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Status |
Public on Nov 27, 2020 |
Title |
Streptomyces coelicolor_M1146_22h |
Sample type |
SRA |
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Source name |
Streptomyces coelicolor
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Organism |
Streptomyces coelicolor |
Characteristics |
strain: M1146 time: 22h
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Treatment protocol |
Before sampling, all materials for sampling was autoclaved two times to avoid RNase contamination. Based on growth curve, expectedly more than 2.0 mg DCW cell cultures was sampled and mixed with the twice amount of RNA protection reagent (Qiagen) immediately. The cells were vortexed for 10 seconds, kept for 5 minutes at room temperature and centrifuged at 10000 rpm, 20°C for 10 minutes. Supernatant was eliminated as much as possible, and the cells were quenched by liquid nitrogen immediately and kept at -80°C until RNA isolation.
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Growth protocol |
Bacterial strains were grown in 50 mL liquid minimum nutrition medium with same composition as previous study in well siliconized 250 mL flasks containing stainless-steel springs. For inoculation, 1×109 CFU of spores were inoculated to the 50 mL medium. Incubation speed and temperature was set to 220 rpm and 30°C respectively.
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Extracted molecule |
total RNA |
Extraction protocol |
Cells were disaggregated by double autoclaved empty tip and 0.17 mL of 15 mg/mL lysozyme was added to the cells and incubated at 30°C for 10 minutes. The solution including cells were transferred to tube containing lysing matrix E (MPbio medicals) and 0.6 mL RLT buffer (Qiagen) supplemented with beta-mercaptoethanol (100: 1, v/v), and vortexed for 5 seconds. Three pulses were applied to the tube by Fast Prep (6.5 m/s, 30s) while the tubes were kept on ice between pulses for 30 seconds. The tubes were centrifuged at 10000 rpm and 4°C for 1 minute and the lysate was recovered. After centrifuging heavy PLG tube to pack the resine for 1min, 0.65 mL recovered lysate was transferred to the heavy PLG tube. 0.65 mL chloroform, isoamyl alcohol and acid phenol mixture was added to the lysate in heavy PLG tube and mixed by inversion for 1 minute. The heavy PLG tubes containing extracts were centrifuged for 5 minutes and superior aqueous phase was recovered. RNA purification was performed with Direct-zol RNA MiniPrep Plus (Zymo research). After 0.6 mL ethanol was added to recovered 0.6 mL of aqueous phase and mixing by pipette, Zymo-spin ⅢCG column was assembled to collection tube, and 0.6 mL mixture solution was transferred to the assembled column-collection tube, and centrifuged at 10000 rpm for 30 seconds. Flow through from the collection tube was discarded and remaining 0.6 mL of mixture of aqueous extract and ethanol was transferred to the assembled column-collection tube, consequently centrifuged at 10000 rpm for 30 seconds. The flow through in the collection tube was discarded again. RNA wash buffer completed with ethanol (0.4 mL) was added to the column, centrifuged at 10000 rpm for 30 seconds and the flow-through was discarded. To the column matrix, 80 µL of DNase (6 U/µL) was added and incubated at 30°C for 15 minutes. After incubation, two cycles of RNA washing (Adding 0.4 mL of Direct-zol RNA prewash to the column, centrifuging at 10000 rpm for 30 seconds and discarding the flow through). To the column, 0.7 mL RNA wash buffer was added and centrifuged for 2 minutes to ensure complete removal of wash buffer. Consequently, the column was transferred to new RNase-free tube, 50 µL of DNase/RNase-free water was added and incubated at room temperature for 1 minute. The column with sample containing water was centrifuged at 10000 rpm for 1 minute, and tube with RNA sample was frozen by liquid nitrogen until next step. The rRNA depleted RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3’ adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase. The TruSeq barcode sequences, which are part of the 5' and 3'TruSeq sequencing adapters, are included in Table 2. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
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Description |
M1146_22h
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Data processing |
Generating reference genome by software STAR ver. 2.7 Read mapping to reference genome by software STAR ver. 2.7 Read counting by R-package feature counts RLE normalized counts were generated by R-package DESeq2 Genome_build: ASM20383v1 Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
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Submission date |
Aug 06, 2020 |
Last update date |
Nov 27, 2020 |
Contact name |
Katsuaki Nitta |
E-mail(s) |
katsuaki_nitta@bio.eng.osaka-u.ac.jp
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Organization name |
Osaka University
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Street address |
Yamadaoka 2-1
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City |
Suita |
State/province |
Osaka |
ZIP/Postal code |
06-6879-4170 |
Country |
Japan |
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Platform ID |
GPL28977 |
Series (1) |
GSE155796 |
Time-course transcriptome analysis of non-antibiotics producer and actinorhodin over-producing Streptomyces coelicolor strain |
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Relations |
BioSample |
SAMN15747763 |
SRA |
SRX8898446 |
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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