|
| Status |
Public on Jan 01, 2015 |
| Title |
70_Day_0_HTKL_2 |
| Sample type |
SRA |
| |
|
| Source name |
Cell culture
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
culture: Exponential phase culture
cell: Whole Cell
experimental day: Day 0
|
| Treatment protocol |
Exponential phase cultures were challenged with D-cycloserine (125 μg/ml). Samples were taken for RNA extraction at Day 0 (prior to antibiotic) and Day 7 of the challenge. RNA Protect Bacteria Reagent (Qiagen) was added to the samples prior to RNA extraction.
|
| Growth protocol |
M. tuberculosis was grown in Middlebrook 7H9 broth at 37°C in a shaking incubator (200 rpm) to exponential phase (optical density at 600 nm of 0.5)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
1. Cells were resuspended in Trizol (Invitrogen) and lysed by bead beating. 2. Total RNA was extracted with chloroform and preciptated with isopropanol.
Libraries were created by the Broad Technology Labs specialized service facility. Briefly, 48 RNA samples were fragmented, and RNA 3’ ends were tagged with a DNA oligonucleotide containing a sample barcode and a partial 5’ Illumina adapter. Resulting barcoded RNAs were then pooled and subjected to rRNA depletion (Ribo-Zero™ Magnetic Kit (Bacteria); Epicentre, Madison, WI), cDNA synthesis and ligation to a second oligonucleotide containing a partial Illumina 3’ adapter. A second barcode specific to this pool was then added by amplification with full-length barcoded Illumina adapter primers, yielding a single strand-specific sequence-ready pooled RNA-seq library.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Low-persister
|
| Data processing |
The pool of 48 Illumina RNA-Seq libraries was quantified using qPCR (KAPA Biosystems, USA) and sequenced with 76 base paired-end reads across 4 lanes using an Illumina HiSeq 2000 sequencer (Illumina, USA) running v3 SBS chemistry.
Reads were aligned to the genome of M. tuberculosis H37Rv (RefSeq NC_000962) using BWA (Li, Durbin (2009)) version 5.9.
Gene annotations were obtained from RefSeq and Rfam (Gardner, Daub et al. (2009)).
Fragment coverage of genomic regions corresponding to features such as ORFs and rRNAs was conducted as described (Mandlik, Livny et al. (2011))
Differential expression analysis was conducted using DESeq (Anders, Huber (2010)).
Genome_build: RefSeq NC_000962
Supplementary_files_format_and_content: Normalized reads per gene (txt)
|
| |
|
| Submission date |
Oct 03, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Kim Lewis |
| Organization name |
Northeastern University
|
| Street address |
360 Huntington Ave
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL18768 |
| Series (1) |
| GSE62025 |
Transcriptome analysis of Mycobacterium tuberculosis clinical isolates. |
|
| Relations |
| BioSample |
SAMN02594456 |
| SRA |
SRX499204 |
