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Status |
Public on Nov 05, 2020 |
Title |
H37Rv_Cholesterol_5 |
Sample type |
SRA |
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Source name |
H37Rv_Cholesterol_5
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Organism |
Mycobacterium tuberculosis |
Characteristics |
strain: H37Rv genotype: Wild type time point: day4 media: Cholesterol agent: No drug
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Growth protocol |
The logphase culture of Mtb strains was washed with PBST twice and grown in “minimal media” (pH6.5; 0.5 g/L asparagine, 1 g/L KH2PO4, 2.5 g/L Na2HPO4, 50mg/L ferric ammonium citrate, 0.5g/L MgSO4*7H2O, 0.5mg/L CaCl2, 0.1 mg/L ZnSO4) containing either 0.1% glycerol (v/v) or 0.01% cholesterol (w/v). To assess the effect of rifampicin (RIF) on the generation of persisters, in some conditions RIF was added. RNA was isolated from the cultures at day4 using Qiagen RNaeasy Minikit according to manufacturer’s protocols (Qiagen 74104). The RNA was DNase treated using Turbo DNA free kit using manufacturer’s protocol (Thermo Fischer scientific) to remove any genomic DNA contamination. All Mycobacterial total RNAs were analyzed using an Agilent Bioanalyser (Agilent, Santa Clara, CA, USA) for quality assessment with RNA Integrity Number (RIN) range of 5.6 to 9.7 and a median of 7.5. Ribosomal RNA (rRNA) were depleted from 500ng of bacterial RNA using RiboMinus™ Bacteria transcriptome isolation kit (Invitrogen Thermo Fisher Scientific Waltham, MA, USA), according to manufacturer's protocol.
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Extracted molecule |
total RNA |
Extraction protocol |
cDNA libraries were prepared from the resultant rRNA depleted RNA and 1 ul of a 1:500 dilution of ERCC RNA Spike in Controls (Ambion® Thermo Fisher Scientific, Waltham, MA, USA) using Lexogen SENSE Total RNA-Seq Library Prep Kit (Lexogen GmnH, Vienna, Austria) according to manufacturer's protocol except with 21 PCR cycles. The length distribution of the cDNA libraries was monitored using a DNA High Sensitivity Reagent Kit on the Perkin Elmer Labchip (Perkin Elmer, Waltham, MA, USA). All samples were subjected to an indexed paired-end sequencing run of 2x51 cycles on an Illumina HiSeq 2000 system (Illumina, San Diego, CA, USA) (16 samples/lane).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 4000 |
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Description |
RBB453 gene_read_counts_mux5708.txt.gz
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Data processing |
Raw reads (FASTQ files) were mapped to the M. tuberculosis H37Rv (GenBank accession AL123456) using bowtie2 using default parameters. The read counts of genes were then counted using featureCounts v1.6.3 using the genome annotations provided in the GenBank file. Computations were done using the R statistical language version 3.3.1. Genome_build: M. tuberculosis H37Rv (GenBank accession AL123456) Supplementary_files_format_and_content: Tab delimited text file with read counts for genes
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Submission date |
Aug 19, 2019 |
Last update date |
Nov 05, 2020 |
Contact name |
Kaibo Duan |
E-mail(s) |
duan_kaibo@immunol.a-star.edu.sg
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Organization name |
Singapore Immunology Network
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Street address |
8A Biomedical Grove, Immunos Building, Level 4
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City |
Singapore |
ZIP/Postal code |
138648 |
Country |
Singapore |
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Platform ID |
GPL25317 |
Series (1) |
GSE135952 |
Host cholesterol modulates the generation and enrichment of persisters during Mycobacterium tuberculosis infection |
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Relations |
BioSample |
SAMN12599106 |
SRA |
SRX6733668 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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