|
|
GEO help: Mouse over screen elements for information. |
|
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 Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
|
|
|
|
|