|
| Status |
Public on Nov 17, 2020 |
| Title |
A4F244_44C_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
A4F244_44C_bacteria cells
|
| Organism |
Escherichia coli K-12 |
| Characteristics |
strain background: MG1655
strain: A4F244
genotype/variation: evolved strain adapted at 45.3C
medium: glucose M9 minimal
phase: mid-log phase
temperature: 44°C
|
| Growth protocol |
Cultures were inoculated from -80°C glycerol stocks into M9 minimal medium with 4g/L glucose supplementation, and grown aerated at 37°C overnight. Physiological adaptation was achieved by growing cell cultures exponentially over 2 passages for 5 to 10 generations at the target temperature. Next, cultures growing at the exponential growth phase were passaged to a 15 mL working volume tube and grown fully aerated until mid-log phase (OD600 ≈ 0.6) for RNA-Seq sample extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
3 mL of cell broth was taken at OD600~0.6 and immediately added to 2 volumes Qiagen RNAprotect Bacteria Reagent (6 mL). Then, the sample was vortexed for 5 seconds, incubated at room temperature for 5 minutes, and immediately centrifuged for 10 minutes at 5000 x g. The supernatant was decanted, and the cell pellet was stored in the -80°C. Cell pellets were thawed and incubated with Readylyse Lysozyme, SuperaseIn, Protease K, and 20% SDS for 20 minutes at 37°C. Total RNA was isolated and purified using the RNeasy Plus Mini Kit (Qiagen) columns following vendor procedures. An on-column DNase-treatment was performed for 30 minutes at room temperature. RNA was quantified using a Nano drop and quality assessed by running an RNA-nano chip on a bioanalyzer. The rRNA was removed using Illumina Ribo-Zero rRNA Removal Kit (Gram-Negative Bacteria). The quantity was determined by Nanodrop 1000 spectrophotometer (Thermo Scientific). The quality was checked using RNA 6000 Pico Kit using Agilent 2100 Bioanalyzer (Agilent). Paired-end, strand-specific RNA-seq library was built with the KAPA RNA Hyper Prep kit (Kapa Biosystems) following manufacturer’s instructions.
RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
ALE_HRS_A4_F244_R1_44C_S65_L004
|
| Data processing |
The base calling was done using Illumina’s Real-Time Analysis (RTA) v2.7.7; data was converted to fastq and demultiplexed using Illumina’s bcl2fastq2 Conversion Software v2.20;
Raw sequencing reads were mapped to the reference genome NC_000913.3 using Bowtie 2.3.4.3 with the following options ``-X 1000 -N 1 -3 3''.
Transcript abundance was quantified using summarizeOverlaps from the R GenomicAlignments package, with the following options ``mode="IntersectionStrict", singleEnd=FALSE, ignore.strand=FALSE, preprocess.reads=invertStrand''.
Transcripts per Million (TPM) were calculated by DESeq2.
Genome_build: reference genome (NC_000913.3)
Supplementary_files_format_and_content: tab-delimited text files containing raw counts of sequencing reads for each sample
|
| |
|
| Submission date |
Nov 15, 2019 |
| Last update date |
Nov 18, 2020 |
| Contact name |
Ke Chen |
| E-mail(s) |
kekechen919@gmail.com
|
| Phone |
9494139582
|
| Organization name |
UCSD
|
| Department |
Bioengineering
|
| Lab |
Bernhard O Palsson
|
| Street address |
Bioengineering Department, 9500 Gilman Dr.
|
| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92093 |
| Country |
USA |
| |
|
| Platform ID |
GPL24377 |
| Series (1) |
| GSE140478 |
A multi-level understanding of the evolutionary trade-offs in thermal adaptation |
|
| Relations |
| BioSample |
SAMN13293105 |
| SRA |
SRX7157068 |
