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Sample GSM3584849 Query DataSets for GSM3584849
Status Public on May 21, 2019
Title 5GB1C-JG15-N-BR1 : LanA mutant, no lanthanum added, biological replicate 1
Sample type SRA
 
Source name exponential phase culture
Organism Methylotuvimicrobium buryatense
Characteristics strain: 5GB1C
genotype/variation: lanA mutant
tissue: exponential phase culture
Treatment protocol Wild-type and lanA- cells were growth in biological duplicate in either 0 µM or 30 µM lanthanum, for a total of 8 samples.
Growth protocol Starter cultures were grown to late logarithmic phase (OD600 ~ 0.8) and a 2% inoculum (v/v) was transferred into 50 mL NMS2 medium, under an atmosphere of 25% methane and 75% air.
Extracted molecule total RNA
Extraction protocol Once samples reached OD600 ~ 0.6-0.7, 2.5 mL of a stop solution of 5% (v/v) phenol in ethanol was added to 22.5 mL of culture. Cells were pelleted for 15 min at 4°C, and resuspended in RNA extraction buffer (a 1:3 ratio of 5% cetrimonium brimide in 2.5 M NaCl to 0.1 M phosphate buffer at pH 5.8). An equal volume of phenol-chloroform-isoamyl alcohol (25:24:1 ratio) was added, and sodium dodecyl sulfate and N-lauroylsarcosine sodium salt were each added at 0.5% (v/v). Cells were lysed in a bead beater with 0.1-mm zirconia-silica beads (Biospec Products, Bartlesville, OK, USA) for 4 min, then centrifuged at 14,000 x g for 5 min. The aqueous fraction was then washed with chloroform-isoamyl alcohol (24:1 ratio), and the aqueous fraction from this wash was precipitated overnight with 150 mM sodium acetate (pH 5.5), 1.5 mM MgCl2, and 50% isopropanol at -80°C. Precipitated RNA was harvested at 14,000 x g for 30 min at 4°C, and treated with DNase I (Ambion, Foster City, CA, USA) before purification with the RNeasy minikit and on-column DNase (Qiagen, Hilden, Germany).
cDNA library preparation and RNA sequencing was performed by GENEWIZ using Illumina HiSeq 2x150 (pair ended) reads.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
 
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: CP035467
Supplementary_files_format_and_content: *.dat.txt files are two columns: locus tag and read count. At the bottom of the file five lines of summary read alignment statistics are given.
 
Submission date Jan 30, 2019
Last update date May 24, 2019
Contact name Mitchell William Pesesky
E-mail(s) [email protected]
Organization name University of Washington
Street address 616 NE Northlake Place
City Seattle
State/province WA
ZIP/Postal code 98105
Country USA
 
Platform ID GPL26131
Series (1)
GSE125909 A mutagenic screen identifies a TonB-dependent receptor required for the lanthanide metal switch in the Type I methanotroph “Methylotuvimicrobium buryatense” 5GB1C
Relations
BioSample SAMN10847876
SRA SRX5311933

Supplementary file Size Download File type/resource
GSM3584849_5GB1C-JG15-N-BR1.summary.dat.txt.gz 20.4 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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