title: NHP17.D005.fresh.a1

description:
Animal: NHP17; Day post infection: D005; Fresh or freeze-thawed: fresh; MDCK Spike in: False; Array number: a1
Data for a given Seq-Well array for a specific biological sample
Combined data from Illumina NovaSeq 6000 and NextSeq 550 High Output
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.

value: Peripheral blood mononuclear cells (PBMC)


title: NHP18.D000.fresh.a1

status: Public on Sep 23 2020

type: SRA

city: Cambridge

first: Dylan

last: Kotliar

state: MA

address: 4 Garnder Rd, Apt 2

department: Viral genomics

country: USA

institute: Broad Institute

description:
Animal: NHP18; Day post infection: D000; Fresh or freeze-thawed: fresh; MDCK Spike in: False; Array number: a1
Data for a given Seq-Well array for a specific biological sample
Combined data from Illumina NovaSeq 6000 and NextSeq 550 High Output
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.

library_source: transcriptomic

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

molecule: total RNA

organism: Macaca mulatta

source_name: Peripheral blood mononuclear cells (PBMC)

extract_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.

tag: fresh_vs_freezethawed (frz)

value: fresh

value: FALSE

tag: library_treatment (standard, dash, dashx2 if dash was applied sequentially, or mixed if dash-treated and standard libraries were combined)

value: mixed

tag: tissue


title: NHP10.D007.fresh.a1

description:
Animal: NHP10; Day post infection: D007; Fresh or freeze-thawed: fresh; MDCK Spike in: False; Array number: a1
Data for a given Seq-Well array for a specific biological sample
Combined data from Illumina NovaSeq 6000 and NextSeq 550 High Output
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.


title: NHP08.D-04.fresh.a1

description:
Animal: NHP08; Day post infection: D-04; Fresh or freeze-thawed: fresh; MDCK Spike in: False; Array number: a1
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.

value: DASH


title: NHP24.D006.fresh.a1

description:
Animal: NHP24; Day post infection: D006; Fresh or freeze-thawed: fresh; MDCK Spike in: False; Array number: a1
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.

value: std


title: NHP23.D006.fresh.a2

description:
Animal: NHP23; Day post infection: D006; Fresh or freeze-thawed: fresh; MDCK Spike in: False; Array number: a2
Data for a given Seq-Well array for a specific biological sample
Combined data from Illumina NovaSeq 6000 and NextSeq 550 High Output
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.


title: NHP15.D003.fresh.a1

description:
Animal: NHP15; Day post infection: D003; Fresh or freeze-thawed: fresh; MDCK Spike in: False; Array number: a1
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.


title: NHP24.D006.fresh.a2

description:
Animal: NHP24; Day post infection: D006; Fresh or freeze-thawed: fresh; MDCK Spike in: False; Array number: a2
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.


title: NHP11.D006.fresh.a1

description:
Animal: NHP11; Day post infection: D006; Fresh or freeze-thawed: fresh; MDCK Spike in: False; Array number: a1
Data for a given Seq-Well array for a specific biological sample
Combined data from Illumina NovaSeq 6000 and NextSeq 550 High Output
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.


title: NHP18.D005.fresh.a1

description:
Animal: NHP18; Day post infection: D005; Fresh or freeze-thawed: fresh; MDCK Spike in: False; Array number: a1
Data for a given Seq-Well array for a specific biological sample
Combined data from Illumina NovaSeq 6000 and NextSeq 550 High Output
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.


title: whole blood, preHD B 16, year 3

type: RNA

city: Leicester

first: Christopher

last: Mitchell

address: University Road

department: Neuroscience, Psychology & Behaviour

country: United Kingdom

institute: University of Leicester

description: Gene expression data from peripheral whole blood of pre-symptomatic Huntingtin gene change carrier

hyb_protocol: Affymetrix Eukaryotic Target Hybridization protocol (Affymetrix GeneChip® Expression Analysis Technical Manual).

scan_protocol: Affymetrix® GeneChip® Scanner 3000 (Affymetrix GeneChip® Expression Analysis Technical Manual).

data_processing: Probe sets were normalised using robust multi-array averaging (RMA) with the R/Bioconductor package affy.

