|
| Status |
Public on Feb 01, 2013 |
| Title |
pifq-RNA-2 |
| Sample type |
SRA |
| |
|
| Source name |
whole seedlings
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
developmental satge: 2-day-old
growth conditions: true dark
genotype: pif1pif3pif4pif5 (pifq)
|
| Growth protocol |
Stratified seeds were irradiated with WL at 21 ºC for 3 h to induce germination, followed by a FR pulse for 15 min to suppress pseudo dark effects, and grown in darkness at 21 ºC for 2 d before harvest.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
The sequencing library construction was adapted from Illumina’s directional mRNA-seq sample preparation protocol. The mRNA was fractionated from 20 µg of total RNA using Dynabeads Oligo (dT)25 (Invitrogen), and fragmented using Fragmentation Reagents (Ambion) at 70 ºC for 2.5 min in 20 µl of reaction. The polyA-tailed 3’-end fragments were captured by another run of mRNA purification as described above, and then treated by Antarctic Phosphatase and T4 Polynucleotide Kinase (NEB) at 37 ºC for 1 h and 2 h, respectively. The sample was purified using RNeasy MinElute Cleanup Kit (Qiagen) according to Illumina’s protocol. The eluted mRNA fragments were ligated with 2.5 µM of Illumina’s SRA 5’ adaptor by T4 RNA Ligase 1 (NEB) at 20 ºC for 4 h. The 3’ cDNA adapter derived from Illumina’s v1.5 sRNA 3’ adapter was conjugated with the anchored oligo (dT)20 primer, and introduced through reverse transcription using the SuperScript III First-Strand Synthesis System (Invitrogen). The first-strand cDNA was purified using the Agencourt AMPure XP system (Beckman Coulter Genomics), and then amplified by PCR reaction using Phusion High-Fidelity DNA Polymerase (NEB) with Illumina’s sRNA PCR primer set. The size of purified library DNA was validated by Bioanalyzer 2000 (Agilent).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
3'-end fragments were captured by polyA purification after mRNA fragmentation
pifq.xlsx
|
| Data processing |
Base-callling was performed using Illumina pipeline CASAVA-1.7.0.
For consistency, 50-nt reads were trimmed from 3’-end by 14 nt before alignment.
Reads that have no unrecognized nucleotides (Ns) and passed quality filtering were aligned to the TAIR10 transcriptome assembly using Bowtie-0.12.7 with the following settings: -v 0 --norc -a --best.
Only reads mapped uniquely to the 3’-end 500-bp region of the single locus were counted for gene expression.
Genome_build: TAIR10
Supplementary_files_format_and_content: Tabular files containing raw reads counts of every locus in each of biological triplicates
|
| |
|
| Submission date |
Jul 10, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Yu Zhang |
| E-mail(s) |
yuzhang@berkeley.edu
|
| Phone |
1-510-559-5889
|
| Organization name |
UC Berkeley
|
| Department |
Plant and Microbial Biology
|
| Lab |
Peter H. Quail
|
| Street address |
800 Buchanan Street
|
| City |
Albany |
| State/province |
California |
| ZIP/Postal code |
94710 |
| Country |
USA |
| |
|
| Platform ID |
GPL13222 |
| Series (2) |
| GSE39214 |
Genome-wide identification of PIF3-binding sites and direct-target genes of PIF3 transcriptional regulation in skotomorphogenesis [RNA-Seq] |
| GSE39217 |
Genome-wide identification of PIF3-binding sites and direct-target genes of PIF3 transcriptional regulation in skotomorphogenesis |
|
| Relations |
| SRA |
SRX159024 |
| BioSample |
SAMN01086894 |
