|
| Status |
Public on Jun 22, 2020 |
| Title |
herlperless_8h_avrRpm1_Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
leaf disc
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype: herlperless
time: 8h
treatment: avrRpm1
rep: 1
|
| Treatment protocol |
Plants for bacterial infiltration assays were grown for 6 weeks under short day conditions (8h light/16h dark at 21°C/18°C and 45% humidity). For bacterial growth curves, Pst DC3000 ∆hrcC and Pst DC3000 expressing either AvrRps4, AvrRpt2, EV, AvrRpm1, grown on KB plates containing appropriate antibiotics, were re-suspended in 10 mM MgCl2 to a final concentration of 5x105 (OD600 0.001). Plants were hand-infiltrated with the bacterial suspension.
|
| Growth protocol |
Arabidopsis plants were grown at short day conditions (8-hour light/16-hour dark cycle at 21°C/18°C and 45% humidity. Arabidopsis sequence data for helper NLRs is available under the following AGI accession numbers: ADR1/At1g33560, ADR1-L1/At4g33300, ADR1-L2/At5g04720, NRG1.1/NRG1A/At5g66900, NRG1.2/NRG1B/At5g66910.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified from plant tissue using the RNeasy 96 Kit (Qiagen). The plant “user developed protocol” was followed except for the addition of a 96% ethanol wash step after the second RPE wash to ensure removal of residual salts. Purified RNAs were kept at -80C.
Illumina-based mRNA-Seq libraries were prepared from 1 μg RNA following [61]. Briefly, mRNAs were selected using Sera-mag oligo(dT) magnetic beads (GE Healthcare Life Sciences). RNAs were washed and fragmented at 94°C for 6 minutes. First-strand cDNA synthesis was performed using random hexamers and reverse transcriptase (Superscript III reverse transcriptase, Invitrogen). Second-strand cDNA synthesis was done using DNA Polymerase I and RNAseH. Double-stranded cDNAs were end-repaired using T4 DNA polymerase, T4 polynucleotide kinase, and Klenow polymerase. The DNA fragments were then adenylated using Klenow exo-polymerase to allow the ligation of Illumina adapters (Kapa Dual-indexed adapter kit, Roche). Unless specified, reagents were purchased from Enzymatics.
Library quality control and quantification were performed using the 5200 Fragment Analyser and the NGS fragment kit (Agilent). Libraries were sequenced using Illumina HiSeq4000 sequencers to generate 50-bp single-end reads.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
C12HPK.8h.M1.1
|
| Data processing |
Initial quality assessment of the Illumina RNA-Seq reads was performed using FastQC version 0.11.7.
Trimmomatic version 0.36 was used to identify and discard reads containing the Illumina adaptor sequence.
The resulting high-quality reads were then mapped against the TAIR10 Arabidopsis reference genome using HISAT2 version 2.1.0 with default parameters.
The featureCounts function from the Subread package was then used to count reads that mapped to each one of the 27,206 nuclear protein-coding genes, we used these counts to construct a raw count table of expression.
Genome_build: TAIR 10
Supplementary_files_format_and_content: Tab delimited file containing the count matrix
|
| |
|
| Submission date |
Jun 05, 2020 |
| Last update date |
Jun 22, 2020 |
| Contact name |
Isai Salas Gonzalez |
| E-mail(s) |
isai@email.unc.edu
|
| Phone |
919-537-3890
|
| Organization name |
The University of North Carolina at Chapel Hill
|
| Street address |
250 Bell Tower Drive, Room 4244
|
| City |
Chapel Hill |
| ZIP/Postal code |
27599 |
| Country |
USA |
| |
|
| Platform ID |
GPL21785 |
| Series (1) |
| GSE151885 |
Two unequally redundant 'helper' immune receptor families mediate Arabidopsis intracellular 'sensor' immune receptor functions |
|
| Relations |
| BioSample |
SAMN15146333 |
| SRA |
SRX8480032 |
