title: Treatment Patient 3 [ATAC-Seq]

city: Clayton

first: Shanti

last: Gurung

state: Vic

address: 27-31 Wright Street

country: Australia

institute: Hudson Institute of Medical Research

data_processing:
ATAC-seq libraries were sequenced with 100 base pair (bp) paired-ends on a HiSeq 2500 (Illumina), and mapped to the hg19 human genome assembly using bowtie2-2.2.6 and samtools-1.2.0.
Peaks were called on a merged mapped file using MACS-2.1.0, leading to the identification of 132,296 peaks. 100,155 of these peaks were deemed high confidence (q-value < 1  10-7) and were used for further analysis.
To determine differential chromatin opening, HTSeq-0.6.1 was used to assign reads to peaks and DESeq2 was used to identify differential peaks.
Genome_build: hg19
Supplementary_files_format_and_content: ATAC-seq peak coordinates and change upon decidualization in .XLSX format

source_name: Endometrial mesenchyme stem cells

growth_protocol: Endometrial biopsies were processed and single-cell suspensions of eMSCs obtained as described previously (Masuba et al. 2012).

extract_protocol: ATAC-seq was performed as described (Buenrostro et al 2013), although with some modifications. Briefly, cells were washed with cold Dulbecco's Phosphate Buffered Saline (PBS) and then lysed using cold EZ lysis buffer (Sigma-Aldrich, UK). The cells were transferred to chilled pre-labelled nuclease-free 1.5 ml micro-centrifuge tubes. Samples were vortexed, left on ice for 5 minutes, then pelleted in a fixed-angle refrigerated centrifuge. Supernatant was carefully removed and the pellet was washed in EZ lysis buffer. The nuclear pellet was re-suspended in the transposase reaction mix, containing 25 μl Tagment DNA (TD) Buffer, 5 μl Tagment DNA Enzyme and 20 μl nuclease free water (Nextera DNA sample preparation kit, Illumina, UK).  The transposition reaction was carried out for either 45 min at 37 °C. Samples were purified using a Zymo DNA Clean and Concentrator-5 Purification kit, according to the manufacturer’s instructions. Briefly, DNA binding buffer was added to the 50 μl sample which was then mixed and transferred to the column. Samples were centrifuged at 17,000 × g for 30 secs at room temperature (RT). Flow-through was discarded and 200 μl DNA wash buffer added, and columns centrifuged as described above. The wash and centrifugation steps were repeated twice. After removing the residual liquid, 23 μl pre-warmed elution buffer was added to the columns. Samples were incubated for 2 min at RT, then centrifuged for 2 min to elute DNA. Following purification, 20 μl sample was added to a 0.2 mL PCR tube containing the following reagents (Nextera DNA sample preparation kit and Nextera index kit, Illumina): 5 μl index 1, 5 μl index 2, 15 μl Master mix (NPM), 5 μl Primer Cocktail (PPC). Amplification was performed in a Veriti 96 Well Thermal Cycler (Applied Biosystems, USA) using the following PCR conditions: 72 °C for 3 min, 98 °C for 30 secs then 15 cycles of 98 °C for 10 secs, 63 °C for 30 secs, 72 °C for 1 min. The libraries were purified using AMPure XP beads according to the Illumina Nextera kit recommended protocol. The amplified libraries were quantified by using Qubit HS DNA Assay on a Qubit 2.0 Fluorometer according to the manufacturer’s instructions. Library sizes were assessed on an Agilent Bioanalyzer using the High Sensitivity DNA chip.

treatment_protocol: eMSCs were cultured in DMEM/F12 medium containing 10% FCS, 1% primocin and 2mM glutamine (Invitrogen), supplemented with 10 ng/ml basic fibroblast growth factor (bFGF) (Peprotech) and slowly scaled-down to an in-house DMEM/F12 serum free medium (SFM) and incubated at 37 C in 5%CO2/5% O2/90% N2 with 1uM TGFbeta-R inhibitor (A83-01 - Trocis Bioscience) or without (0.01% DMSO). Cells were seeded at 5,000 cells/cm2 with medium changed every 48h and passaged on days 15, 22, 29 and 36 onto fibronectin-coated clusture flasks as described previously (Gurung et al. 2015). Treated and untreated eMSC cultures were subjected to ATAC-seq at passage 4 after 36 days in culture.

value: Endometrial mesenchyme stem cells

tag: patient id

value: Patient 3

tag: treatment

value: A83-01 treated


title: NHP09.D008.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: NHP09; Day post infection: D008; 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.

