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| Status |
Public on Jul 18, 2014 |
| Title |
SynH_LT_Exp_CCOG |
| Sample type |
SRA |
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| Source name |
Escherichia coli GLBRCE1
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| Organism |
Escherichia coli |
| Characteristics |
strain: GLBRCE1
medium: SynH_LT
growth phase: Exp
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| Treatment protocol |
Cells (10 ml) for transcriptomic analysis were collected into tubes containing 1.25 ml ice-cold 5% (vol/vol) unbuffered phenol in ethanol (EP) and pelleted by centrifugation (10,000 g, 4°C, 3 min). To remove residual traces of hydrolysate, cell pellets were twice resuspended in ice-cold GMM plus 0.125 volume of EP, repelleted, then flash frozen in dry ice-ethanol, and stored at -80°C.
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| Growth protocol |
Fermentations were carried out in 3 L bioreactors (Applikon Biotechnology) containing 2.45 L of ACSH or SynH media, and cultures were diluted into ACSH or SynH with initial OD at 0.2, grown anaerobically overnight, and then inoculated into bioreactors to a starting OD600 of 0.2
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was obtained from cells pellets by lysozyme treatment and phenol-chloroform extraction, analyzed by agarose gel electrophoresis to confirm integrity, quantified using a Nanodrop spectrophotometer (Thermo Scientific), and stored at -80°C
Total RNA (2ug) was subjected to rRNA depletion using Ribozero rRNA removal kit – Bacteria (Epicenter, illumina). rRNA-depleted RNA was purified using Ampure XP beads. Purified RNA was then fragmented using RNA Fragmentation Reagents (Ambion) at 70C for 2mins, targeting fragments ranging from 200-300bp. Fragmented RNA was then purified using Ampure XP beads (Agencourt). Reverse transcription was performed using SuperScript II Reverse Transcription (Invitrogen) with an initial annealing of random hexamers (Fermentas) at 65C for 5mins, follow by an incubation of 42C for 50mins and an inactivation step at 70C for 10mins. cDNA was then purified with Ampure XP beads. This was followed by second strand synthesis using dNTP mix where dTTP is replaced by dUTP. Reaction was performed at 16C for 1h. Double stranded cDNA fragments were purified and selected for targeted fragments (200-300bp) using Ampure XP beads. The dscDNA were then blunt-ended, the 3' ends were adenylated with a single A, and ligated with library adapters using Kapa Library Amplification Kit (Kapa Biosystems). Adapter-ligated DNA was purified using Ampure XP beads. Digestion of dUTP was then performed using AmpErase UNG (Applied Biosystems) to remove second strand cDNA. Digested cDNA was again cleaned up with Ampure SPRI beads. This was followed by amplification by 10 cycles of PCR using Kapa Library Amplification Kit (Kapa Biosystems). The final library was cleaned up with Ampure SPRI beads. Sequencing was done on the Illumina HiSeq platform generating paired end reads of 100bp each. Note: Target fragments here refers to the insert only, that is the cDNA. The actual size we use in selection will be larger, typically the target fragment size + 125bp adaptors (~300-450bp).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
NA
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| Data processing |
Basecalling was done on the sequencers using the sequencer's Real-Time Analysis (RTA) software. CASAVA was used to process the basecalled data into FASTQ format.
FASTQ formatted sequence files from strand-specific Illumina RNA-Seq reads were aligned to the GLBRCE1 reference genome NC_000913 using Bowtie version 0.12.7(Langmead et al., 2009) with '--nofw' strand-specific parameter and maximal distance between the paired reads of 1000 bp. NOTE: Genome NC_000913 (ASM584v2) represents the parental strain. The strain used in our study was modified from the parental strain by replacing gene pflB with an insertion that contained 3 different genes.
Probabilistic expression counting was performed using the RNA-Seq by Expectation-Maximization (RSEM) version 1.2.4 (Li and Dewey, 2011)
Genome_build: ASM584v2
Supplementary_files_format_and_content: CSV file contains counts generated using RSEM software. Ribosomal RNA transcripts excluded.
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| Submission date |
Jul 14, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Robert C Landick |
| E-mail(s) |
landick@bact.wisc.edu
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| Phone |
(608) 265-8475
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| Organization name |
University of Wisconsin - Madison
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| Department |
Great Lakes Bioenergy Research Center
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| Street address |
1550 Linden Dr
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| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
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| Platform ID |
GPL14548 |
| Series (2) |
| GSE58927 |
Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and detoxification |
| GSE59397 |
Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and detoxification (V) |
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| Relations |
| BioSample |
SAMN02911533 |
| SRA |
SRX652182 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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