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Sample GSM7698576

Query DataSets for GSM7698576

Status

Public on Jun 24, 2024

Title

0 min, replicate 1, 3'-end-seq, wildtype

Sample type

SRA

Source name

BW25113

Organism

Escherichia coli BW25113

Characteristics

strain: BW25113
genotype: wildtype
treatment: None
seq method: 3' -end-seq

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.

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.

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

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

Submission date

Aug 09, 2023

Last update date

Jun 24, 2024

Contact name

Jintao Liu

E-mail(s)

jintaoliu@tsinghua.edu.cn

Organization name

Tsinghua University

Street address

Haidian District