|
| Status |
Public on Mar 02, 2016 |
| Title |
Artificial Sputum 2 |
| Sample type |
SRA |
| |
|
| Source name |
M. abscessus grown in artificial sputum medium
|
| Organism |
Mycobacteroides abscessus |
| Characteristics |
experimental group: Group 1
experimental status: Group 1 Treatment
grown in: artificial sputum medium
|
| Treatment protocol |
For Artificial Sputum: The cultures were washed 2x with PBS and transferred to Artificial Sputum (stress condition) or Middlebrook 7H9 medium (control condition). Hypoxia was induced by 40 min incubation in the presence of 50 μM diethylenetriamine/nitric oxide (DETA/NO) adduct. For macrolide treatment, 1 μM Erythromycin was added to bacterial cultures and the exposure was carried out for 1 h. Kanamycin was added at a final concentration of 2 μM and cultures were incubated for 1 h.
|
| Growth protocol |
For Artificial Sputum and Sputum Control: bacterial cultures grown in Middlebrook 7H9 were grown till OD 1; for all other other conditions: bacterial cultures were grown till OD 0.8
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Bacteria were recovered by centrifugation (10 min at 4000 x g) and re-suspended in TRI-Reagent (Sigma Aldrich). Following physical disruption using 0.1 mm zirconia-silica beads in a MagNA Lyzer (Roche Diagnostics). RNA was isolated using miRNeasy spin columns with an additional on-column DNase treatment, according to manufacturer’s instructions (Qiagen). RNA was quantified using NanoDrop 1000 spectrophotometer (Thermo Scientific) and sample quality was assessed using RNA Nano Chip on a Bioanalyzer 2100 instrument.
Sequencing libraries were generated with the TruSeq Stranded RNA kit according to manufacturer’s instructions (Illumina Inc.), validated using High Sensitivity DNA chip (Bioanalyzer 2100) and quantified using High Sensitivity DNA Quantification assay (Qubit Fluorometer).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
S1ASp2_GTTTCG_L001_R1_001
|
| Data processing |
Adapters (first 10 bases of reads) were trimmed from Fastq files using the fastx_trimmer utility from FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/)
Trimmed reads were reverse-complemented using the fastx_reverse_complement utility from FASTX-Toolkit, to obtain reads in the correct orientation (Illumina outputs the reverse complement)
Trimmed, reverse-complemented reads were aligned to the M. abscessus genome using bowtie, with the following parameters: --best -S -p 2
SAM format files were converted to BAM format using the import function of samtools
BAM files were sorted and indexed using the sort and index functions of samtools, respectively
Genome_build: NC_010397.1
Supplementary_files_format_and_content: Read counts per gene obtained using a custom python script, invoking samtools to count the numbers of reads from aligned BAM files. Genomic coordinates of genes were as specified in the PTT-format annotation file for M. abscessus from NCBI. Columns represent experimental conditions, rows represent genomic features.
|
| |
|
| Submission date |
Mar 01, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Brendan J. Loftus |
| E-mail(s) |
brendan.loftus@ucd.ie
|
| Organization name |
University College Dublin
|
| Department |
School of Medicine & Medical Science
|
| Street address |
Conway Institute, University College Dublin
|
| City |
Dublin |
| ZIP/Postal code |
Dublin 4 |
| Country |
Ireland |
| |
|
| Platform ID |
GPL21540 |
| Series (1) |
| GSE78787 |
RNA sequencing of Mycobacterium abscessus under infection-relevant conditions |
|
| Relations |
| BioSample |
SAMN04525397 |
| SRA |
SRX1607052 |
