|
| Status |
Public on Oct 02, 2014 |
| Title |
pif145-RNA |
| Sample type |
SRA |
| |
|
| Source name |
whole seedlings
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
developmental stage: 2-day old
growth conditions: true dark
genotype: pif145
tissue: seedling
genetic background: Columbia
|
| Treatment protocol |
2d continuous darknes (48 hours post-stratification) at 21ºC
|
| Growth protocol |
Stratified seeds were irradiated with WL at 21ºC 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 |
total RNA |
| Extraction protocol |
Rneasy Plus mini kit was used to prepare total RNA from ~100 mg ttissue (fw). 20 ug total RNA was used for construction of each library. There are technical duplicate runs for each biological triplicate sample.
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).
Since only short fragments from the 3' end of all mRNA's are sequenced in this protocol there is no need to adjust (RPKM) for length of message. Construction strategy is directional - mRNA's are sequenced only 5' to 3' to avoid ambiguities in overlapping ORFS.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
3'-end polyA RNA
|
| Data processing |
single-end 50-nt reads
Reads 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.
differential expression between two genotypes was determined using EdgeR (Bioconductor)
Genome_build: TAIR10
Supplementary_files_format_and_content: Tab-delimited text files containing raw reads counts of every locus in each biological triplicate sample - reads from technical duplicates are combined.
|
| |
|
| Submission date |
Dec 01, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
james m tepperman |
| E-mail(s) |
jmtepp@berkeley.edu
|
| Phone |
510-559-5935
|
| Organization name |
Plant Gene Expression Center/UC Berkeley
|
| Department |
Plant and Microbial Biology
|
| Lab |
QUAIL
|
| Street address |
800 Buchanan St.
|
| City |
Albany |
| State/province |
CA |
| ZIP/Postal code |
94710 |
| Country |
USA |
| |
|
| Platform ID |
GPL13222 |
| Series (2) |
| GSE42665 |
Transcriptome-wide analysis of gene expression in dark-grown WT and pif mutant seedlings |
| GSE43286 |
Genome-wide identification of PIF-binding sites and direct-target genes of PIF transcriptional regulation in skotomorphogenesis |
|
| Relations |
| SRA |
SRX207960 |
| BioSample |
SAMN01821951 |
