|
| Status |
Public on Apr 01, 2023 |
| Title |
Col_biorep_2 |
| Sample type |
SRA |
| |
|
| Source name |
whole seedling
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: whole seedling
developmental stage: 10-day-old
genotype: Col-0
treatment: constant light (55 umol m-2 s-1) and temperature (22 C)
time: sampled every 2h over 24 hours and pooled
|
| Treatment protocol |
At day 11, approximately 60 mg of whole seedlings of each genotype were collected every 2h over 24 hours and pooled. Three independent biological replicates were collected for each genotype.
|
| Growth protocol |
Arabidopsis seeds were surface sterilized in 70% ethanol prepared in 0.1% (v/v) Triton X-100 (Sigma) for 5 minutes and then in 100% Ethanol for 20 minutes. After air drying, seeds were plated on 1x Murashige and Skoog (MS) growth media containing 0.7% agar, pH 5.7. After 3d stratification in dark at 4˚C, plates were transferred to a growth chamber with constant light (55 µmol m-2 s-1) and temperature (22˚C) to germinate.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were flash-frozen in liquid nitrogen, and ground into fine powder using a beadbeater. Total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen) followed by purification using the RNA Clean & Concentrator Kit (Zymo Research). Purified RNAs were quantified using a Nanodrop (ThermoFisher) and quality control was performed by an Agilent Bioanalyzer 2100.
PacBio Sequel II library preparation and RNA-sequencing (Iso-Seq) was performed by UC Davis DNA Technologies & Expression Analysis Core (https://dnatech.genomecenter.ucdavis.edu/pacbio-library-prep-sequencing/).
PacBio Sequel II
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Sequel II |
| |
|
| Data processing |
Raw reads generated by the PacBio Sequel II sequencer were imported into PacBio SMRT Link v 8.0 for Circular Consensus Sequence (CCS) calling and demultiplexing.
Next, poly(A) tails and concatemers were removed using the refine command from isoseq3 package via Bioconda (v 3.10) with the option ‘--require-polya’.
Then the fastq files containing full-length non-concatemer reads were mapped to Arabidopsis TAIR 10 genome assembly using minimap2 (v 2.17) with the parameter ‘-ax splice -t 30 -uf --secondary=no -C5’.
For differential splicing analysis, counts of exonic regions and known/novel splice junctions were generated using QoRTs software package (v 1.3.6; Hartley and Mullikin, 2015) with the parameter ‘--stranded --singleEnded --stranded_fr_secondstrand --keepMultiMapped’. To adapt to the long-read data, the ‘maxPhredScore’ was set to 93 and the ‘maxReadLength’ was adjusted manually for each library. To increase the power of detecting novel splice junctions, the '--minCount' threshold was set to 5.
The raw count data was then loaded to JunctionSeq R package (v 1.5.4; Hartley and Mullikin, 2016) to determine differential usage of exons or splice junctions relative to the overall expression of the gene with a false discovery rate (FDR) of 0.05.
Assembly: Arabidopsis TAIR 10 genome assembly
Supplementary files format and content: The raw count files are gzip compressed tab-delimited text files that include raw counts for each exon/known junction/novel junction
Supplementary files format and content: The normalized abundance files are gzip compressed tab-delimited text files that include normalized expression levels, statistic significance of the differential usage and corresponding location information of each of each exon/known junction/novel junction
|
| |
|
| Submission date |
Dec 13, 2022 |
| Last update date |
Apr 03, 2023 |
| Contact name |
Stacey L Harmer |
| E-mail(s) |
[email protected]
|
| Phone |
(530) 752-8101
|
| Organization name |
UC Davis
|
| Department |
Plant Biology
|
| Lab |
Stacey Harmer
|
| Street address |
1002 Green Hall, One Shields Ave.
|
| City |
DAVIS |
| State/province |
California |
| ZIP/Postal code |
95616 |
| Country |
USA |
| |
|
| Platform ID |
GPL32947 |
| Series (1) |
| GSE220902 |
XAP5 CIRCADIAN TIMEKEEPER regulates RNA splicing and the circadian clock by genetically separable pathways |
|
| Relations |
| BioSample |
SAMN32207378 |
| SRA |
SRX18680725 |
