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| Status |
Public on Apr 17, 2013 |
| Title |
ddm1-2 |
| Sample type |
SRA |
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| Source name |
AT roots 3 weeks Columbia-0
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| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Columbia-0
developmental stage: roots 3 weeks
genotype: ddm1
function: Snf2
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| Growth protocol |
Arabidopsis plants were grown according to one of the following methods: 1) For shoot tissues, seeds were planted on 1x Murashige and Skoog Media with micronutrients and 1.5% Sucrose (Caisson Laboratories) and grown under 16h light/ 8h dark for 14 or 21 days in a growth chamber. 2) For roots, seeds were grown in Gamborg's B-5 liquid Media with 1.5% Sucrose (Caisson Laboratories) and grown shaking under 16h light/ 8h dark for 21 days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For bisulfite sequencing (Bisulfite-Seq) About 500 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. The bisulfite-converted libraries were then 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 either gel-purified (~300 bp band) or purified with solid phase reversible immobilization (SPRI) method using AM-Pure beads (Beckman Coulter) prior to quantification with a Bioanalyzer (Agilent). For RNA-Sequencing, approximately 30 ug total RNA was isolated from 21 days roots using the RNEasy Plant Extraction Kit (Qiagen) with the optional on-column DNAse treatment. mRNA was purified from total RNA by two treatments of poly-A enrichment using the Oligotex kit (Qiagen #72022), followed by a rRNA removal step using the RiboMinus Plant Kit for RNA-sequencing (Invitrogen #A1083702). Precipitated mRNA samples were eluted with 9 μl of RNase free water and fragmented with 1 μl of 10X fragmentation buffer (Ambion, #AM8740) at 70oC. Reactions were stopped after 5 minutes by adding 1 μl Stop buffer and RNA was purified by ethanol precipitation. cDNA was synthesized from 100-300 ng of mRNA using Superscript III reverse transcriptase (Invitrogen #18080-051). Double-stranded DNA was synthesized as described (3). DNA was cleaned with a QIAquick PCR spin column (Qiagen, #28106), sequencing adapters were ligated according to the Illumina protocol and library was amplified by 18 cycles of PCR using Phusion DNA polymerase (NEB, #F-530). Bands around 300 bp were gel-purified and cloned for validation. Traditional sequencing confirmed that the libraries were properly constructed, showing high percentage of mRNA over rRNA. The libraries were sequenced at the UC Berkeley Genomic Sequencing Laboratory, generating single ends (SE) 36 base reads. For MNase-sequencing, Arabidopsis roots (1 g) were ground in liquid nitrogen, resuspended in 20 ml of HBM buffer (25mM Tris, pH 7.6, 0.44M Sucrose, 10 mM MgCl2, 2 mM spermine and 0.1% Triton X-100), homogenized, filtered through miracloth, transferred to a 30 ml round bottom glass tube, centrifuged at 2000g (40C) for 10 min and resuspended in 1 ml HBB buffer (25mM Tris, pH 7.6, 0.44M Sucrose, 10 mM MgCl2 and 0.1% Triton X-100). Nuclei were further spun down at 200g, 40C for 2 min and resuspended in 1 ml of TNE buffer (10 mM Tris, pH 8.0, 100 mM NaCl and 1 mM EDTA). MNase digestion was done with 4 ul of 1M CaCl2 and 1 ul of diluted (1/20) MNase (200ul/ml; Sigma #N-3755) per 100 ul of pellet nuclei. Nuclei were then divided to several tubes and digestion was stopped at 45 sec intervals with 10mM EDTA. Digested nuclei were spun down at maximum speed for 5 min at 40C, and released soluble nucleosomes were collected from the supernatant. Following RNase A and proteinase K digestion, DNA was purified using phenol/chloroform. Purified DNA samples were run on a 2% agarose gel, digested samples with most enriched intact mononucleosomes were chosen and bands corresponding to ~150 bp were cut and purified with a Gel Purification kit (Qiagen). Illumina libraries were constructed and sequenced at the UC Berkeley Genomic Sequencing Laboratory, generating paired ends (PE) 36 base reads. For MNase sequencing, we aligned 36 bases Illumina paired reads to the TAIR8 Arabidopsis genomic scaffold, 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. To measure nucleosome occupancy, we summed the aligned reads (all bases between both paired reads got a score of 1) for every single nucleotide throughout the genome.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
Bisulfite-Seq: 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 reference scaffold, allowing up to two mismatches per read. 76 base reads were divided into the first 45 and the last 31 bases, 100 base reads were divided in half and 50 base reads were processed as single reads. 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.
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).
All files are in GFF format. Files contain fractional methylation data either for individual cytosines (single-c) or in 50 bp windows.
RNA-seq: Illumina reads from cDNA libraries were aligned to genomic DNA. The file lists the log2 of the number of reads (first base) matching each 50-bp window, normalized to the total number of aligned reads in each library.
Mnase-seq: We aligned 36-base Illumina paired ends reads to genomic DNA. To measure nucleosome occupancy, we counted the number of sequenced molecules corresponding to each nucleotide of the genome. To reduce the size of the file we collapsed all adjacent positions with equal scores to one window.
Genome_build: TAIR8
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| Submission date |
Oct 03, 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 |
GPL9302 |
| Series (1) |
| GSE41302 |
DDM1 and RdDM are the major regulators of transposon DNA methylation in Arabidopsis |
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| Relations |
| SRA |
SRX190984 |
| BioSample |
SAMN01757896 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1014117_ddm1-2_bs-seq_CG_single-c.gff.gz |
32.6 Mb |
(ftp)(http) |
GFF |
| GSM1014117_ddm1-2_bs-seq_CG_w50.gff.gz |
18.3 Mb |
(ftp)(http) |
GFF |
| GSM1014117_ddm1-2_bs-seq_CHG_single-c.gff.gz |
35.4 Mb |
(ftp)(http) |
GFF |
| GSM1014117_ddm1-2_bs-seq_CHG_w50.gff.gz |
18.2 Mb |
(ftp)(http) |
GFF |
| GSM1014117_ddm1-2_bs-seq_CHH_single-c.gff.gz |
154.9 Mb |
(ftp)(http) |
GFF |
| GSM1014117_ddm1-2_bs-seq_CHH_w50.gff.gz |
22.3 Mb |
(ftp)(http) |
GFF |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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