label: biotin

organism: Homo sapiens

source_name: Peripheral whole blood

label_protocol: Year 1 Samples: The Ovation Whole Blood Kit and Encore Biotin Module (Nugen, Inc.) was used according to the manufacturer’s protocol.    Year 3 Samples: The Ovation RNA Amplification System V2 (Nugen, Inc.) was used according to the manufacturer’s protocol.

extract_protocol: Blood was stored in PAX Gene tubes from which RNA was extracted using the PAX RNA isolation kit according to manufacturer’s protocol.

tag: year

tag: Sex

value: Female

tag: age at year 1

tag: location

value: london

tag: disease stage

value: preHD B

tag: expanded allele cag repeat count

tag: uhdrs tms

tag: uhdrs tfc


title: whole blood, preHD B 17, year 1

value: vancouver


title: whole blood, preHD B 17, year 3


title: whole blood, preHD B 16, year 1


title: Primary human Sertoli cells

city: Tucson

first: Nathan

middle: J.

last: Cherrington

state: AZ

address: 1703 E. Mabel St.

department: Pharmacology & Toxicology

institute: The University of Arizona

description:
CTRL (Primary hSerC)
Cell line

hyb_protocol: Experiment performed at: University of Arizona Cancer Center Genomics Shared Service Core, University of Arizona, Tucson, Arizona, USA.

data_processing:
Data were analyzed using the Affymetrix TAC software performed by University of Arizona Cancer Center Genomics Shared Service Core, University of Arizona, Tucson, Arizona, USA.
probe group file: Clariom_D_Human.r1.pgf
meta-probeset file: Clariom_D_Human.na36.hg38.probeset.csv

source_name: primary hSerCs

growth_protocol: Primary hSerCs were grown in DMEM/F12 supplemented with 10 μg/mL human insulin, 2.5 ng/mL EGF, 10% fetal bovine serum, and 1% penicillin and streptomycin. hTERT hSerCs were grown in similar conditions as the primary hSerCs with the added constant selection pressure of 1 μg/mL puromycin.

extract_protocol: Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol.

treatment_protocol: pBABE-puro-hTERT plasmid was transfected into Phoenix-AMPHO packaging cells to produce retroviruses encoding the plasmid. Retroviruses encoding the plasmid in the cell culture media supernatant were used to transduce primary human Sertoli cells (hSerCs), thus resulting in an hTERT-immortalized human Sertoli cell line (hTERT hSerC).

tag: gender

value: male

value: testis

tag: cell type

value: Sertoli cell

tag: genotype/variation

value: Wild-type


title: Human TERT-immortalized human Sertoli cells

description:
hTERT hSerC
Cell line

scan_protocol: Experiment performed at: University of Arizona Cancer Center Genomics Shared Service Core, University of Arizona, Tucson, Arizona, USA.

source_name: hTERT-immortalized hSerCs

value: + hTERT


title: NHP14.D003.fresh.a1

description:
Animal: NHP14; Day post infection: D003; Fresh or freeze-thawed: fresh; MDCK Spike in: False; Array number: a1
Data for a given Seq-Well array for a specific biological sample
Combined data from Illumina NovaSeq 6000 and NextSeq 550 High Output
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.


title: NHP14.D000.FRZ.a3

description:
Animal: NHP14; Day post infection: D000; Fresh or freeze-thawed: FRZ; MDCK Spike in: False; Array number: a3
Data for a given Seq-Well array for a specific biological sample
Combined data from Illumina NovaSeq 6000 and NextSeq 550 High Output
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.

value: FRZ


title: low response profile cell 18

city: Valbonne - Sophia-Antipolis

first: Kevin

last: Lebrigand

address: 660 route des lucioles

country: France

institute: IPMC/CNRS

description:
Hela cells in culture were treated with TRAIL for 50min, then isolated using laser capture microdissection.
Low.18

data_processing:
Mapping of cDNA reads was performed using STAR against human hg19 build following encode RNA-seq recommendations. Drop-seq tools (http://mccarrolllab.com, Macosko et al, 2015) was then used to tag SAM records with cell barcode (XC) and molecule identifier (XM) and produce the digital expression count matrix.
ERCC were removed from the count table, then size factors were calculated for each cell using the TMM method, and librairies were normalized and converted to log2(CPMs+1). All processing was performed using the R package edgeR.
Genome_build: hg19
Supplementary_files_format_and_content: Processed data files includes a raw count table and  a filtered and normalized count table. Normalized counts are provided as log2(CPM + 1).

