 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 22, 2024 |
| Title |
25_SMM_Rep1_T5 |
| Sample type |
SRA |
| |
|
| Source name |
bacterial cultures
|
| Organism |
Streptomyces coelicolor A3(2) |
| Characteristics |
tissue: bacterial cultures
medium: SMMS
treatment: control
time (min): 5
replicate: 1
|
| Treatment protocol |
Liquid medium cultures were exposed to cold-shock by the addition of 3 volumes of fresh media at 4°C to reduce the temperature to 10°C, whilst control cultures had fresh media at 30°C added. Solid medium cultures were exposed to cold-shock by transfering the mycelium growing on cellophane discs to a fresh plate pre-chilled plates to 4°C, whilst control cultures were transferred to pre-warmed plates at 30°C.
|
| Growth protocol |
Rich liquid medium cultures were inoculated at a concentration of approx. 1,000,000 cfu / ml in 500 ml conical flasks containing springs and 120 ml yeast extract-malt extract (YEME) medium (Practical Streptomyces Genetics, Kieser et al. 2000, ISBN 9780708406236) and incubated for 19 h 19 min in a shaking incubator at 30°C and 250 rpm. Solid medium cultures were pre-germinated in 2 x YT medium (Kieser et al., 2000) for 9hrs at 30°C and 250 rpm then plated on supplemented minimal medium, solid (SMMS) media (Kieser et al., 2000) overlayed with cellophane discs at a concentration of approx 1,000,000 germ tubes per plate and incubated for 42 h at 30°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
10 ml of bacterial cultures were resuspended in 200 µl of lysozyme dissolved in TE buffer at a concentration of 0.015 g per ml and incubated at room temperature for 15 min. 600 µl of RNeasy Lysis Buffer (RLT buffer, Qiagen #79216) containing 10 µl/ml b-mercaptoethanol was added and the mixture mixed by vortex mixer then transferred to a 2ml microfuge tube containing 5 mm stainless steel beads (Qiagen 69989) and placed in a Tissuelyser LT (Qiagen #85600) for efficient mechanical cell disruption / homogenisation for 2 min at 30 rpm. The suspension was then centrifuged at 16,627 RCF for 2 min, the supernatant was recovered and its volume estimated. One volume of neutral pH phenol:chloroform:isoamyl alcohol (25:24:1, Sigma #77617) was added, and the mixture mixed by vortex mixer for 30 seconds, then centrifuged at 4°C for 5 min at 16,627 RCF. The top phase containing the nucleic acids was removed and retained leaving the interface, and the phenol/chloroform extraction repeated twice more. Then one volume of chloroform was added, the mixture mixed by vortex mixer then centrifuged at 4°C for 2 min at 16,627 RCF, and the top phase was again transferred to a fresh RNase-free microfuge tube. From this point onward the standard manufacturer’s instructions were followed for the miRNAeasy RNA extraction kit (Qiagen #217004). The total RNA was then eluted in 30 µl of nuclease free water and stored at -80°C. Genomic DNA was removed using the Turbo DNA free kit (Invitrogen AM1907).
NEBNext® Ultra™II Directional RNA Library Prep Kit for Illumina® (cat. E7760) according to manufacturer’s instructions
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Reads were quantified against S. coelicolor transcriptome using kallisto quant version 0.46.2. Transcriptome gene annotations used from NCBI reference NC_003888.3 and sRNA annotations from 'The dynamic transcriptional and translational landscape of the model antibiotic producer Streptomyces coelicolor A3(2)', Jeong et al. 2016 https://doi.org/10.1038/ncomms11605
Kallisto quantifications were converted to gene level by tximport version 1.20.0 then analysed for differential expression using DESeq2 version 1.32.0.
lfcShrink used to calculate differential expression contrasts with adaptive shrinkage (type='ashr').
Assembly: GCF_000203835.1_ASM20383v1 NC_003888.3
Supplementary files format and content: CSV-delimited text file. DE contrasts log2-fold changes and padj for contrasts cold-shock at 15, 30 and 60 min vs cold-shock at 5 min, and cold-shock vs control at 5 and 60 min by media type.
Supplementary files format and content: tab-delimited text files. Matrix table of counts for every gene and sRNA generated by kallisto quant.
|
| |
|
| Submission date |
Dec 14, 2022 |
| Last update date |
Feb 22, 2024 |
| Contact name |
Ruslan Evans |
| Organization name |
University of Brighton
|
| Department |
School of Applied Science
|
| Street address |
Lewes Road
|
| City |
Brighton |
| State/province |
East Sussex |
| ZIP/Postal code |
BN2 4GJ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL26763 |
| Series (1) |
| GSE220990 |
Establishing the conditions required to initiate a cold-shock response in Streptomyces coelicolor |
|
| Relations |
| BioSample |
SAMN32234872 |
| SRA |
SRX18701054 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6836443_25-SMM-Rep1-T5.tsv.gz |
107.7 Kb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
