all.w50-CHH.merged.gff.gz |
24.5 Mb |
(ftp)(http) |
GFF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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