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| Status |
Public on Sep 23, 2020 |
| Title |
NHP19.D004.FRZ-MDCK.a6 |
| Sample type |
SRA |
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| Source name |
Peripheral blood mononuclear cells (PBMC)
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| Organism |
Macaca mulatta |
| Characteristics |
donor_animal: NHP19
day_post_infection: 4
fresh_vs_freezethawed (frz): FRZ
mdck_spike_in: TRUE
library_treatment (standard, dash, dashx2 if dash was applied sequentially, or mixed if dash-treated and standard libraries were combined): std
tissue: Peripheral blood mononuclear cells (PBMC)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Library construction and sequencing was performed with the S3 protocol (Hughes et al., 2019). We sequenced all libraries on either NextSeq 550 High Output or NovaSeq 6000 S2 flowcells (Illumina, San Diego, CA, USA), with 20 cycles for Read 1 (cell barcode and unique molecular index [UMI]) and 88 cycles for Read 2 (cDNA of interest). In some cases, we merged fastq files from multiple sequencing runs for increased coverage.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Animal: NHP19; Day post infection: D004; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a6
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.
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| Data processing |
Library strategy: scRNA-Seq
Raw sequencing files were demultiplexed and converted to fastq using bcl2fastq version 2.20.
Reads were then trimmed, aligned to a reference transcriptome, and parsed into a digital gene expression matrix using the previously published Dropseq-tools pipeline (Macosko et al., 2015)
The scRNA-Seq data was preprocessed, clustered, and visualized using Scanpy (Wolf et al., 2018). We removed cells with <300 genes detected, >10% of their UMIs derived from mitochondrial genes, or >95% of UMIs mapped to non-genic regions. We excluded ribosomal genes, genes correlated with the percentage of UMIs assigned to mitochondrial genes (Pearson R > 0.1), and HBB as these were largely driven by the technical covariate of whether cells had loaded into Seq-Well arrays fresh or had undergone a freeze-thaw cycle with cryoprotectant. We also excluded EBOV genes and cell-cycle genes (defined by correlation with TOP2A, Pearson R > 0.1) prior to clustering so that these signals would not influence identification of cell types.
See https://www.biorxiv.org/content/10.1101/2020.06.12.148957v2 for details
Genome_build: hybrid of Mmul_8.01 and KU182905.1
Supplementary_files_format_and_content: digital gene expression matrix were generated using the previously published Dropseq-tools pipeline
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| Submission date |
Sep 23, 2020 |
| Last update date |
Sep 24, 2020 |
| Contact name |
Dylan Kotliar |
| E-mail(s) |
Dylan_Kotliar@HMS.Harvard.edu
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| Phone |
7323790215
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| Organization name |
Broad Institute
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| Department |
Viral genomics
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| Lab |
Sabeti Lab
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| Street address |
4 Garnder Rd, Apt 2
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL27943 |
| Series (2) |
| GSE158390 |
Single-cell profiling of Ebola virus infection in vivo reveals viral and host transcriptional dynamics |
| GSE158442 |
Single-cell profiling of Ebola virus infection in vivo reveals viral and host transcriptional dynamics (InVivo scRNA-seq) |
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| Relations |
| BioSample |
SAMN16246619 |
| SRA |
SRX9178177 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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