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| Status |
Public on Aug 21, 2017 |
| Title |
HR_2 |
| Sample type |
SRA |
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| Source name |
Uninfected mouse heart controls
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| Organism |
Mus musculus |
| Characteristics |
sample type: uninfected mouse heart
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| Growth protocol |
Pneumococci were grown in Todd Hewitt Broth (THB) (Acumedia, Neogen) with 0.5% yeast extract (THY) at 37°C in 5% CO2 until reaching mid-exponential phase [Optical density, O.D.620nm = 0.5; ~1.0 x 108 colony forming units (CFU)/mL] and serially diluted in sterile phosphate buffered saline (PBS). Blood was collected from anesthetized mice retro-orbitally and transferred to heparin-coated collection tubes. Following euthanasia, hearts were surgically excised and washed in PBS to remove blood. Isolated hearts were homogenized in 5ml of PBS followed by filtration of the homogenate through a 40µm cell strainer. Paired blood (BIP) and strained heart (HIP) samples were flash-frozen at -80° C in working aliquots with 10% glycerol.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For HIP-RNA, hearts were excised from HIP-infected mice (n=3) when deemed moribund. Hearts were rinsed, diced, fragments washed with ice cold PBS, and the fragments homogenized in RNAprotect bacteria reagent (Qiagen). For BIP-RNA, 10 mice pre-depleted for neutrophils using anti-Ly6G antibody (BioXCell, clone RB6-8C5), were infected with TIGR4 and blood was collected in RNAprotect bacteria reagent (Qiagen) when the mice were deemed moribund such that blood from 5 mice were pooled as one sample (n=2 BIP samples). These heart homogenates were stored at -80°C. On the day of total RNA isolation from heart homogenates, the samples were thawed and spun down to discard the supernatant. The pellets were further homogenized in 600 μL RLT with B-ME buffer using a motorized mortar for 30 seconds. The re-homogenized samples were then disaggregated in a Qiashredder followed by RNA extraction with the RNeasy Micro Kit (Qiagen) with DNase treatment on column and in solution. The isolated RNA was quantitated using Nanodrop and Bioanalyzer. Samples were then depleted of rRNAs using the Ribo-Zero rRNA Removal Kit for Gram-positive bacteria and human/mouse/rat (Illumina, San Diego, CA). For the in vitro biofilm and planktonic samples: planktonic mid-log phase (OD620nm = 0.5) TIGR4 grown in THB were used to seed continuous once-flow through biofilm reactors. Biofilms were allowed to grow for 48 hours prior to collection of bacteria. RNA was isolated from the planktonic seed cultures and their respective biofilms. In total, 3 independent biological samples were collected with planktonic and biofilm RNAs from each experiment paired. Total RNA was extracted from each replicate separately using enzymatic lysis of pneumococcal cells (10 μL mutanolysin, 20 μL proteinase K, 15 μL lysozyme, and 55 μL TE) followed by RNA extraction using the same protocol mentioned above. Blood samples were isolated in a similar manner as biofilm and planktonic samples, including enzymatic lysis, except that cells were first disaggregated with the Qiashredder like for the heart samples.
Strand specific RNA libraries were prepared for sequencing using standard Illumina protocols
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Mus musculus cells
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| Data processing |
Illumina reads were mapped to the Mus musculus GRCm38 genome using TopHat v. 1.4.0
The mapped reads were counted using HTSeq and normalized and then the differential expression of each gene for one sample compared to another was determined using DESeq v. 1.5.24, and statistical analyses were performed using R-2.15.2, and was represented as the log2 of the fold-change values.
RPKMs for each gene were calculated for each sample.
Genome_build: Mus musculus GRCm38
Supplementary_files_format_and_content: Tab-delimited text files include RPKMs
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| Submission date |
May 02, 2017 |
| Last update date |
Jun 03, 2020 |
| Contact name |
Suvarna Nadendla |
| Organization name |
University of Maryland School of Medicine
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| Department |
Institute for Genome Sciences
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| Street address |
670 W.Baltimore Street
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21201 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (1) |
| GSE86118 |
Pneumococci in the heart subvert the host response through biofilm-mediated macrophage killing |
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| Relations |
| Alternative to |
GSM4589967 |
| BioSample |
SAMN06883695 |
| SRA |
SRX2779378 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2597299_HR2.accepted_hits.sorted_by_position.genic.coverage.stats.rpkm.txt.tpm.txt.gz |
1.3 Mb |
(ftp)(http) |
TXT |
| GSM2597299_HR_2.sorted_by_name.genic.coverage.stats.rpkm.txt.gz |
1.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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