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Sample GSM1362154

Query DataSets for GSM1362154

Status

Public on Apr 21, 2014

Title

Rery_PR4_Hex2

Sample type

SRA

Source name

bacterial cell, hexadecane

Organism

Rhodococcus erythropolis PR4

Characteristics

strain/background: PR4
growth condition: n-hexadecane
sample type: test

Growth protocol

Rhodococcus erythropolis PR4 cells were inoculated into 5L minimal medium (1L medium consist of 50mL 'A' solution: 4.35 g/L KH2PO4, 29.2 g/L K2HPO4, 50mL 'B' solution: 11.7g/L NaCl, 2.5g/L MgSO4 x 7 H2O, 0.88g/L CaCl2 x 2 H2O, 2mL 'D' solution: 0.1g/L ZnSO4 x 7 H2O, 0.03g/L MnCl2 x 4 H2O, 0.3g/L H3BO3, 0.2g/L CoCl2 x 6 H2O, 0.01g/L CuCl2 x 2 H2O, 0.02g/L NiCl2 x 6 H2O, 0.023g/L NaMoO4 x 4 H2O, 1mL 'E' solution: 14g FeSO4, 9.3g EDTA x 2 H2O, 10mL 'F' solution: 120g/L NH4NO3) in a Biostat C Fermenter (B. Braun Biotech International GmbH, Melsungen, Germany) in 4% (V/V). The medium was supplemented with 30 g/L sodium acetate, or 1% (V/V) n-hexadecane, or diesel oil as carbon and energy source. All media was also supplemented with 0.5g/L amino acid mix (26.3 mg/L of each amino acids except glycine) to accelerate cell growth in the early phase of the fermentation. Temperature was kept at 25°C, pH was set to 7.5 and maintained with the addition of 1M NaOH and HCL. Pure oxygen 4.5 (Linde Gas Hungary Co. Cltd., Budapest) was used for the aeration at a gas flow of 100mL/min. Oxygen saturation was kept at 20% and stirring was set to 200-360 rpm. Initial stir velocity was set to 100 rpm in the case of cultures grown on sodium acetate. Samples for RNA extraction were taken in the late exponential phase (after 21 hours in the case of cultures grown on n-hexadecane and diesel oil and 31 hours in the case of cultures grown on acetate).

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated with Qiagen RNeasy Mini Kit, followed by DNase I treatment using Invitrogen DNase I. RNA quality and quantity measurements were performed on Bioanalyzer (Agilent Technologies).
Libraries were processed according to the SOLiD transcriptome sequencing protocol (Publication Part Number 4452437). Briefly, total RNA was DNase I treated and rRNA-depleted using RiboMinus Plant Kit. After the fragmentation of RNA, the 50-200nt fraction was cleaned and was the subject of adaptor hybridization and reverse transcription. The cDNA library was cleaned with Qiagen MinElute PCR purification Kit and size-selected on a 6% TBE-Urea denaturing polyacrylamide gel. The 150-250nt cDNA fraction was amplified to generate sufficient template for SOLiD sequencing. To determine the accurate concentration of each library, a SOLiD Library TaqMan Quantitation Kit was used according to the manufacturer's instructions. After, each library was clonally amplified on SOLiD P1 DNA Beads by emulsion PCR (ePCR). ePCR beads were enriched for template-positive beads by hybridization with capture beads. Template-enriched beads were extended at the 3' end in the presence of terminal transferase and 3' bead linker. Approximately 60 million beads per sample with clonally amplified DNA were deposited onto one lane of the 5500 SOLiD sequencing flowchip. The flowchip was then loaded onto a SOLiD 5500xl Instrument and the 50-base sequences were obtained according to manufacturer's protocol.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

AB 5500xl Genetic Analyzer

Description

Hex2

Data processing

Basecalls performed using ABI SOLiD 5500xl.
Data were filtered using the following specifications: minimum number of nucleotides in reads = 50.
RNA reads were aligned to the NC_007486, NC_007487, NC_007491, NC_012490 genome assembly using CLC Genomics Workbench version 7 with the following configurations:
-- Use annotations for gene and transcript identification = Yes
-- References = NC_007486, NC_007487, NC_007491, NC_012490
-- Additional upstream bases = 0
-- Additional downstream bases = 0
-- Maximum number of mismatches allowed (applies to short reads) = 2
-- Unspecific match limit = 10
-- Use colorspace encoding = Yes
-- Use strand specific assembly = No
-- Organism type = PROKARYOTE
-- Minimum number of reads = 10
-- Create report = No
-- Expression level = Genes
-- Calculate RPKM for genes without transcripts = No
-- Expression value = Read Per Kilobase of exon Model value
-- Normalization method = Total
-- Counts reported as = reads per 1000000
-- Normalized data = Original expression values
Calculate expression levels using CLC Genomics Workbench version 7.0.
Genome_build: NC_007486.1, NC_007487.1, NC_007491.1, NC_012490.1
Supplementary_files_format_and_content: Excel file 'GSE56474_Datasets_Rery_1.xls' contains normalized expression data and the fold change values of 3 samples: R. erythropolis PR4 grown on acetate, n-hexadecane or diesel oil.
Supplementary_files_format_and_content: In the Excel file 'GSE56474_Full_Datasets_Rery.xls', the data was supplemented with a preliminary experiment marked as Hex2, where the sampling point was at the end of the exponential phase.

Submission date

Apr 02, 2014

Last update date

May 15, 2019

Contact name

Balazs Horvath

Organization name

SeqOmics Biotechnology Ltd.

Department

NGSP

Street address

Vallalkozok u. 7.