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| Status |
Public on Feb 02, 2023 |
| Title |
ro6_nv_12h_1_R1 |
| Sample type |
SRA |
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| Source name |
R06 bat cell culture_infected with NAIVE
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| Organism |
Chiroptera |
| Characteristics |
tissue/cell line: R06 bat cell culture
treatment: infected with NAIVE
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| Treatment protocol |
Myotis lucifugus bats infected with RV. For a separate rabies study, bats were inoculated at 104 , 50% tissue culture infectious dose (TCID50) with a Myotis lucgus RV variant in their deltoid muscle. Bats were inoculated, maintained, and euthanized as described earlier [28]. Brain, lung, and spleen tissue were aseptically collected from control and RV-inoculated bats following euthanasia due to clinical RV infection or at the end of the study (Table 2). Tissues were placed in 800 µl Trizol (Ambion) and stored at -80 oC until further processing could occur.
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| Growth protocol |
Two immortalized cell lines, derived from R. aegyptiacus bats, were used in the study, including R06EJ (fetus body) and RoNi/7 (adult bat kidney). The R06EJ cells were maintained in Dulbecco's modified Eagle's medium (DMEM)?F-12 GlutaMAX medium, and RoNi/7 cells were maintained in DMEM (Life Technologies, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (HyClone, Thermo Fisher Scientific, Waltham, MA) and antibiotic-antimycotic (Life Technologies, Thermo Fisher Scientific, Waltham, MA) as previously described. Cells were seeded in 6-well plates (Corning) in the density assuring formation of 80-100% confluent monolayer in 24 hours. Cells either transfected by PolyI:C (using Trans-IT mRNA reagent [Mirrus Bio] per manufacturer?s recommendation), or infected with filoviruses at multiplicity of infection (MOI) of 2 plaque forming units (PFU) per cell as determined previously in Vero E6 cells. After one hour of adsorption, the inoculum was removed, cell monolayers washed three times with phosphate buffered saline (PBS), and two ml of fresh culture medium was added into each well. Cells transfected with PolyI:C were harvested after 6, 12 and 24 hours whereas cells infected with filoviruses were harvested after 8, 12 and 24 hours. Prior to harvesting, cell medium was removed, the monolayer washed twice with PBS, and lysed in 1 ml of TRIzol reagent. Productive filovirus infections in cell culture were confirmed by the presence of eGFP expression [15]. Active replication in the cells was also evidenced by expression of all viral genes (from sequencing data), including MARV, which could not be visualized and did not produce cytopathic effect at harvesting (Table 1)
Viruses: Recombinant wild-type EBOV, strain Mayinga, was recovered from the full-length clone and support plasmids in HEK 293T cells and passaged twice in Vero E6 cells for amplification, as described previously29. Recombinant wild-type MARV, strain Uganda, was recovered similarly in BHK-21 cells as described previously30 and was also passaged twice in Vero E6 cells for amplification.
Bat experimental protocol: Adult Egyptian rousettes were obtained from a commercial source and quarantined for 30 days under ABSL-2 conditions. Animals were implanted with microchip transponders for animal ID and temperature data collection. For studies with EBOV and MARV, animals were transferred to the Galveston National Laboratory ABSL-4 facility. Animals were segregated into groups of three. Except for one MARV-infected male, all bats were female. Each group was housed in a separate cage for inoculation with the same virus. After acclimation to the facility, animals were anesthetized with isoflurane and infected subcutaneously in the scapular region with 105 focus forming units (FFU; titrated on Vero E6 cells) of EBOV or MARV. Every other day, animals were anesthetized by isoflurane, weighed, temperature was determined via transponder, and 100-150 µL of blood was collected from the propatagial vein. Nucleases in blood were inactivated in 1 mL of TRIzol reagent (Thermo-Fisher Scientific). Samples were then removed from ABSL-4 containment, and RNA was extracted. Droplet-digital RT-PCR (ddRT-PCR) with primers specific to the nucleoprotein (NP) gene was used to detect viremia. If fewer than 106 MARV RNA copies/mL viremia were detected in a MARV-inoculated bat, the animal was observed for additional 2 days to allow the animal to reach a higher viral RNA load. In 48-96 hours after first observation of viremia, the animal was euthanized under deep isoflurane sedation via cardiac exsanguination confirmed by bilateral open chest. All EBOV-inoculated bats were euthanized 48 hours after the first detection of viremia, independent of viral RNA load. Tissues were collected (listed in Table S1) and immediately homogenized in an appropriate volume of TRIzol reagent and stored at -80°C. Tissue sections were also homogenized in minimal essential media (MEM) supplemented with 10% fetal bovine serum and stored at -80°C. Additional tissue sections were fixed in 10% neutral buffered formalin for histopathology.
All animal procedures were performed in compliance with protocols approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch at Galveston.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Tissues and PBMCs were also collected from three uninfected control animals. Given that the course of infection appears to be relatively short in these animals21, we sacrificed them shortly after onset of viremia in the infected animals to ensure adequate capture of changes in transcriptional dynamics. As such, animals were bled every other day, and viral loads were assessed via ddRT-PCR. In addition, animals were weighed, and temperature was determined with each bleed.
Total RNA was isolated from samples using Ambion's RNA isolation and purification kit. PolyA-tailed mRNA was selected using beads with oligo-deoxythymidine and then fragmented. cDNA was synthesized using random hexamers and ligated with bar-coded adaptors compatible with Illumina's NextSeq 500. A total of 88 samples, as 75 nucleotide-long single-end reads, were sequenced on the NextSeq
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
From cell line infected with NAIVE
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| Data processing |
Kallisto was used to determine transcript expression levels from reads. Kallisto uses pseudo-alignments and is relatively more accepting of errors/variants in reads31
kallisto version 0.44.0 was used to generate the processed files.
A custom reference database of RNA sequences was used, it is attached to this dataset (bat_master_rna.fa)
The reference rna database was indexed using the command: kallisto index -i bat_23 -k 23 bat_master_rna.fa
The processed files were generated using the following commands,
For Single end reads:
kallisto quant -i ref/bat_23 --single -t 30 -l 250 -s 60 -o outfile infile
For Paired-end reads;
kallisto quant -i ref/bat_23 -t 30 -o outfile infileR1 infileR2"
Genome_build: bat_master_rna.fa; a database of bat transcripts were assembled from a variety of sources, and the genes were names on the basis of mouse and/or human homologues
Supplementary_files_format_and_content: Expression values from Kallisto
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| Submission date |
Jun 18, 2020 |
| Last update date |
Feb 02, 2023 |
| Contact name |
Ravi Sachidanandam |
| E-mail(s) |
ravi.mssm@gmail.com
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| Organization name |
Girihlet Inc.
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| Street address |
2945 Webster St
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| City |
Oakland |
| State/province |
CA |
| ZIP/Postal code |
94609 |
| Country |
USA |
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| Platform ID |
GPL28706 |
| Series (1) |
| GSE152728 |
Infection of bats and cell cultures with Marburg, Ebola and Rabies viruses |
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| Relations |
| BioSample |
SAMN15313483 |
| SRA |
SRX8572420 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4625304_ro6_nv_12h_1.csv.gz |
279.0 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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