|
| Status |
Public on Nov 23, 2022 |
| Title |
Group_3_inoculum |
| Sample type |
SRA |
| |
|
| Source name |
culture
|
| Organism |
[Haemophilus] ducreyi |
| Characteristics |
tissue: culture
treatment: culture
|
| Growth protocol |
The inocula consisted of H. ducreyi cultured in GC broth supplemented with 1% IsoVitaleX, 50 𝜇g/mL hemin, and 5% heat-inactivated fetal bovine serum and shaken at 33°C in ambient air to mid-log phase (OD660 0.200-0.250). 10 mL of mid-log (OD660 0.200-0.250) culture was pelleted and stored in 2 mL of RNALater at -80°C. The culture was processed and diluted in PBS so that ~70 CFU of H. ducreyi would be delivered to the upper arm skin of volunteers using a allergy testing device. Volunteers were both infected with the inoculum and sham inoculated with PBS. The infection progressed for 6-8 days, at which point painful pustules had developed at the infected sites. A 6 mm punch biopsy was taken of both the pustule and wound. Biopsies were placed in 2 mL of RNALater at the bedside and then stored at -80°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from samples the day after collection using the Qiagen RNeasy Fibrous Tissue Mini Kit with on-column DNase digestion. RNA was eluted from the column with nuclease free water and then treated with Turbo DNase (Invitrogen) for an in-solution DNase digestion.
Purified RNA was treated with Qiagen FastSelect for bacteria and human rRNA depletion. Libraries were prepared with a Roche KAPA kit.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
G3J_inoc
|
| Data processing |
Basecalling was performed with Illumina RTA v3.4.4 and bcl2fastq v2.20.
Sequenced reads were aligned to the Haemophilus ducreyi 35000HP genome (AE017143.1) using Bowtie2 or human genome (hg38) using STAR.
Human reads in pustule samples were subsampled to 125 million reads. H. ducreyi reads in cultures were subsampled to 1 million reads.
Count matricies were made for human and H. ducreyi reads using featureCounts.
Library normalization and statistical analysis was performed in R v4.1.0 with the DESeq2 package.
Assembly: hg38; AE017143.1
Supplementary files format and content: txt file for the count matrix of the 12 H. ducreyi samples from this study and the reanalyzed samples from 8 H. ducreyi samples from GSE130901
Supplementary files format and content: txt file for the count matrix of the 16 human samples from this study and the reanalyzed samples from 8 human samples from GSE130901
|
| |
|
| Submission date |
Oct 01, 2022 |
| Last update date |
Nov 23, 2022 |
| Contact name |
Julie Brothwell |
| E-mail(s) |
jbrothwe@iu.edu
|
| Phone |
3172781027
|
| Organization name |
Indiana University
|
| Department |
Department of Microbiology & Immunology
|
| Street address |
635 Barnhill Dr., MS420
|
| City |
Indianapolis |
| State/province |
IN |
| ZIP/Postal code |
46202 |
| Country |
USA |
| |
|
| Platform ID |
GPL31188 |
| Series (1) |
| GSE214586 |
Haemophilus ducreyi infection induces oxidative stress, central metabolic changes, and a mixed pro- and anti-inflammatory environment in the human host |
|
| Relations |
| BioSample |
SAMN31117572 |
| SRA |
SRX17771342 |