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)

tag: tissue


title: NHP08.D006.fresh.a1

description:
Animal: NHP08; 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: Peripheral blood mononuclear cells (PBMC)


title: Control Patient 3 [ATAC-Seq]

library_source: genomic

molecule: genomic DNA

organism: Homo sapiens

tag: cell type

value: control


title: Treatment Patient 1 [ATAC-Seq]

value: Patient 1


title: Control Patient 2 [ATAC-Seq]

value: Patient 2


title: WT2: U2OS_WT_2

type: genomic

city: Grand Rapids

first: Scott

last: Rothbart

state: MI

address: 333 Bostwick Avenue NE

institute: Van Andel Research Institute

hyb_protocol: Bisulfite converted D was amplified, fragmented and hybridized to Illumina Infinium Human MethylationEPIC Beadchip using standard Illumina protocol

scan_protocol: Standard Illumina Protocol

data_processing: Raw IDAT files for each sample were processed using the Bioconductor package “SeSAMe” (Version 1.2.0) for extraction of probe signal intensity values, normalization of probe signal intensity values, and calculation of β-values from the normalized probe signal intensity values. CpG probes with a detection p-value > 0.05 in any one sample were excluded from the analysis.

label: Cy5 and Cy3

source_name: U2OS Bone Osteosarcoma epithelial cells

label_protocol: Standard Illumina protocol

growth_protocol: U2OS cells were grown in 10% FBS with high glucose DMEM without antibiotics (GIBCO cat no. 11965092). Cell culturing utilized standard laboratory practices and cells were grown and incubated at 37°C containing 5% CO2.

extract_protocol: Cells were lysed overnight at 37°C in 2 mls of TE-SDS buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 0.5% SDS), supplemented with 100 μl of 20 mg/ml proteinase K. D was purified by phenol:chloroform extraction in three phases: (1) 100% phenol, (2) phenol:chloroform:isoamyl alcohol (25:24:1), and (3) chloroform:isoamyl alcohol (24:1). For each phase, the aqueous layer was combined with the organic layer in a 1:1 ratio. Samples were quickly shaken, allowed to sit on ice for approximately 5 minutes, and then separated by centrifugation at 1,693 RCF for 5 minutes at 4°C. The top aqueous layer was then transferred to a new tube for the next organic phase. Following extraction, DNA was precipitated with 1/10 volume 3 M sodium acetate pH 4.8 and 2.5 volumes 100% ethanol and stored overnight at -20°C. Precipitated DNA was pelleted by centrifugation at 17,090 RCF for 30 minutes at 4°C. The pelleted DNA was washed twice with 70% ethanol, allowed to dry for 15 minutes, and resuspended in TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA). Samples were then treated with 1 mg/ml RNAse A at 37°C for 30 minutes and then re-purified by ethanol precipitation as described above.

treatment_protocol: GFP-UHRF1WT and GFP-UHRF1KEN cells were generated by transducing U2OS cell lines with pHAGE-GFP lentivirus that had been produced in HEK293T cells. Infections were performed in the presence of 8µg/mL polybrene for 48 hours prior to antibiotic selection. Cells were selected for 5-7 days with 8ug/mL.

value: U2OS Bone Osteosarcoma epithelial cells

tag: genotype/variation

value: GFP-UHRF1-WT overexpression


title: KEN1: U2OS_KEN_1

value: GFP-UHRF1-KEN/AAA overexpression


title: P3: U2OS_parental_3

value: Parental cell line


title: NHP19.D004.FRZ-MDCK.a3

description:
Animal: NHP19; Day post infection: D004; 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.

value: FRZ

value: TRUE

value: std


title: whole blood, preHD A 14, year 1

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

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: Male

tag: age at year 1

tag: location

value: vancouver

tag: disease stage

value: preHD A

tag: expanded allele cag repeat count

tag: uhdrs tms

tag: uhdrs tfc


title: whole blood, preHD A 14, year 3


title: P1: U2OS_parental_1


title: whole blood, preHD A 13, year 1


title: NHP24.D006.FRZ.a3

description:
Animal: NHP24; Day post infection: D006; Fresh or freeze-thawed: FRZ; MDCK Spike in: False; 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: Control Patient 1 [ATAC-Seq]

value: Endometrial mesenchyme stem cells


title: whole blood, preHD A 12, year 1

value: london


title: NHP23.D-30.fresh.a1

description:
Animal: NHP23; Day post infection: D-30; 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: fresh

value: FALSE

value: DASH


title: NHP16.D000.FRZ.a2

description:
Animal: NHP16; Day post infection: D000; Fresh or freeze-thawed: FRZ; 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.

value: Peripheral blood mononuclear cells (PBMC)


title: NHP14.D000.FRZ.a2

description:
Animal: NHP14; Day post infection: D000; Fresh or freeze-thawed: FRZ; 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: NHP10.D-04.fresh.a1

description:
Animal: NHP10; 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.


