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| Status |
Public on Jul 09, 2021 |
| Title |
M5a1 WT Nfix rep2 |
| Sample type |
SRA |
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| Source name |
bacterial cells_nitrogen starved
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| Organism |
Klebsiella oxytoca |
| Characteristics |
strain: M5aI
medium: NFDM + 0.5 mM NH4Cl
od600nm: 0.57
illumina truseq i5 adapter barcode: GCCTCTAT
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| Growth protocol |
M5aI cells were cultured in 100 mL Nitrogen-Free David and Mingioli (NFDM) medium [69 mM K2HPO4, 25 mM KH2PO4, 0.1 mM Na2MoO4, 90 μM FeSO4, 0.8 mM MgSO4, 2% w/v glucose] supplemented with NH4Cl (Nfix=0.5 mM; Nrich=10 mM) as a nitrogen source from an initial OD600 of 0.1. Culture vessels were sealed and flushed with N2 gas for 45 minutes to initiate micro-aerobic conditions, prior to incubation at 25°C and 200 rpm. Cultures were cultured for a further 3 hours following exhaustion of NH4 in the supernatant, measured by colorimetric assay.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cell cultures were fixed with a 1/10 volume of 5% phenol/95% ethanol (v/v), harvested by centrifugation at 4000 x g and stored at -80°C. Cell pellet samples were shipped on ice to Vertis Biotechnologie AG (Freising, Germany) for RNA extraction and sample processing.Total RNA was isolated via lysozyme lysis and the mirVana microRNA isolation kit (Ambion). DNase was used to specifically remove contaminant DNA and ribosomal RNA was depleted using the Ribo-Zero rRNA Removal Kit for bacteria (Illumina).
The primary RNA transcripts were fragmented using ultrasound (4 pulses of 30 s each at 4°C) prior to ligation of 3’ oligonucleotide adapters. First-strand cDNA synthesis via Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase was primed using the 3’ adapter. Following purification of first-strand cDNA, a 5' Illumina TruSeq sequencing adapter (Illumina), inclusive of an 8 nt barcode sequence, was ligated to the 3' end of the antisense cDNA prior to PCR-amplification of double-stranded cDNA to about 10-20 ng/μl (12-13 thermocycles) using a high fidelity DNA polymerase. cDNA was purified, pooled in equimolar quantities and size-fractionated (200-500 bp) using the Agencourt AMPure XP kit (Beckman Coulter Genomics) prior to analysis by capillary electrophoresis. cDNA pools were quality checked on a Shimadzu MultiNA microchip electrophoresis system prior to strand-specific sequencing on an Illumina NextSeq 500 system, utilising a read length of 75 nt and a depth of 100 million reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
M5a1-fix-B_S2
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| Data processing |
Sequencing reads were trimmed to remove adapter sequences and filtered to remove duplicates
Reads were mapped to the ICL_KoM5aI template sequence (GenBank WGS accession: JAFHKG010000000; BioSample: SAMN17288411), resulting in binary alignment map (bam) files and a matrix of normalised gene counts (reads per kilobase per million; RPKM)
Differential gene expression analysis was performed using the R/Bioconductor statistical package DEseq2 (version 1.22.2), utilising settings of regularized logarithm (rlog) normalisation of read counts, Wald hypothesis testing and p-value adjustment using a False Discovery Rate threshold of 0.05. Log2 fold changes between conditions were calculated for differentially expressed genes (DEGs) with an adjusted p value of <0.05
Genome_build: Klebsiella oxytoca M5aI (GenBank WGS accession: JAFHKG010000000; BioSample: SAMN17288411)
Supplementary_files_format_and_content: M5aI_NfixNrich_processed_RPKM_DESeq.xls [xls file containing two tabs: Expression_matrix=a matrix of total mapped reads and RPKM values for all gene ids and samples; DESeq_DEGs=all differentially expressed genes (where FDR adjusted p-values <0.05) in Nfix samples compared to Nrich control, with log2 fold changes]; .bam files [Binary Alignment Map (BAM) for each sample. Read alignment to the ICL_KoM5aI template sequence (GenBank WGS accession: JAFHKG010000000; BioSample SAMN17288411)]
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| Submission date |
Jan 12, 2021 |
| Last update date |
Jul 09, 2021 |
| Contact name |
Martin Buck |
| Organization name |
Imperial College London
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| Department |
Life Sciences
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| Lab |
Sir Alexander Fleming Building
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| Street address |
Imperial College Road, South Kensington
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| City |
London |
| ZIP/Postal code |
SW7 2AZ |
| Country |
United Kingdom |
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| Platform ID |
GPL29593 |
| Series (1) |
| GSE164668 |
The transcriptome of Klebsiella oxytoca M5a1 during onset of nitrogen fixation |
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| Relations |
| BioSample |
SAMN17294105 |
| SRA |
SRX9833005 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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