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| Status |
Public on Jun 16, 2022 |
| Title |
RNA seq of Oryzae sativa cv BPT control plants |
| Sample type |
SRA |
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| Source name |
Leaf
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| Organism |
Oryza sativa |
| Characteristics |
tissue: Leaf
cultivar: BPT5204
treatment: untreated
age: 51 days
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted according to the protocol described by Datta et al. (1989) with some modifications. Solutions were prepared with 0.1% (v/v) diethylpyrocarbonate (DEPC). Leaf material (1 g) was collected, flash frozen in liquid nitrogen, and finely ground. 10ml of extraction buffer [0.1 M tris HCl pH 9.0, 0.25M sucrose, 0.2 M NaCl and 10 mM MgCl2] was added and homogenized properly. To the extraction buffer, 10 ml of phenol (water saturated): chloroform mixture (1:1) v/v was added and homogenized, 1 ml of 0.5 M potassium EDTA and 1 ml of 20% (w/v) SDS were added sequentially. The extract was transferred to Oakridge tube and 144 μl of ß- mercaptoethanol was added and kept on rocking shaker in ice for 20 min. The extract was centrifuged at 16000 rpm for 30 min and the supernatant was transferred to the Oakridge tube. An equal volume of chloroform: isoamyl alcohol (49:1) was added to the aqueous phase, vortexed and spin at 15000 rpm for 15 min at 40C. The aqueous phase was transferred to a new Oakridge tube and 8 M LiCl2 was added to a final concentration of 3 M and kept in -200C for 12-18 h. After precipitation, the tubes were centrifuged at 16000 rpm for 30 min at 40C. The supernatant was discarded and the precipitate was washed with 5 ml of 2 M LiCl2 and subsequently with 5 ml of 75 % (v/v) ethanol by centrifuging at 15000 rpm for 20 min at 4oC. The pellet was air dried and dissolved in 100μl of autoclaved DEPC treated water and RNA quantity was estimated spectrophotometrically (Nanodrop®, USA). The purity was checked by analysing the 260/280 ratio and visualizing on formaldehyde denaturing agarose (1% (w/v)) gel. Three micrograms of total RNA was reverse transcribed to single stranded cDNA in a 20 μl reaction mixture consisting of 200 units MMLV reverse transcriptase (RT) (MBI Fermentas), 1X MMLV- RT buffer, 40 picomoles oligo-dT primer and 100 mM dNTP mix. Reverse transcription was performed at 42oC for 1 h. Synthesized cDNA was confirmed by semi-quantitative Reverse Transcription PCR (RT-PCR) using actin as housekeeping gene.
RNA libraries were prepared for sequencing using standard Illumina protocols
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Rice BPT5204 control
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| Data processing |
Read alignment was carried out using Hisat2 v2.1.0
The quantification of the gene expression level in FPKM (Fragments per kilobase of the transcript sequence per million base pairs sequenced) were done by StringTie (1.3.4) by counting the number of reads mapped to each gene.
The differential expression analysis was performed using the DESeq2 R package (2.11.38).
Genome_build: RGAP 7
Supplementary_files_format_and_content: tab-delimited gene abundance files whcih include FPKM values for each Sample
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
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| Submission date |
Feb 21, 2022 |
| Last update date |
Jun 17, 2022 |
| Contact name |
Ramu Vemanna |
| E-mail(s) |
ramu.vemanna@rcb.res.in
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| Organization name |
Regional Centre for Biotechnology
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| Street address |
NCR Biotech Science Cluster, Faridabad
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| City |
Faridabad |
| ZIP/Postal code |
121001 |
| Country |
India |
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| Platform ID |
GPL23013 |
| Series (1) |
| GSE197133 |
RNA Seq analysis of tolerant (BPT5204) and susceptible (TN1) variety of Oryza sativa cv. Indica under drought, pathogen, and combined stress |
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| Relations |
| BioSample |
SAMN26137930 |
| SRA |
SRX14241917 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5909711_B-C_gene_ab.tsv.gz |
1.1 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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