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Status |
Public on Jun 24, 2024 |
Title |
Gentamicin, 60 min, 3'-end-seq, wildtype |
Sample type |
SRA |
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Source name |
BW25113
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Organism |
Escherichia coli BW25113 |
Characteristics |
strain: BW25113 genotype: wildtype treatment: 6 mg/L, gentamicin seq method: 3' -end-seq
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Treatment protocol |
Overnight seed cultures were diluted by 1:350 into 700 μL of fresh medium and grown in a 48-well plate in a microplate reader (Tecan, Spark). When the cultures grew to OD600 ~ 0.2, we carried out another dilution of 1:50 into fresh medium and cultivated with the same condition. When subcultures reached an OD600 of ~ 0.25, 10 μL gentamicin (Selleck, S4030) was supplemented to make a final concentration of 6 mg/L.
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Growth protocol |
In all experiments, E. coli K12 BW25113 was used (originally from CICC, 23872) as the wildtype strain. Single colony was picked up from a stock LB agar plate and was grown aerobically with shaking at 220 rpm in Luria-Bertani (LB) broth (10 g/L tryptone (Oxoid, LP0042), 10 g/L NaCl (Macklin, S805275), 5 g/L yeast extract (Oxoid, LP0021)) medium overnight (typically 12 h) as the seed culture. All cultures were carried out at 37 °C with shaking at 200 rpm.
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Extracted molecule |
total RNA |
Extraction protocol |
For RNA exactraction, bacteria lysate was prepared using lysozyme and proteinase K. Total RNA was then purified by RNAClean XP beads (Beckman, A63987). For genomic DNA, TIANamp Bacteria DNA kit (TIANGEN, DP302) were used. See decrisption in our paper for details. 3' -end-seq library preparation method was adopted from a previous multiplexed RNA-seq library preparation method reported by Avraham et al. Systematic modifications were introduced to repurpose it to keep 3’ end information of bacterial transcripts. To prepare RNA-seq library, here we did not follow routine RNA-seq library construction method, in order to minimize the batch effect introduced when applying different library preparation protocols.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Data processing |
For 3’ -end-seq, firstly, sequencing raw data was subjected to FastQC (v0.11.9) for quality control and cutadapt (2.10) for adaptor trimming. In detail, fastqc was used with default parameters, cutadapt command was used with parameters -q 10,10 --minimum-length 100:100 --max-n 3 --pair-filter=any -o. The data was then demultiplexed by cutadapt based on the unique RNA adaptors. Paired-end reads were aligned to E. coli reference genome (NC_000913.3) by bowtie2 (2.4.1) with parameters -X 1000 -I 18 --no-mixed --no-discordant. The mapped reads were converted to bam files by samtools (1.10). To minimize the effect of sequencing depth on further analysis, we subsampled every bam file to the equal number of 4.5 million paired-end reads. Reads were counted for bam files using R (4.0.2) method summarizeOverlaps from GenomicAlignments package (v1.26.0) in a strand specific manner, with the following options “mode = "Union", ignore.strand = FALSE, singleEnd = FALSE, preprocess.reads = invertStrand”. For RNA-seq data, data processing was similar to our previous report (Wang et al., Environmental Microbiology 2022). Adaptor trimming, alignment to genome and gene read count table were all the same with 3’-end-seq as shown above. Assembly: NC000913.3 Supplementary files format and content: comma-delimited text file for expression matrix Supplementary files format and content: xlsx file for 3' -end-seq CPM of PRSEs
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Submission date |
Aug 09, 2023 |
Last update date |
Jun 24, 2024 |
Contact name |
Jintao Liu |
E-mail(s) |
jintaoliu@tsinghua.edu.cn
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Organization name |
Tsinghua University
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Street address |
Haidian District
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City |
Beijing |
ZIP/Postal code |
100084 |
Country |
China |
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Platform ID |
GPL27123 |
Series (1) |
GSE240474 |
RNA polymerase stalling-derived genome instability underlies ribosomal antibiotic efficacy and resistance evolution |
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Relations |
BioSample |
SAMN36909002 |
SRA |
SRX21311719 |
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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