|
| Status |
Public on Jun 06, 2011 |
| Title |
Input |
| Sample type |
SRA |
| |
|
| Source name |
aerial tissue of two-week-old plants
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
age: 2 wk
tissue: aerial tissue
ecotype: Col-0
chip antibody: none
|
| Growth protocol |
For plants used for ChIP-seq, Arabidopsis plants were germinated and grown for two weeks in 0.5X MS media solidified with 0.8% agar. Plants used for RNA-seq were grown in soil for 21-28 days.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For the construction of ChIP library, the purified ChIPed DNA was fragmentated by the Covaris S2 system to ~125 bp-long. After recovering the DNA with ethanol precipitation, samples were treated with DNA End-Repair Kit. End-repaired DNA was then purified with MinElute Reaction Cleanup Kit. Adaptors were synthesized as single-strand oligos (P1 HPLC grade; P2 desalted grade) and annealed in 1X PCR buffer with a thermal cycler. P2 adaptors were resolved on a 3% agarose gel and the dominant 50 bp band was purified. After ligating both P1 and P2 adaptors, a phenol/chloroform extraction followed by ethanol precipitation was performed to remove ligase and salts. The purified DNA was resolved on a 6% native PAGE gel to separate ligation products from adaptors. The gel piece corresponding to where 150-250 bp DNA migrate was sliced and stored frozen. The optimal cycle for library amplification was empirically determined with 1/3 of the gel piece. Library prepared with the rest 2/3 of gel slice was resolved on a 3% agarose gel to recover the 150-250 bp smear. For the construction of RNA-seq libraries, ribosomal RNAs were depleted from the total RNA with two rounds of treatment with the RibominusTM Plant Kit (Invitrogen). RNA-seq fragment libraries were constructed using the SOLiDTM Whole-Transcriptome Analysis Kit following the manufacturerâs protocol. Briefly, the RNA was fragmented using RNase III and subsequently purified using the Qiagen gel extraction minelute kit. The RNA fragments were then ligated to barcoded RNA adaptors supplied in the Whole Transcriptome Kit, and then reverse transcribed. The resulting cDNA was then resolved on a 3% agarose gel, size selected and purified, and then amplified by PCR. The libraries were quantitated using a NanoDrop and pooled.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB SOLiD System 2.0 |
| |
|
| Description |
input DNA
SRX017605
SRA010097
|
| Data processing |
The barcodes were decoded allowing zero mismatches. The color-space reads for each barcoded library were aligned to TAIR 8 Arabidopsis thaliana reference genome using Corona Lite 4.2.1 (Life Technologies) allowing 3 colorspace mismatches in the 35 bps reads. For ChIP-seq tags that mapped to more than one position in the genome, the reported position was randomly chosen from up to 1,000 potential matches. For RNA-seq data, only tags mapped to unique location were used for analyses. Tags mapped to an identical genomic position were removed except for one in order to compensate for the potential over-amplification of the library.
|
| |
|
| Submission date |
Apr 05, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Chongyuan Luo |
| E-mail(s) |
chongyuanluo@gmail.com
|
| Organization name |
University of California Los Angeles
|
| Department |
Human Genetics
|
| Lab |
Chongyuan Luo
|
| Street address |
695 Charles E Young Drive South
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platform ID |
GPL10272 |
| Series (1) |
| GSE28398 |
Histone modifications of Arabidopsis thaliana (aerial tissue) |
|
| Relations |
| SRA |
SRX017605 |
| BioSample |
SAMN02198589 |
