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Sample GSM4873501

Query DataSets for GSM4873501

Status

Public on Oct 29, 2021

Title

E. coli 042 wt (aggR3UTRFRT control)

Sample type

SRA

Source name

E. coli 042 wt

Organism

Escherichia coli 042

Characteristics

genotype: wild type

Growth protocol

LB medium at 37ºC with 200rpm agitation.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated from the cell pellets using the RNAsnap protocol developed by Stead et al. 2012. The total RNA preparations were examined by capillary electrophoresis. From the total RNA samples, ribosomal RNA molecules were depleted using the in-house developed depletion probes.
The ribodepleted RNAs were first fragmented using ultrasound (2 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' ends of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3’ adapter as primer. The first strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. For Illumina NextSeq sequencing, the cDNAs were pooled in approximately equimolar amounts. The cDNA pool was fractionated in the size range of 200 – 500 bp using a differential clean-up with the Agencourt AMPure kit. An aliquot of the size fractionated cDNA pool was analyzed by capillary electrophoresis. The cDNAs have a size range of 200 - 500 bp. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina. The cDNA pool was paired-end sequenced on an Illumina NextSeq 500 system using 75 bp read length.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

We used an Illumina NextSeq 500 system and a MID 150 Kit with 1x75 bp read length. Base-calling was perform online during the sequencing procedure with the Real-Time Analysis (RTA) software version 2.4.11 and System Suite Version 2.1.2.1.
Illumina sequencing instruments generate per-cycle BCL base call files as primary sequencing output in bcl2 format. Conversion of the bcl2 file to gzipped fastq files was performed using the bcl2fastq script v. 2.18.0.12 provided by Illumina.
Quality and adapter trimming was performed with the CLC Genomics Workbench 9.0 software package using the 'Trim Sequences' tool with standard parameters.
Mapping of the trimmed reads to the reference sequences was also performed with the CLC Genomics Workbench 9.0 using the 'Map Reads to Reference' tool with standard parameters.
For quantification of gene expression (read counting), the alignments generated with the Genomics Workbench were exported in BAM format. Read counting was then performed with the FeatureCounts v. 1.5.0-p1 program using the following parameters; Level : meta-feature leve. Paired-end : no. Strand specific : yes. Multimapping reads : counted (as fractions). Multi-overlapping reads : not counted. Overlapping bases : 30. Read orientations : fr
Genome_build: NCBI reference sequence (NC_017626 for the E. coli 042 chromosome) and (NC_017627 for the pAA plasmid).
Supplementary_files_format_and_content: The expression_1_FN554766_7_GEO.xlsx archive contains expression values in RPKM (Reads Per Kb exon (contig) per Million mapped reads ( see Mortazavi et al. 2008, Nat Methods. 5(7):621-8 ) and can be directly compared to each other) tab-delimited text files include RPKM values for each Sample.

Submission date

Oct 29, 2020

Last update date

Oct 29, 2021

Contact name

Mario Huttener Queiroz

Organization name

Universitat de Barcelona

Street address

Av. Diagonal 643

City

Barcelona