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Sample GSM3755976 Query DataSets for GSM3755976
Status Public on May 30, 2019
Title Inoculum 462
Sample type SRA
 
Source name Mid-log phase growth H. ducreyi
Organism [Haemophilus] ducreyi
Characteristics time: Day of infection
strain: 35000HP
tissue: N/A
Treatment protocol N/A
Growth protocol For samples 1-4, RNA was directly extracted from H. ducreyi-infected pustules on human volunteers. For samples 5-8, RNA was directly extracted from human wound-control sites. For samples 9-12, H. ducreyi was grown under GMP conditions to mid-log phase in a proteose peptone broth-based medium with 1% IsoVitaleX, 5% heat-inactivated fetal calf serum, and 50 ug/ml hemin and grown at 33°C for 5-6 hours.
Extracted molecule total RNA
Extraction protocol Biopsy samples immediately after collection and the extra inocula immediately after inoculation were placed in 2 mL RNAlater, incubated for 30 minutes at room temperature, and stored in RNAlater for one day at -80°C. Using a mini-Beadbeater (Biospec Products), we next homogenized the samples and extracted total RNA using a RNeasy Fibrous Tissue Mini kit according to manufacturer’s instructions along with addition of lysozyme (20 ug/mL) added at the proteinase K step. The RNA was treated twice with TURBO DNA-free DNase (Ambion) following manufacturer’s instructions. RNA concentrations and integrities were measured using a NanoPhotometer P330 (Implen). RNA samples were stored at -80°C until all samples were ready for sequencing. We removed 23S, 16S, and 5S rRNA before RNA-seq using the Ribo-Zero Gold rRNA Removal Kit (Epidemiology) (Epicentre Biotechnologies) following manufacturer’s instructions and confirmed their removal using the Agilent 2100 Bioanalyzer.
Twelve libraries (four mid-log phase growth H. ducreyi cultures, four infected sites, and four wounded sites) were constructed using the TruSeq Stranded Total RNA Library kit (Illumina) following manufacturer’s instructions.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 4000
 
Description Sample 9
H_ducreyi_Raw_Read_Counts.txt
Data processing For the human samples, the sequence data were mapped to the human reference genome hg19 with STAR-2.5.2b
For the bacterial samples, the sequence data were mapped to the regerene genome Haemophilu ducreyi 35000hp ASM794v1 with bowtie 2 (version 2.3.2)
featureCounts (subread-1.6.0) was used to quantify gene expression (parameters applied: "“-s 2 –p –Q 10")
Differential expression analysis was performed using edgeR
Genome_build: Haemophilu ducreyi 35000hp reference genome ASM794v1 and human reference genome hg19
Supplementary_files_format_and_content: Two files (one for H. ducreyi and one for human) containing raw counts of sequencing reads
 
Submission date May 08, 2019
Last update date May 30, 2019
Contact name Brad Griesenauer
E-mail(s) bgriesen@iu.edu
Organization name Indiana University School of Medicine
Department Microbiology and Immunology
Lab Dr. Stanley M Spinola
Street address 635 Barnhill Dr, MS 420
City Indianapolis
State/province Indiana
ZIP/Postal code 46202
Country USA
 
Platform ID GPL26640
Series (1)
GSE130901 Determination of an Interaction Network between an Extracellular Bacterial Pathogen and the Human Host
Relations
BioSample SAMN11604901
SRA SRX5807098

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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