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Sample GSM3402792

Query DataSets for GSM3402792

Status

Public on Sep 27, 2018

Title

Co-culture-2-Pectin

Sample type

SRA

Source name

Cultured cells

Organisms

Butyrivibrio proteoclasticus; Butyrivibrio hungatei

Characteristics

strain: MB2003 & B316T
growth phase: Stationary phase
growth environment: Anaerobic - Co-culture
growth substrate: Pectin

Treatment protocol

Bacterial cultures were snap-frozen with liquid N2 immediately upon collection and stored at -85 ºC.

Growth protocol

B. hungatei MB2003 and B. proteoclasticus B316T were cultured anaerobically under a CO2 atmosphere at 39 ºC in pre-warmed RM02 medium supplemented with 0.5% (w/v) cellobiose. Culture purity was verified via wet mounts and Gram staining. Cells were sub-cultured two times consecutively in triplicate with the respective growth media to ensure complete adaptation to the carbohydrate growth source. The optical density (OD) at 600 nm wavelength was monitored using an Ultrospec 1100 Pro Spectrophotometer (GE Healthcare) over a period of 24 h. At mid-log phase of growth (OD600nm = 0.4), cell counts of each mono-culture were quantified using a Petroff-Hausser chamber (Hausser Scientific) to the manufacturer’s instructions. Cell density-equilibrated cultures were sub-cultured into 70 mL pre-warmed RM02 media with 0.5% (v/v) inoculum and supplemented with either 0.5% (w/v) of xylan from oat spelts (Sigma-Aldrich) or pectin isolated from apple (Sigma-Aldrich) as the main carbohydrate source. For the mono-cultures, B316T or MB2003 were inoculated separately into the media and for the co-culture samples, 0.25% (v/v) inocula of each equilibrated culture was added. Inoculations were done in triplicate per organism (or co-culture) and harvested after 12 h of growth.

Extracted molecule

total RNA

Extraction protocol

RNA was extracted using a modified version of a liquid N2 and grinding method. Briefly, 10 g of each frozen culture sample was combined with two volumes of RNA Bacterial Protect Reagent (Qiagen). The thawed sample was centrifuged at 15,000 × g for 10 min at 4 ºC and cell pellet resuspended in 1 mL of Extraction buffer A (200 mM NaCl, 20 mM EDTA), 420 µL of 20% (w/v) SDS and 1 mL of a mixture of phenol: chloroform: isoamyl alcohol solution. The cells were disrupted by five rounds of bead-beating using a Mini-Beadbeater-96 (BioSpec) for 4 min at full speed. An equal volume of isopropanol and 0.1 volume of 3 M sodium acetate (pH 5.5) were added, gently mixed and stored at -20 ºC overnight (54). The cell pellets were ethanol precipitated, followed by DNase treatment using the Baseline-ZERO DNase (Epicentre Technologies) kit and RNA was purified using the MEGAClear kit (Thermo Fisher Scientific), based on the manufacturer’s instructions. RNA yield and quality were assessed using the Bioanalyzer 2100 with a RNA 6000 Nano Assay reagent kit from Agilent (Santa Clara, CA), and stored at -85 ºC. RNA was sequenced on the Illumina HiSeq 2000 platform at the Beijing Genomics Institute, BGI (Shenzen, China).
RNA libraries were prepared for sequencing using standard Illumina protocols.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

CP017831_Pectin_transcripts.txt
CP017830_Pectin_transcripts.txt
CP017832_Pectin_transcripts.txt
CP017833_Pectin_transcripts.txt
NC_014387_Pectin_transcripts.txt
NC_014388_Pectin_transcripts.txt
NC_014389_Pectin_transcripts.txt
NC_014390_Pectin_transcripts.txt

Data processing

Sequence data from BGI had been filtered using DynamicTrim (Cox MP, Peterson DA, Biggs PJ. 2010) to remove reads containing: ≥ 10% unreadable bases, ≥ 20% low quality (≥ Q20) bases, any reads with a quality score ≤ Q28, adapter contamination or duplicate read pairs, and quality of the sequence data before analysis was assessed using FastQC (Andrews S, 2010).
Bowtie 2 (Langmead B & Salzberg SL, 2012) was used with default parameters, to remove any sequence reads aligning to ribosomal RNA, transfer RNA and non-coding RNA sequences.
Reference-based transcriptome assembly and differential expression between mono- and co-culture growth of B. hungatei MB2003 and B. proteoclasticus B316T on xylan and pectin conditions separately was performed using Rockhopper (Tjaden B. 2015) with default parameters.
Genome_build: CP017831
Genome_build: CP017830
Genome_build: CP017832
Genome_build: CP017833
Genome_build: CP001810
Genome_build: CP001811
Genome_build: CP001812
Genome_build: CP001813
Supplementary_files_format_and_content: Tab-delimited text files include RPKM values for each Sample

Submission date

Sep 26, 2018

Last update date

Sep 27, 2018

Contact name

Nikola Palevich

E-mail(s)

nik.palevich@agresearch.co.nz

Phone

021972511

Organization name

AgResearch