molecule: polyA RNA

source_name: HeLa cells, modified with stable retroviral transformation of plasmid IC-RP: Addgene Plasmid #24542

growth_protocol: DMEM Glutamax supplemented with 10% fetal bovine serum and penicillin / streptomycin (Thermo Fisher Scientific, France)

extract_protocol:
Laser-captured single-cells were catapulted into 0.2ml domed caps tube (strips) containing 4.3 µl of lysis buffer (0,2% w/v Tween 20, 1.5U/µl Promega RNAseIN Plus). Tubes where kept on ice and frozen in dry ice directly after collection. Samples were thawed on ice and 2.43μl of a mix containing 10,000 molecules Life Technologies ERCC spike-In Mix 1, 2.5mM dNTPs, and 3.7µM single barcoded reverse transcription primer was added to each tube, incubated for 10 min at room temperature, 3 min at 70°C and kept at 10°C. Immediately after lysis, 8.27 µl 1.8X reverse transcription buffer (Invitrogen), 4.53mM DTT, 1.8M Betaïne, 10.8 mM MgCl2, 3.62 µM template switching oligonucleotide, 0.6U/µl Promega RNAse IN Plus, 7.5 U/µl Invitrogen SuperScript II reverse transcriptase were added and samples were reverse transcribed 10 min at 25°C, 90 min at 42°C, 15 min at 70°C and kept at 10°C until PCR amplification. Samples were PCR amplified (KAPA HiFi Hotstart) 20 cycles in 31 μl with 0.8µM forward PCR primer, 0.8µM biotinylated reverse PCR primer. Samples were incubated 3 min at 98°C followed by 20 cycles at 98°C for 20 sec, 64°C for 15 sec, 72°C for 6 min followed by a final extension for 5 min at 72°C. After PCR amplification, samples were pooled and purified with 0.8X Beckman Coulter SPRIselect beads, washed once with 85% EtOH and eluted in 30 μl.
Batches of barcoded cDNAs were pooled, purified with SPRIselect beads (0.8x) and eluted in 12µl. Nine µl (typically 10 – 20 ng of cDNA) were used for tagmentation and Illumina library preparation as described in Arguel MJ et al. NAR 2017

treatment_protocol: Recombinant human (rh) TRAIL (R&D Systems Europe - Lille, France) was added to culture media for 50 minutes at a final concentration of 25 nanograms per millilitre.

tag: cell line

value: HeLa

tag: cell response

value: Low

tag: treatment

value: TRAIL 25ng/ml for 50min


title: NHP22.D006.fresh.a2

description:
Animal: NHP22; Day post infection: D006; Fresh or freeze-thawed: fresh; MDCK Spike in: False; Array number: a2
Data for a given Seq-Well array for a specific biological sample
Combined data from Illumina NovaSeq 6000 and NextSeq 550 High Output
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.

tag: fresh_vs_freezethawed (frz)

value: fresh

value: FALSE

value: Peripheral blood mononuclear cells (PBMC)


title: NHP13.D003.fresh.a1

description:
Animal: NHP13; Day post infection: D003; Fresh or freeze-thawed: fresh; MDCK Spike in: False; Array number: a1
Data for a given Seq-Well array for a specific biological sample
Combined data from Illumina NovaSeq 6000 and NextSeq 550 High Output
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.

tag: library_treatment (standard, dash, dashx2 if dash was applied sequentially, or mixed if dash-treated and standard libraries were combined)

value: mixed


title: NHP15.D003.FRZ-MDCK.a4

description:
Animal: NHP15; Day post infection: D003; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a4
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.

value: TRUE

value: std

tag: tissue


title: NHP14.D000.FRZ-MDCK.a1

description:
Animal: NHP14; Day post infection: D000; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a1
Data for a given Seq-Well array for a specific biological sample
Combined data from Illumina NovaSeq 6000 and NextSeq 550 High Output
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.


