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Sample GSM2879508

Query DataSets for GSM2879508

Status

Public on Dec 20, 2017

Title

T60-Xyl-2

Sample type

SRA

Source name

cell culture

Organism

Clostridium acetobutylicum ATCC 824

Characteristics

media: CGM + 0.5% xylose
added sugar: 0.25% xylose
time (min): 60
replicate: 2

Treatment protocol

All cultures were incubated at 37°C under anaerobic conditions open to the environment of the anaerobe chamber, without shaking. Spores were heat shocked at 80°C for 10 minutes and inoculated into CGM supplemented with 0.5% xylose. Starter cultures were subcultured into 0.5% xylose CGM and incubated overnight. At OD600 = 0.2 the culture was aliquoted into milk dilutions bottles (70 mL each; four conditions in triplicate independent biological replicates ) and incubated until OD600 = 0.4. Pre-carbohydrate supplementation RNA and metabolite analysis samples were collected. Cultures were then supplemented with arabinose (0.25% final), glucose (0.25% final), or xylose (0.25% final), from a 50% stock solution of the respective sugars in water, or equal volume of water (control). RNA, metabolites, and OD600 readings were taken at 0 min, 15 min, 30 min, and 60 min. Cultures were monitored for an additional 9 hours and metabolites and OD600 reading were taken every hour. Final OD600 readings and metabolite samples were taken the following day at 21 hours post supplemental sugar addition. Samples collected for RNA isolation were immediately incubated with 0.03 mg/mL rifampicin (final concentration) on ice, pelleted and treated with RNA Protect (Qiagen, Valencia, CA) according to the manufacturer’s protocol, and stored at -80°C until RNA extraction.

Growth protocol

Clostridium acetobutylicum ATCC 824 was utilized for all studies. All growth was conducted anaerobically in a Coy anaerobic chamber (Coy Lab Products) at 37°C in an atmosphere containing 5% H2, 5% CO2, and 90% N2. Cells were maintained as spore suspensions in potato glucose medium (PGM) containing, per liter: 150 g potato (shredded, boiled 1 hour, and filtered through cheesecloth), 1% glucose, 30 mM CaCO3, and 4 mM (NH4)2SO4. Experimental cultures were grown in clostridium growth medium (CGM) containing, per liter: 0.5% yeast extract, 15 mM (NH4)2SO4, 15 mM L-Asparagine, 17 mM NaCl, 5 mM KH2(PO4), 3 mM Mg(SO4)*H2O, 0.7 mM Mn(SO4)*H2O, 0.4 mM Fe(SO4)*7H2O.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated using the miRNeasy Mini Kit (Qiagen; 217004) according to the manufacturer’s protocol, with an additional homogenization and mechanical disruption step using a bead beater (BioSpec, Bartlesville, OK, USA) with Zirconia/Silica beads (BioSpec; 11079101z). RNA quality was assessed using the 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) and quantified using a spectrophotometer (DeNovix, Wilmington, DE, USA) and was stored at -80°C until DNase treatment. DNA was removed using the TURBO DNA-free kit (Life Technologies; AM1907) according to the manufacturer’s protocol. RNA was quantified and quality assessed, as stated above, and genomic DNA depletion was confirmed using qPCR with 16S rRNA gene primers [19] and iQ SYBR Green Supermix (Bio-Rad; 170-8882) according to the manufacturer’s instructions. Ribosomal RNA was removed using the Ribo-Zero rRNA Removal Kit (Gram-Positive Bacteria) (Illumina; MRZGP126) according to the manufacturer’s protocol. The quality of rRNA depleted samples was assessed using a 2100 BioAnalyzer prior to processing for sequencing library generation.
TruSeq Stranded mRNA Sample Preparation Kit (Illumina; RS-122-2101) was used to prepare the rRNA depleted RNA for sequencing according to the manufacturer’s protocol. Libraries were quantified using the Kapa Library Quantification Kit (KapaBiosystems; KK4854) according to the manufacturer’s instructions, then normalized and pooled for sequencing according to the Denature and Dilute Libraries Guide for the NextSeq 500 (Illumina; Doc No – 15048776 v02).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

Data processing

Pooled libraries were 1 x 75 bp sequenced on a NextSeq 500 (Illumina, San Diego, CA, USA) in two sequencing runs. Tpre and T15 in one run. T30 and T60 in another run.
Base calling with RTA version 2.4.11
Concatenate and compress reads from different lanes with zcat and gzip
Quality filter and trim reads with Trimmomatic-0.36 (parameters: ILLUMINACLIP:Trimmomatic-0.36/adapters/TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36)
Reverse complement reads with FASTX toolkit version 0.0.13 (fastx_reverse_complement -Q33)
Determine gene expression levels with EDGE-pro version 1.3.1
Genome_build: GCF_000008765.1_ASM876v1
Supplementary_files_format_and_content: Concatenate mapped read counts for all genes and samples into table (pentose_hierarchy_EdgePro_Reads_table.csv)
Supplementary_files_format_and_content: Differential expression testing with DESeq2 version 1.10.1 in R version 3.4.0 (default parameters, rlog transformed counts in: pentose_hierarchy_EdgePro_rlog_Reads_table.csv)
Supplementary_files_format_and_content: RPKM values calculated using R (pentose_hierarchy_EdgePro_RPKM_table.csv)

Submission date

Dec 07, 2017

Last update date

May 15, 2019

Contact name

Matthew Perisin

Organization name

US Army Research Laboratory

Department

Biotechnology Branch

Street address

2800 Powder Mill Rd