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Sample GSM2794753

Query DataSets for GSM2794753

Status

Public on Oct 27, 2024

Title

pACYC177 plasmid / no insert rep 3

Sample type

SRA

Source name

pACYC177 plasmid / no insert rep 3

Organism

Escherichia coli str. K-12 substr. MG1655

Characteristics

strain: K-12 MG1655
vector backbone: pACYC177
insert: N/A

Growth protocol

MOPS minimal medium (Neidhardt et al., 1974) plates (0.6% glycerol, 0.2% casamino acids, 100 μg/ml ampicillin) were streaked with E. coli MG1655 (Yale CGSC # 6300; F-, λ-, rph-1) cells bearing one of the pACYC177-derived plasmids and grown at 37°C (Table S1). Glycerol and casamino acids, rather than glucose, were added to minimize catabolite repression while avoiding amino acid starvation. Overnight cultures were prepared by inoculating 2.5 ml of MOPS medium, with 1 mM IPTG, from a plate, and grown overnight, shaking at 37°C at 250 RPM. Under these conditions, cells typically grow with a doubling time of 50 min.
200 ml of minimal medium medium, in pre-warmed 2 L flasks, were inoculated by diluting the overnight cultures more than one thousandfold and set to shake at 37°C in an Innova 40 incubator shaker (New Brunswick Scientific) at 200 RPM.

Extracted molecule

total RNA

Extraction protocol

Once the cultures reached an OD600 of 0.40 - 0.65 (measured using a Shimadzu UV-1800 spectrophotometer; 1 OD600 = 6 x 108 cells / mL) they were filtered through a pre-warmed 0.22 μm nitrocellulose filter paper and 90 mm glass filtration system (Kontes) with vacuum (Whatman cat. no. 7182-009); the filtration took about 1 min. The apparatus was rapidly disassembled, and the cells were immediately scraped from the filter paper, using a pre-warmed scoopula, and submerged into a 50 ml Falcon tube (Corning cat. no. 352070) filled with liquid nitrogen (LN2). The cells were then transferred to a mortar immersed in, and containing, LN2 and 600 μl of cell lysis buffer (10 mM MgCl2, 100 mM NH4Cl, 20 mM Tris-HCl pH 8.0, 0.1% Nonidet P40, 0.4% Triton X-100, 500 μg/ml chloramphenicol) were dripped in. The cells were pulverized with the chilled mortar and pestle, whereupon the LN2 was allowed to boil off. The remaining powder was scraped with a chilled spatula into a chilled 1.5 ml Eppendorf tube and stored at -80°C overnight.
Cell lysates were centrifuged 15 min at 14,000 RPM and 4°C, and the clarified supernatant was transferred to a 1.5 ml Eppendorf tube. Between 250 μl and 500 μl of supernatant were then treated with 2.0 μl of micrococcal nuclease (Life Technologies cat. no. EN0181) and 5 mM CaCl2 and incubated 1 h while gently rotating at room temperature. The reactions were quenched using 6 mM EGTA and then transferred to 3.5 ml thickwall polycarbonate ultracentrifuge tubes (Beckman Coulter cat. no. 349622). The samples were underlaid with 2.0 ml of 1 M sucrose and centrifuged 6.5 h on a Beckman XL-80K ultracentrifuge at 55,000 RPM and 4°C in a SW55 rotor. Following removal of the liquid, the invisible pellets were re-suspended with 700 μl of Qiazol reagent, and RNA was extracted as directed by the miRNEasy kit (Qiagen cat. no. 217004).
Samples were denatured at 70°C and then subjected to electrophoresis on a 15% polyacrylamide TBE-urea gel 65 min at 200 V. The gel was then stained using SYBR gold and imaged with a blue light transilluminator. Gel slices were taken from the regions corresponding to lengths between 20 to 45 nt, as indicated by 10 bp ladder (Invitrogen cat. no. 10821015) and RNA size markers (5′-AUGUACACGGAGUCGAGCUCAACCCGCAACGCGA-(Phos)-3′ and 5′-AUGUACACGGAGUCGACCCAACGCGA-(Phos)-3′). The samples were then prepared as directed in Ingolia et al. (Ingolia et al., 2012), with modifications: ProtoScript II reverse transcriptase (New England Biolabs cat no. M0368) was used for reverse transcription, and different biotinylated oligonucleotides were used for the removal of E. coli rRNA sequences (Li et al., 2014). The library was sequenced on an Illumina HiSeq 2500 by the Johns Hopkins University Genetic Resources Core Facility. Each bacterial strain was processed in duplicate or triplicate.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

Replicate 3
RNA treated with MNase
C_3

Data processing

Library strategy: Ribo-Seq
fastx_clipper 0.0.13.2 to remove the adapter from the end with parameters: -Q33 -a CTGTAGGCACCATCAAT -l 21 -c -n -v -i input_file -o trimmed.fq
rRNA and tRNA sequences were removed using bowtie2 2.2.9 with parameters: bowtie2 -x rrna_db -U trimmed.fq --very-sensitive -t --quiet -p 8 --un filtered.fq > /dev/null
Reads were mapped to the E. coli genome (NC_000913.3) using bowtie2 2.2.9 with parameters: bowtie2 -x genome_db -U filtered.fq -S aligned.sam -D 20 -R 3 -N 1 -L 20 -i S,1,0.5 -p 8 --no-hd -t -a --un unaligned.fq
Procedure, written in Perl, which corrects sequencing errors and merges identical reads (Martens et al., 2015). Using the list of proteins and their corresponding genomic map points from PTT files, each gene was scanned for mapped reads and the totals were recorded to a new file. Reads mapped to tufA and tufB were merged, and an additional scan was performed to assign reads to ssrA.
Differential gene expression analysis was performed using DeSeq2 1.12.3 and R 3.3.1 (Love et al., 2014).
Genome_build: E. coli genome: NC_00913.3
Genome_build: Additional files for pACYC177 vectors (3 genes: ampR (bla), Luc (optional), kanR)
Supplementary_files_format_and_content: Integer sequencing reads assigned to each genomic feature, formatted as a matrix of values, with rows corresponding to genomic features and columns corresponding to experiments

Submission date

Sep 27, 2017

Last update date

Oct 27, 2024

Contact name

Vincent J Hilser

E-mail(s)

[email protected]

Phone

410-516-6072

Organization name

The Johns Hopkins University