|
| Status |
Public on Oct 19, 2022 |
| Title |
24h_WT_2DG_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
BMDMs
|
| Organisms |
Mus musculus; Salmonella enterica subsp. enterica serovar Typhimurium |
| Characteristics |
bacteria status: WT
2-dg status: with 2-DG
time post-infection (hours): 24
replicate: 2
|
| Treatment protocol |
800,000 cells/well of BMDM cells were plated in non-tissue culture treated 6 well plates supplemented with DMEM with 20% FBS and 25ng/ml rmM-CSF. Infected BMDMs samples were infected with WT or the ∆manZ mutant strains harboring a GFP expressing plasmid (pFPV25.1; Addgene) at MOI of 10:1. In the 2-DG treated samples, 2-DG (Sigma D8375-5G) in a concentration of 1mM was supplemented to the media containing 15 μg/ml gentamicin after the wash of the uninternalized bacteria. Uninfected samples were treated under the same conditions. 20,000 cells per sample of uninfected controls or infected (GFP positive) cells were sorted under continuous cooling to 4°C by a BD FACS Aria III into a low bind eppendorfs contained 700μl QIAzol Lysis reagent, over a time curse of 0.5h, 6h and 24h post-infection.
|
| Growth protocol |
Bone marrow was extracted from 6-8 week old female C57BL/6 mice as previously described (Falk et al., 1988). In brief, 6-8 week old female C57BL/6 mice were euthanized and femurs and tibias were harvested. Bone marrow was flushed from the cells, resuspended in DMEM, and plated in non-tissue culture treated dishes in DMEM media containing 5% sodium pyruvate, 20% fetal bovine serum, and 25 ng/ml recombinant mouse M-CSF. Cells were harvested at day 6 after bone marrow isolation and were frozen untill use. All bone marrows extractions were performed in accordance with the Institutional Animal Care and Use Committee at the Weizmann Institute. (IACUC Approval number 27410516-3)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Gentle bead beating was done using OmniLyse (disposable cell disruptors) to break the bacterial cell envelope without harming the macrophage RNA. miRNeasy kit (QIAGEN) was used for further RNA extraction
RNAtag-seq cDNA libraries for simultaneous detection of host and intracellular bacterial transcripts was done as previously described in Avraham et al (2016) (Avraham et al., 2016). In brief, samples contain murine RNA transcripts (uninfected controls) or a mix of bacterial and murine RNA transcripts (infected) were barcoded by ligation with RNA adaptors and pooled together. Murine rRNA was depleted using the RiboZero Gold (Human/Mouse/Rat) kit (Illumina) according to the manufacturer’s guidelines. Strand-specific cDNA libraries were prepared and sequenced as Paired-end (R1: 30, R2: 50) using Nextseq machine and 75 high throughput kit with total coverage of ~1.1B reads and a mean of ~19.5M reads per library, ranges from ~4.5M to ~45.5M. For each condition, biological triplicates were analyzed
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Fastq reads were demultiplex to their respective sample by using fastq-multx (version 1.3.1) and aligned to a composite genome of S.Tm 14028S and mouse (mm10) using STAR (version 2.5.3a).
For the host, read counts were calculated using STAR. For S.Tm read counts, R2 reads were calculated by HTSeq.
Data of each organism was normalized to a library size factor. Factors were calculated by dividing the total number of reads from each sample to the median of the total number of reads across all samples.
Data was transformed to log2 scale. Host minimal expression threshold was set to 4.5 and bacteria minimal expression threshold was set to 2.
Genome_build: composite genome of S.Tm 14028S and mouse (mm10)
Supplementary_files_format_and_content: tab-delimited text files include normalized number of reads (log2 scale) for each sample
|
| |
|
| Submission date |
Oct 21, 2019 |
| Last update date |
Oct 19, 2022 |
| Contact name |
Roi Avraham |
| E-mail(s) |
roi.avraham@weizmann.ac.il
|
| Organization name |
Weizmann Institute of Science
|
| Street address |
Herzl St 234
|
| City |
Rehovot |
| ZIP/Postal code |
7610001 |
| Country |
Israel |
| |
|
| Platform ID |
GPL24107 |
| Series (2) |
| GSE139205 |
Host metabolic reprogramming during immune activation promotes intracellular bacterial survival (host and bacteria) |
| GSE139208 |
Host succinate is a signal for activation of Salmonella virulence during intracellular infection |
|
| Relations |
| BioSample |
SAMN13071296 |
| SRA |
SRX7031434 |
