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| Status |
Public on Jun 16, 2020 |
| Title |
EC_0_3 |
| Sample type |
SRA |
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| Source name |
E. coli K12 strain MG1655
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| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
treatment: untreated
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| Treatment protocol |
treated with 2 μg/mL ciprofloxacin for 20min
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| Growth protocol |
E. coli K12 strain MG1655 was grown in 250 mL flasks with Luria-Bertani (LB) medium in a shaking incubator at 37 ℃ and 250 rpm overnight.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolation from E. coli cells was performed using the Qiagen RNA protect bacterial reagent (Qiagen, Hilden, Germany) and purification was conducted using Qiagen RNeasy Plus Mini Kit (Qiagen) following the manufacturer’s instruction. After assessment of RNA purity and integrity, RNA samples were treated using Epicentre Ribo-zero rRNA Removal Kit (Epicentre, Madison, WI, USA), followed by RNA fragmentation, synthesis of the first and second strands DNA, and appendence of sequencing adapter. Then the selected fragments by Agencourt AMPure XP Beads (Illumina Inc , San Diego, CA, USA) were amplified, followed by library purification, enrichment and quality determination. Accordingly, 6 cDNA libraries (3 from ciprofloxacin-treated E. coli, and 3 from non-ciprofloxacin-treated E. coli) were constructed and subjected to Illumina Hiseq 2500 sequencing platform.
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 2500 |
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| Data processing |
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to NC_000913.3 whole genome using Bowtie2-2.2.3 with parameters -p 8 -x index -1reads1.fq -2 reads2.fq -S out.sam
HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, 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.
(For DESeq with biological replicates) Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq R package (1.18.0). Differential expression analysis of two conditions was performed using the DEGSeq R package (1.20.0).
Picard-tools v1.96 and samtools v0.1.18 were used to sort, mark duplicated reads and reorder the bam alignment results of each sample. GATK2 software was used to perform SNP calling
Genome_build: NC_000913.3
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for all Samples
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| Submission date |
Jun 15, 2020 |
| Last update date |
Jun 16, 2020 |
| Contact name |
Rui Sui |
| E-mail(s) |
sunruibba@126.com
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| Phone |
+8615245068743
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| Organization name |
Harbin medical University
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| Department |
Microbiology
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| Street address |
Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Nangang District, Harbin, China, 150086
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| City |
Harbin |
| ZIP/Postal code |
150086 |
| Country |
China |
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| Platform ID |
GPL18956 |
| Series (1) |
| GSE152445 |
Genome-wide screening and characterization of genes involved in response to high dose of ciprofloxacin in Escherichia coli |
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| Relations |
| BioSample |
SAMN15236475 |
| SRA |
SRX8545691 |
| 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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