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| Status |
Public on Jan 29, 2019 |
| Title |
wild type, with 3-OH-C10-HSL, replicate 2 |
| Sample type |
SRA |
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| Source name |
exponential phase culture
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| Organism |
Methylomonas sp. LW13 |
| Characteristics |
genotype/variation: wild type
treatment: with 3-OH-C10-HSL
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| Treatment protocol |
Growing cultures of Methylomonas sp. strain LW13 were subdiluted to OD600nm 0.01 and added to bottles containing dried 3-OH-C10-HSL (or solvent control) resulting in a final concentration of 2 µM signal. RNA was extracted during log phase (OD600nm 0.4-0.6). Briefly, cultures were chilled on ice and then centrifuged at 5000 rpm for 15 minutes at 4ºC. Pellets were subsequently flash frozen in liquid nitrogen and stored at -80ºC until further processing.
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| Growth protocol |
Methylomonas sp. strain LW13 was cultured in an atmosphere of 25% methane in air. For routine culturing plates were incubated at room temperature in sealed jars (Oxoid Limited), while liquid cultures were grown at 18ºC in 250-ml glass serum bottles (Kimble Chase) or 18- by 150-mm tubes (Bellco Glass) sealed with rubber stoppers and aluminum seals (Wheaton) shaken at 200 rpm. Cultures were grown in nitrate mineral salts (NMS) medium (38) containing 0.2 g/L MgSO4·7H2O, 0.2 g/L CaCl2·6H2O, 1 g/L KNO3, and 30 µM LaCl as well as 1X trace elements. 500X trace elements contains 1.0 g/L Na2-EDTA, 2.0 g/L FeSO4·7H2O, 0.8 g/L ZnSO4·7H2O, 0.03 g/L MnCl2·4H2O, 0.03 g/L H3BO3, 0.2 g/L CoCl2·6H2O, 0.6 g/L CuCl2·2H2O, 0.02 g/L NiCl2·6H2O, and 0.05 g/L Na2MoO·2H2O. A final concentration of 5.8 mM phosphate buffer, pH 6.8 was added immediately before use.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellets were lysed by bead beating with 0.1-mm zirconia-silica beads (Biospec Products) in 1 mL TRIzol (Thermo Fisher Scientific). The lysate was then separated according to the TRIzol instructions, and subsequently 1.5 volumes of 100% ethanol was added to the aqueous phase of the extract which was then used as the input for an RNeasy RNA isolation kit (Qiagen). The resulting elution was then digested using Ambion DNase I (Thermo Fisher Scientific) for 30 minutes at 37ºC before being re-purified via RNeasy RNA isolation kit with the addition of an on-column DNase (Qiagen) digestion step for 15 minutes at room temperature. The resulting purified RNA was checked for DNA contamination by PCR using the universal 16S primers 27F and 1492R.
cDNA library preparation and RNA sequencing were performed by GENEWIZ using Illumina HiSeq 2x150 (pair ended) reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
LW13-WT-Y-BR2
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| Data processing |
Alignment was performed using BWA version 0.7.12-r1044 using the BWA-MEM algorithm and default parameters.
The alignments were post-processed into sorted BAM files with SAMTools version 1.2-232-g87cdc4a.
Reads were attributed to open reading frames (ORFs) using the htseq-count tool from the ‘HTSeq’ framework version 0.6.1p1 in the ‘intersection-nonempty’ mode.
Differential abundance analysis was performed with DESeq2 1.2.10 using R 3.3.0.
Genome_build: CP033381
Supplementary_files_format_and_content: *.dat files are two columns: locus tag and read count. At the bottom of the file, a few summary read alignment statistics are given.
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| Submission date |
Nov 07, 2018 |
| Last update date |
Feb 01, 2019 |
| Contact name |
Mitchell William Pesesky |
| E-mail(s) |
[email protected]
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| Organization name |
University of Washington
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| Street address |
616 NE Northlake Place
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98105 |
| Country |
USA |
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| Platform ID |
GPL25782 |
| Series (1) |
| GSE122293 |
Interspecies chemical signalling in a methane-oxidizing bacterial community |
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| Relations |
| BioSample |
SAMN10393022 |
| SRA |
SRX4993761 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3463548_LW13-WT-Y-BR2.summary.dat.gz |
22.3 Kb |
(ftp)(http) |
DAT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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