title: NHP09.D-04.fresh.a1

description:
Animal: NHP09; 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.


title: NHP20.D-30.fresh.a1

description:
Animal: NHP20; Day post infection: D-30; 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: whole blood, preHD A 12, year 3


title: whole blood, preHD A 10, year 1

value: Female


title: whole blood, preHD A 13, year 3


title: N57-E15-Ts1Cje-Apigenin

city: Bethesda

first: Faycal

last: Guedj

state: Maryland

address: 35A Convent DR

department: NHGRI

institute: National Institute of Health

hyb_protocol: Arrays were washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Hybridization, Wash and Stain Kit according to the manufacturer's instructions (Affymetrix, Santa Clara, CA).

scan_protocol: Arrays were scanned on a GeneArray Scanner 3000 7G, using the GeneChip Microarray Suite 5.0 (Affymetrix, Santa Clara, CA).

data_processing: Quality control and normalization were performed using the pipeline at the www.arrayanalysis.org website (Maastricht University, the Netherlands). Normalization was done using the Robust Multichip Average (RMA) algorithm and the MBNI custom CDF (http://brainarray.mbni.med.umich.edu/) version #14 for the mouse gene 1.0 ST array. Normalization output consisted of data for 21225 probe sets each corresponding to unique Entrez Gene IDs.

organism: Mus musculus

source_name: Forebrain from day 15 embryo

label_protocol: Samples were labeled with Biotin Allonamide Triphosphate according to the GeneChip Whole Transcriptome Labeling and Hybridization protocol (http://www.affymetrix.com) the Affymetrix GeneChip WT PLUS Reagent Kit (Thermo Fisher Scientific, Waltham, MA).

growth_protocol: Mice were housed in standard cages with food and water ad libitum under a controlled environment (temperature = 20°C; humidity = 60%) and a light/dark cycle of 12h. Ts1Cje (Jax Stock 004838) males were crossed to C57Bl/6J (Jax Stock 000664) females. For embryonic studies, the presence of a vaginal plug was defined as embryonic day 0.5 (E0.5) and 10-15% weight gain at embryonic day 10 was used to confirm pregnancy. Pregnant females were anesthetized with 2.5% isoflurane in a 3/7 02/N2O mixture and euthanized by decapitation at embryonic day E15.5 (E15.5). Embryos were extracted and decapitated in ice-cold phosphate-buffered-saline (PBS1X). Embryonic brains were rapidly removed and brain hemispheres dissected on a cold platform and snap frozen in liquid nitrogen before storage at -80°C. For adult studies, 6-7 month old animals were euthanaized as described above and brains were removed and dissected on a cold platform. Cerebral cortex, hippocampus and cerebellum were snap frozen in liquid nitrogen before storage at -80°C.

extract_protocol: Total RNA was isolated using the NucleoSpin RNA II kits following the manufacturer’s instructions (Macherey-Nagel, Bethlehem, PA). RNA concentrations were measured as absorbance at 260 nm on the Nanodrop instrument (Thermo Fisher Scientific, Waltham, MA). RNA quality was analyzed with the Bioanalyzer (Agilent Biotechnologies, Santa Clara, CA) using the RNA 6000 Nano kit according to the manufacturer’s instructions. Only samples displaying an A260/A280 ratio of 2.0-2.1 and a RIN (RNA Integrity Number) > 9 were used for hybridization to the arrays.

treatment_protocol: Breeding pairs received either purified powdered (Pow) chow F3197 (Bioserv, Flemington, NJ) or 333-400 mg/kg/day of apigenin (Apig) (2 g of apigenin in 1 kg of purified powdered chow) (SelleckChem, Houston, TX). Treatment was given as powdered chow with apigenin starting at the time of mating and continuing throughout pregnancy and lactation until all behavioral and biochemical studies in adulthood were completed.

value: forebrain

tag: developmental stage

tag: genotype

value: Apigenin


title: N28-E15-Ts1Cje-Apigenin


title: whole blood, preHD A 10, year 3


title: NHP11.D-04.fresh.a1

description:
Animal: NHP11; 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.


