|
| Status |
Public on Jan 20, 2021 |
| Title |
kpsM mt 2 |
| Sample type |
SRA |
| |
|
| Source name |
Synechocystis
|
| Organism |
Synechocystis sp. PCC 6803 |
| Characteristics |
strain: sp. PCC 6803
optical density: 1.5
genotype: kpsM mutant
|
| Growth protocol |
Cells were grown in BG11 at 30 °C under a 12 h light (50 μE m−2 s−1)/12 h dark regimen, with orbital shaking at 150 r.p.m.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA extraction, the TRIzol Reagent (Ambion) was used in combination with the PurelinkTM RNA Mini Kit (Ambion). Briefly, cells were disrupted in TRIzol containing 0.2 g of 0.2 mm-diameter glass beads (acid washed, Sigma) using a FastPrepH-24 (MP Biomedicals) and the following extraction steps were performed according to the manufacturer’s instructions. DNase treatment was performed according to the On-column PureLink® DNase Treatment Protocol (Life Technologies/Invitrogen). RNA was quantified on a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc.) and the quality and integrity was checked using the ExperionTM RNA StdSens Analysis Kit (Bio-Rad).
RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Sample 5
|
| Data processing |
Sequencing libraries were generated using NEBNext® Ultra TM RNA Library Prep Kit for Illumina® (NEB, USA)
library quality was assessed on the Agilent Bioanalyzer 2100 system. Raw data (raw reads) of FASTQ format were firstly processed through in-house scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter and poly-N sequences and reads with low quality from raw data.
Paired-end clean reads were mapped to the reference genome using HISAT2 software. HISAT2 uses a large set of small GFM indexes that collectively cover the whole genome. These small indexes (called local indexes), combined with several alignment strategies, enable rapid and accurate alignment of sequencing reads. HTSeq was used to count the read numbers mapped of each gene, including known and novel genes. And then RPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. RPKM, Reads Per Kilobase of exon model per Million mapped reads, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels.
Differential expression analysis between two conditions/groups (three biological replicates per condition) was performed using DESeq2 R package. DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P values were adjusted using the Benjamini and Hochberg’s approach for controlling the False Discovery Rate (FDR).
genome build: GCF_000009725.1
Supplementary_files_format_and_content: Excel files include FPKM values for each Sample
Supplementary_files_format_and_content: DEA between the wild type and the mutant
|
| |
|
| Submission date |
Jan 19, 2021 |
| Last update date |
Jan 20, 2021 |
| Contact name |
Marina Patrícia Santos |
| E-mail(s) |
[email protected], [email protected]
|
| Phone |
+351 965502568
|
| Organization name |
i3S - Instituto de Investigação e Inovação em Saúde
|
| Department |
Host Pathogen Interaction
|
| Lab |
Bioengineering and Synthetic Microbiology
|
| Street address |
R. Alfredo Allen 208
|
| City |
Porto |
| State/province |
Porto |
| ZIP/Postal code |
4200-135 Porto |
| Country |
Portugal |
| |
|
| Platform ID |
GPL29624 |
| Series (1) |
| GSE165073 |
Absence of KpsM (Slr0977) impairs the secretion of extracellular polymeric substances (EPS) and impacts carbon fluxes in Synechocystis sp. PCC 6803 |
|
| Relations |
| BioSample |
SAMN17379532 |
| SRA |
SRX9895144 |
