GSE95575-GSM2516627-GPL15010-PMID:29338696.tsv 5.45 KB
"treatment: MG1655 + 3µM PMA at t30"	"characteristics_ch1.1"
"Quality control processing of sequence data was performed using Galaxy (https://galaxyproject.org). The FASTX tools in Galaxy (http://hannonlab.cshl.edu/fastx_toolkit) were used for filtering by quality (80% of sequence > quality score of 20), then reads were trimmed at both 5’ and 3’ ends using a window and step size of 1 with quality score > 20."	"data_processing.1"
"Forward- and reverse-read mate-pairs were assembled and aligned to the Escherichia coli MG1655 K-12 genome using Bowtie2 (Langmead and Salzberg, 2012, Nat Methods). SAMtools (Li et al., 2009, Bioinformatics) was used to convert Bowtie2 output (.bam file) to SAM format."	"data_processing.2"
"The number of sequence reads that aligned to features in annotation file (Escherichia_coli_str_k_12_substr_mg1655.GCA_000005845.2.24.gtf from http://bacteria.ensembl.org) were tabulated from the resulting SAM alignment files using the HTSeq-count program (Anders et al., 2015, Bioinformatics) with intersection non-empty mode."	"data_processing.3"
"Mapped read counts were analyzed for differential expression (false discovery rate of < 0.01, fold-change > 2) using the baySeq package in R (Hardcastle and Kelly, 2010, BMC Bioinformatics). Within baySeq, two-way comparisons using quantile normalization were made for all three biological replicate transcriptomes over time for HgCl2 exposure or PMA exposure versus the unexposed control."	"data_processing.4"
"Genome_build: ASM584v2 (Escherichia_coli_str_k_12_substr_mg1655.GCA_000005845.2.24.gtf )"	"data_processing.5"
"Supplementary_files_format_and_content: raw read counts as determined by HTseq-Count, provided in .csv format"	"data_processing.6"
"One cell pellet from each condition and sampling time was thawed on ice; total RNA was isolated by RNAsnap™ (Stead et al., 2012, Nucleic Acids Res). DNA contamination was removed by two treatments with Turbo-DNase (Ambion; Life Technologies). Ribosomal RNA depletion was performed with the Ribo-Zero™ rRNA removal kit for Gram-negative bacteria (Epicentre) and concentrated using RNA Clean and Concentrator™ -5 columns (Zymo Research) following the manufacturer’s instructions."	"extract_protocol_ch1.1"
"The quality and quantity of rRNA-depleted RNA was assessed on a 2100 Bioanalyzer RNA pico chip (Agilent Technologies) using manufacturer’s recommendations. Next generation sequencing (NGS) libraries were prepared using the Kapa biosystems NGS stranded library prep kit for RNA-Seq with dual indexed Illumina adapters. The mode library insert size was ~150 bp, as determined by high sensitivity NGS fragment analysis kit for Fragment Analyzer™ (Advanced Analytical Technologies) using manufacturer’s instructions. Quantification of each library was done by qPCR and all 30 libraries were pooled in equal concentrations."	"extract_protocol_ch1.2"
"For each biological RNA-Seq replicate E coli K-12 MG1655 was subcultured from cyrostorage on Luria-Bertani (LB) agar overnight at 37°C. A half-dozen well-isolated colonies were used to inoculate a 20 ml starter culture in Neidhardt MOPS Minimal Medium (NM3) (Neidhardt et al., 1974, J Bacteriol) (0.2% final glucose concentration) supplemented with 20 mg/L uracil and 500 µg/L thiamine, which was incubated at 37°C with shaking at 250 rpm overnight (~18 hr). The overnight starter culture was diluted 1:30 to initiate the experimental culture and divided into three 500 ml flasks with 100 ml NM3 in each, which were incubated at 37°C with shaking at 250 rpm."	"growth_protocol_ch1.1"
"Escherichia coli str. K-12 substr. MG1655"	"organism_ch1.1"
"E. coli"	"source_name_ch1.1"
"9B_MG+PMA_t30"	"title.1"
"When cultures reached OD595 ≈ 0.470 (~ 200 min), two cultures were made 3 µM mercuric chloride (HgCl2) or 3 µM phenylmercuric-acetate (PMA) and the third was left as an unexposed control. Duplicate 1 ml aliquots of each culture were collected at 0 (unexposed control only), 10, 30, 60 min after mercurial exposure and immediately centrifuged at 21 krpm, for 3 min at 4°C. Spent medium was aspirated and cell pellets were frozen at -70°C within 5 min after collection."	"treatment_protocol_ch1.1"
"RNA-Seq"	"library_strategy.1"
"treatment: MG1655 + 3µM PMA at t30"	"characteristics_ch1.1"
"For each biological RNA-Seq replicate E coli K-12 MG1655 was subcultured from cyrostorage on Luria-Bertani (LB) agar overnight at 37°C. A half-dozen well-isolated colonies were used to inoculate a 20 ml starter culture in Neidhardt MOPS Minimal Medium (NM3) (Neidhardt et al., 1974, J Bacteriol) (0.2% final glucose concentration) supplemented with 20 mg/L uracil and 500 µg/L thiamine, which was incubated at 37°C with shaking at 250 rpm overnight (~18 hr). The overnight starter culture was diluted 1:30 to initiate the experimental culture and divided into three 500 ml flasks with 100 ml NM3 in each, which were incubated at 37°C with shaking at 250 rpm."	"growth_protocol_ch1.1"
"Escherichia coli str. K-12 substr. MG1655"	"organism_ch1.1"
"E. coli"	"source_name_ch1.1"
"When cultures reached OD595 ≈ 0.470 (~ 200 min), two cultures were made 3 µM mercuric chloride (HgCl2) or 3 µM phenylmercuric-acetate (PMA) and the third was left as an unexposed control. Duplicate 1 ml aliquots of each culture were collected at 0 (unexposed control only), 10, 30, 60 min after mercurial exposure and immediately centrifuged at 21 krpm, for 3 min at 4°C. Spent medium was aspirated and cell pellets were frozen at -70°C within 5 min after collection."	"treatment_protocol_ch1.1"