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| Status |
Public on May 01, 2013 |
| Title |
Oryza Sativa L Endosperm F1 Cultivar Kitaake x Cultivar Nipponbare KNNS1 |
| Sample type |
SRA |
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| Source name |
Oryza Sativa L Endosperm F1 Cultivar Kitaake x Cultivar Nipponbare
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| Organism |
Oryza sativa |
| Characteristics |
genetic background: F1 Cultivar Kitaake x Cultivar Nipponbare
tissue: enodsperm
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| Treatment protocol |
No specific treatments were applied, other than controlled growth conditions for seedling tissue, and emasculation and artificial pollination of rice flowers to obtain F1 seeds.
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| Growth protocol |
For root and shoot samples, Nipponbare rice seedlings were grown in sterile flask culture with Gamborg’s B-5 medium with sucrose (Caisson Laboratories) for 21 days with 24 hours of light and constant shaking. Seed samples were obtained at 7 to 8 DAF from rice plants grown under greenhouse conditions at the University of California, Davis.
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| Extracted molecule |
total RNA |
| Extraction protocol |
DNA and RNA was extracted from snap-frozen tissues using CTAB and TRIzol reagent (Invitrogen), respectively.
For bisulfite sequencing, about 1µg 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. For small RNA libraries, 10 µg of total RNA was loaded on a 15% polyacrylamide, 7M urea gel, and small RNA in the 17-30 nt size range were excised. The 3' miRNA cloning linker 1 (IDT) was ligated to the gel-excised small RNA using truncated T4 RNA ligase 2 (NEB), and ligation products were purified on a 15% polyacrylamide, 7M urea gel. The 5' Illumina RNA linker (5'-rArCrArCrUrCrUrUrUrCrCrCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrU-3') was added using T4 RNA ligase (NEB) and ligation products were again purified on a 15% polyacrylamide, 7M urea gel. Purified ligated small RNAs were reverse transcribed using SuperScriptIII (Invitrogen) and PCR-amplified with Phusion High-Fidelity DNA Polymerase (NEB) for 25 cycles (Forward primer: 5'-AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACG-3', Reverse primer: 5'-CAAGCAGAAGACGGCATACGATTGATGGTGCCTACAG-3'). Sequencing was performed on the Illumina HiSeq 2000 (crossed F1 embryo and endosperm) and GA-II (self-fertilized embryo, endosperm, seedling root and seedline shoot) platforms. Small RNA reads are either 36 or 50 bp long, while BS-seq reads are 100 bp long.
Bisulfite sequencing and small RNA sequencing with Illumina technology
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
small RNA
Oryza Sativa L Endosperm 7 DAF F1 Cultivar Kitaake x Cultivar Nipponbare
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| Data processing |
Alignment of BS-seq reads: 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 Nipponbare and Kitaake scaffolds using the aligner Bowtie, allowing up to two mismatches per read. Reads that aligned to either Nipponbare or Kitaake with fewer mismatches were assigned to that ecotype.
Alignment of small RNA-seq reads: We used Perl and Python scripts to trim adapter sequences in the reads, sorted reads according to size, and aligned the size-sorted reads to the Nipponbare and Kitaake scaffolds using the aligner Bowtie, allowing up to one mismatch per read. Reads that aligned to either Nipponbare or Kitaake 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: For BS-seq data, we used a Perl script to calculate fractional methylation (#C/(#C+#T)) within 50 bp windows of the genome for each sequence context (CG, CHG, CHH). For small RNA-seq data, we used a Perl script to calculate the number of reads mapping within 50 bp windows of the genome.
Genome_build: Oryza sativa L. cv Nipponbare rice reference genome (MSU 6.1)
Supplementary_files_format_and_content: All files are in GFF format. BS-seq files contain fractional methylation data either for individual cytosines (single-c) or in 50 bp windows. Small RNA-seq files have 24 nt abundance calculated in 50 bp windows.
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| Submission date |
Mar 05, 2013 |
| 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 |
GPL13160 |
| Series (1) |
| GSE44898 |
Imprinted expression of genes and small RNA is triggered by localized demethylation of the maternal genome in rice endosperm |
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| Relations |
| SRA |
SRX248524 |
| BioSample |
SAMN01974822 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1093511_KNNS1-w50.gff.gz |
7.4 Mb |
(ftp)(http) |
GFF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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