title: low response profile cell 16

description:
Hela cells in culture were treated with TRAIL for 50min, then isolated using laser capture microdissection.
Low.16

source_name: HeLa cells, modified with stable retroviral transformation of plasmid IC-RP: Addgene Plasmid #24541


title: low response profile cell 9

description:
Hela cells in culture were treated with TRAIL for 50min, then isolated using laser capture microdissection.
Low.9

source_name: HeLa cells, modified with stable retroviral transformation of plasmid IC-RP: Addgene Plasmid #24540


title: NHP23.D006.FRZ-MDCK.a6

description:
Animal: NHP23; Day post infection: D006; 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.


title: low response profile cell 3

description:
Hela cells in culture were treated with TRAIL for 50min, then isolated using laser capture microdissection.
Low.3

source_name: HeLa cells, modified with stable retroviral transformation of plasmid IC-RP: Addgene Plasmid #24537


title: NHP23.D006.FRZ-MDCK.a3

description:
Animal: NHP23; Day post infection: D006; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a3
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.


title: NHP22.D006.FRZ-MDCK.a6

description:
Animal: NHP22; Day post infection: D006; 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.


title: low response profile cell 7

description:
Hela cells in culture were treated with TRAIL for 50min, then isolated using laser capture microdissection.
Low.7

source_name: HeLa cells, modified with stable retroviral transformation of plasmid IC-RP: Addgene Plasmid #24539


title: low response profile cell 4

description:
Hela cells in culture were treated with TRAIL for 50min, then isolated using laser capture microdissection.
Low.4

source_name: HeLa cells, modified with stable retroviral transformation of plasmid IC-RP: Addgene Plasmid #24538


title: NHP22.D006.FRZ-MDCK.a5

description:
Animal: NHP22; Day post infection: D006; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a5
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.


title: NHP23.D006.FRZ-MDCK.a5

description:
Animal: NHP23; Day post infection: D006; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a5
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.


title: NHP22.D006.FRZ-MDCK.a4

description:
Animal: NHP22; Day post infection: D006; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a4
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.


title: NHP19.D004.FRZ-MDCK.a6

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.


title: low response profile cell 1

description:
Hela cells in culture were treated with TRAIL for 50min, then isolated using laser capture microdissection.
Low.1

source_name: HeLa cells, modified with stable retroviral transformation of plasmid IC-RP: Addgene Plasmid #24536


title: NHP19.D004.FRZ-MDCK.a5

description:
Animal: NHP19; Day post infection: D004; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a5
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.


title: NHP13.D000.fresh.a1

description:
Animal: NHP13; Day post infection: D000; Fresh or freeze-thawed: fresh; MDCK Spike in: False; Array number: a1
Data for a given Seq-Well array for a specific biological sample
Combined data from Illumina NovaSeq 6000 and NextSeq 550 High Output
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.


title: NHP19.D004.FRZ-MDCK.a4

description:
Animal: NHP19; Day post infection: D004; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a4
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.


title: NHP15.D003.FRZ-MDCK.a3

description:
Animal: NHP15; Day post infection: D003; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a3
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.


title: NHP15.D003.FRZ-MDCK.a2

description:
Animal: NHP15; Day post infection: D003; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a2
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.


title: whole blood, preHD B 14, year 3

description: Gene expression data from peripheral whole blood of pre-symptomatic Huntingtin gene change carrier

tag: year

value: Male

value: preHD B


title: whole blood, preHD B 13, year 3

tag: Sex

value: vancouver


title: whole blood, preHD B 15, year 3

value: Female

tag: age at year 1


title: NHP23.D006.FRZ-MDCK.a2

description:
Animal: NHP23; Day post infection: D006; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a2
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.


title: NHP15.D003.FRZ-MDCK.a1

description:
Animal: NHP15; Day post infection: D003; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a1
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.


title: whole blood, preHD B 12, year 1

tag: location

value: london


title: NHP23.D006.FRZ-MDCK.a1

description:
Animal: NHP23; Day post infection: D006; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a1
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.