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Sample GSM2190877

Query DataSets for GSM2190877

Status

Public on Nov 15, 2016

Title

18.2A

Sample type

SRA

Source name

18.2A_Earth_75

Organism

Escherichia coli

Characteristics

strain: ATCC 4157
location: Earth
gentamycin concentration (ug/ml): 75

Treatment protocol

Nineteen hours after experiment start, antibiotic (Gentamicin Sulfate (MP Biomedical, Cat No. 1676045, Santa Ana, CA, USA)) was introducing into the cultures. Antibiotic concentration varied from one sample to the next from 0 to 150 ug/mL in 25 ug/mL steps. Thirty hours later, samples were fixed in RNA Later II ((ACROS, Cat. No. 41678, New Jersey, USA)). Samples were stored at -75C until return to Earth for analyses.

Growth protocol

Samples were sent to the International Space Station at 4C in FPAs, as described above. Temperature was increased to 37C and 23 hours later, the experiment was started by introducing the inoculum (E. coli ATCC 4157) into the growth medium (Medium E as described in Vogel & Bonner (1956) supplemented with 5 g/L glucose), yielding 3.25 mL of a culture with a starting cell concentration of 1.22e6 cell/mL.

Extracted molecule

total RNA

Extraction protocol

Four ml of the bacterial suspension in RNALater II and Medium E growth medium were spun down at 200g for 5 min. The supernatant was discarded and the pellet were resuspended in 200ul PBS and mixed by pipetting. 100ul aliquots were taken out for DNA and RNA extraction from the same sample. RNA extraction was done using Qiagen RNeasy mini kit (Qiagen, Hilden, Germany) with on-column DNase digestion. Final elution was made in 30 μL dH2O. The concentration and integrity of the total RNA was estimated by Quant-iT™ RiboGreen® RNA Assay Kit (ThermoFisher Scientific, Waltham, MA), and Agilent 2100 Bioanalyzer (Applied Biosystems, Carlsbad, CA), respectively.
Ribosomal RNA (rRNA) was removed using Ribo-Zero™ Gold (Yeast) kit (Epicenter, Madison, WI) using manufacturer's recommended protocol. Immediately after the rRNA removal the RNA was fragmented and primed for the first strand synthesis using the NEBnext First Strand synthesis module (New England BioLabs Inc., Ipswich, MA). Directional second strand synthesis was performed using NEBNExt Ultra Directional second strand synthesis kit. Following this the samples were taken into standard library preparation protocol using NEBNext® DNA Library Prep Master Mix Set for Illumina® with slight modifications. Briefly, end-repair was done followed by polyA addition and adapter ligation. Post-ligated materials were individually barcoded with primers and amplified through 12 cycles of PCR. Final library quantity was assessed by Quant-iT™ PicoGreen® dsDNA Assay Kit and the library quality was estimated on LabChip® GX (Caliper, PerkinElmer, Waltham, MA). Accurate quantification of the final libraries for sequencing applications was determined using the qPCR-based KAPA Biosystems Library Quantification kit (Kapa Biosystems, Inc., Woburn, MA). Each library was diluted to a final concentration of 12.5 nM and pooled equimolar prior to clustering. Paired-End (PE) sequencing was performed on an Illumina HiSeq2500 sequencer (Illumina, Inc.).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

SL83402

Data processing

Post-processing of the sequencing reads from RNA-seq experiments for each sample was performed using HudsonAlpha’s unique in-house RNA-seq data analysis pipeline. Briefly, quality control checks on raw sequence data for each sample were performed using FastQC (Babraham Bioinformatics, Cambridge, UK). Data-analysis was performed using the CLC Genomics Workbench (Version 7.5.1, CLC Bio, Aarhus, Denmark). The reference genome Escherichia coli (DH10B) sequence was downloaded from the UCSC genome browser. For read mapping, the following parameters were used: mismatch cost = 2, insertion and deletion cost = 3, length fraction: 0.8, similarity fraction = 0.8, global alignment = no, auto-detect paired distances = yes. Samples were grouped and differential expression of genes was calculated on the basis of fold changes (using the default cut-off ≥ ±2.0) observed in comparisons between defined conditions.
Genome_build: Escherichia coli (DH10B)
Supplementary_files_format_and_content: excel file include RPKM values for each Sample

Submission date

Jun 07, 2016

Last update date

May 15, 2019

Contact name

Luis Zea

E-mail(s)

Luis.Zea@colorado.edu

Organization name

University of Colorado Boulder

Street address

429 UCB ECAE 1B02