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| Status |
Public on Feb 12, 2018 |
| Title |
R16 MG1655GFP_pLys_M1_1 |
| Sample type |
SRA |
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| Source name |
Bacterial cells
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| Organism |
Escherichia coli |
| Characteristics |
strain: MG1655GFP
plasmid: PLYS
synthetic circuit: M1
treatment: 15 min after induction with arabinose
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| Treatment protocol |
After 60 minutes into the incubation, the plate was briefly removed so inducer could be added to wells, and this time point was set as time 0. Samples were instead removed from wells at 15 and 60 minutes after induction for processing.
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| Growth protocol |
E. coli cells with construct and control plasmids were grown at 37°C overnight with aeration in a shaking incubator in 5 ml of defined supplemented M9 medium with the appropriate antibiotic. In the morning, 60 μl of each sample were diluted into 3 ml of fresh medium and grown at 37°C with shaking for another hour (outgrowth). 200 μl of each sample were then transferred in 8 wells of a 96-well plate (Costar) at approximately 0.1 OD (600 nm). The samples were placed in a Synergy HT Microplate Reader (BioTek) and incubated at 37°C with orbital shaking at 1,000 rpm for 1 h, performing measurements of GFP (excitation (ex.), 485 nm; emission (em.), 528 nm) and OD (600 nm) every 15 minutes.
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| Extracted molecule |
total RNA |
| Extraction protocol |
170 μl were taken from each of four wells per time point and collected into a fresh tube were 1.360 ml of RNA protection buffer had previously been added. Samples were left for 5 minutes at RT and then centrifuged at 4°C at maximum speed. Supernatant was discarded and pellets frozen at -20°C. RNA extraction was performed using RNeasy Mini Kit from Qiagen [Cat No 74104]. To remove possible traces of genomic DNA contamination, 2 μg of each sample were treated for a second time with DNAseI from Qiagen [Cat No 79254]. Total RNA quality and integrity was assessed using Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano kit [Cat No 5067-1511]. Samples had an average RIN of 9.5. Enrichment of mRNA was performed using MicrobExpress rRNA removal kit from Thermo Scientific [Cat No AM1905]. Successful rRNA depletion was assessed with analysis on Bioanalyzer. Retrotranscription was then performed starting from 50 ng total enriched mRNA using Tetro cDNA synthesis kit from Bioline [Cat No BIO-65043] and 6 μl of Random Hexamers [Cat No BIO-38028] per reaction. Second cDNA synthesis was performed adding to the first strand synthesis mix 5 μl of Second strand synthesis buffer [Cat No B6117S], 3 μl of dNTPs [Cat No N0446S], 2μl of RNAseH [Cat No M0297L] all from NEB, 2 μl of Polymerase I from Thermo Scientific [Cat No 18010025] and 18 μl of water, per reaction. Samples were incubated at 16°C for 2.5 h. Purification of cDNA was performed using MiniElute PCR purification kit [Cat No 28004] with final elution in 10 μl of DEPC-treated free water. cDNA was quantified using a Qubit fluorometer (Invitrogen).
Library preparation was performed using the Nextera XT kit from Illumina [Cat No FC-131-1096] starting from 1 ng of total cDNA. The original protocol was modified where 3 min tagmentation and 13 cycles of step-limited PCR were used. Ampure beads from Beckman Coulter [Cat No A63880] were used for library purification. Library quality assessment and quantification was performed with Agilent 2100 Bioanalyzer and Agilent high sensitivity DNA analysis kit [Cat No 5067-4626]. Finally all 90 samples were pooled together in the same reaction tube at a final concentration of 1 nM.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
MG1655GFP_pLys_M1_1
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| Data processing |
paired end sequencing with Illumina HiSeq 2500 Sequencer
Sequenced reads were trimmed for adaptor sequence and low-quality sequence using TrimGalore version 0.4.1, then mapped to mm8 whole genome using bwa mem version 0.7.12 with default parameters
Numer of reads per gene were counted using Bioconductor Rsubread package v1.12.6
Genome_build: Samples from strain MG1655GFP were mapped to the reference geneome Escherichia coli str. K-12 substr. MG1655, assembly ASM584v2.31 complemented with the GFP sequence, and the sequence of corresponding synthetic circuit.
Genome_build: Samples from strain DH10BGFP were mapped to the reference geneome Escherichia coli str. K-12 substr. DH10B, assembly ASM1942v1.31 complemented with the GFP sequence, and the sequence of corresponding synthetic circuit.
Supplementary_files_format_and_content: CSV files providing the number of reads mapping to each genomic gene
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| Submission date |
Nov 17, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Simone Furini |
| E-mail(s) |
simone.furini@unisi.it
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| Phone |
+390577585297
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| Organization name |
University of Siena
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| Department |
Department of Medical Biotechnologies
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| Street address |
viale Mario Bracci, 16
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| City |
Siena |
| ZIP/Postal code |
53035 |
| Country |
Italy |
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| Platform ID |
GPL18133 |
| Series (1) |
| GSE107093 |
Burden- driven feedback control of gene expression |
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| Relations |
| SRA |
SRX3405646 |
| BioSample |
SAMN08044979 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2861142_R16.csv.gz |
17.2 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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