title: NHPC1.D000.fresh.a1

description:
Animal: NHPC1; 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
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.D000.fresh.a1

description:
Animal: NHP15; 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
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: N58-E15-Ts1Cje-Apigenin


title: whole blood, preHD A 11, year 1


title: NHP19.D004.fresh.a2

description:
Animal: NHP19; Day post infection: D004; 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: NHP16.D000.fresh.a1

description:
Animal: NHP16; 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
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: N1-E15-Ts1Cje-Apigenin


title: N9-E15-Ts1Cje-Apigenin

value: WT


title: N20-E15-Ts1Cje-Apigenin


title: whole blood, preHD A 09, year 3


title: P2: U2OS_parental_2

value: U2OS Bone Osteosarcoma epithelial cells

value: Parental cell line


title: N10-E15-Ts1Cje-Apigenin


title: whole blood, preHD A 08, year 3


title: whole blood, preHD A 05, year 3


title: NHP22.D006.fresh.a1

description:
Animal: NHP22; 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.


title: whole blood, preHD A 06, year 3


title: whole blood, preHD A 08, year 1


title: N59-E15-Ts1Cje-Apigenin


title: whole blood, preHD A 06, year 1


title: N93E15-Ts1Cje-Powder

value: Powder


title: N55-E15-Ts1Cje-Apigenin


title: N13-E15-Ts1Cje-Apigenin


title: Treatment Patient 2 [ATAC-Seq]

tag: patient id

value: Patient 2

value: A83-01 treated


title: whole blood, preHD A 05, year 1

value: vancouver


title: whole blood, preHD A 11, year 3

value: preHD A


title: whole blood, preHD A 04, year 1

value: Male


title: whole blood, preHD A 04, year 3


title: NHP23.D006.fresh.a1

description:
Animal: NHP23; 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: NHP19.D004.fresh.a1

description:
Animal: NHP19; Day post infection: D004; 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: fresh


title: N110E15-Ts1Cje-Powder

tag: treatment


title: NHP17.D000.fresh.a1

description:
Animal: NHP17; 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
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: FALSE


title: NHP2.H024.a4.live

description:
Donor replicate 2; 24 hours post infection; Treatment: live; Seq-well array 4
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: live


title: NHP2.H024.a3.live

description:
Donor replicate 2; 24 hours post infection; Treatment: live; Seq-well array 3
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: Peripheral blood mononuclear cells (PBMC)


title: NHP2.H024.a2.live

description:
Donor replicate 2; 24 hours post infection; Treatment: live; Seq-well array 2
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: NHP12.D008.fresh.a1

description:
Animal: NHP12; Day post infection: D008; 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.

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

value: DASH


title: NHP2.H024.a1.media

description:
Donor replicate 2; 24 hours post infection; Treatment: media; Seq-well array 1
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: media

tag: tissue


title: NHP2.H024.a1.live

description:
Donor replicate 2; 24 hours post infection; Treatment: live; Seq-well array 1
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: Teosinte WGBS_JRIAL2T

city: Lincoln

first: GEN

last: XU

state: Nebraska

address: 383 Plant Science Hall  Department of Agronomy and Horticulture

institute: University of Nebraska-Lincoln

description:
Whole genome bisulfite sequencing
Table_S1_DMR.bed

data_processing:
Whole genome sequencing reads were aligned to the B73 reference genome using BWA; Bisulfite sequencing data analysis was conducted using Bismark
Duplicate reads were removed by picard
We conducted SNP calling using Genome Analysis Toolkit’s(GATK, version 4) HaplotypeCaller
We used a software package ‘metilene’ for DMR detection between two populations
We calculated XPCLR to detect selective signals
Genome_build: B73 reference genome
Supplementary_files_format_and_content: bed files include selective sweeps and differentially methylated regions.

organism: Zea mays

growth_protocol: Plants were growed in greenhouse

extract_protocol:
CTAB method for DNA extraction
Libraries were prepared for sequencing using standard Illumina protocols

value: 3rd leaf

tag: Stage

value: seedling

tag: population

value: Teosinte


title: NHP2.H024.a1.irrad

description:
Donor replicate 2; 24 hours post infection; Treatment: irrad; Seq-well array 1
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: irrad


title: whole blood, preHD A 03, year 1

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

tag: year

value: Female

value: london


title: Teosinte WGBS_JRIAL2S


title: Teosinte WGBS_JRIAL2R


title: NHP2.H004.a4.live

description:
Donor replicate 2; 04 hours post infection; Treatment: live; Seq-well array 4
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: N103E15-Ts1Cje-Powder

value: forebrain

value: Powder


title: NHP2.H004.a3.live

description:
Donor replicate 2; 04 hours post infection; Treatment: live; Seq-well array 3
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: NHP2.H004.a2.live

description:
Donor replicate 2; 04 hours post infection; Treatment: live; Seq-well array 2
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: N88E15-Ts1Cje-Powder

value: WT


title: N87E15-Ts1Cje-Powder


title: whole blood, preHD A 02, year 1

tag: Sex

value: Male

value: vancouver

value: preHD A


title: N108E15-Ts1Cje-Powder