title: NHP22.D006.FRZ-MDCK.a3

description:
Animal: NHP22; Day post infection: D006; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a3
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.


title: whole blood, preHD B 12, year 3

status: Public on Sep 23 2020

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

molecule: total RNA

organism: Homo sapiens

source_name: Peripheral whole blood

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

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

tag: year

tag: Sex

value: Male

tag: age at year 1

tag: location

value: london

tag: disease stage

value: preHD B

tag: expanded allele cag repeat count

tag: uhdrs tms

tag: uhdrs tfc


title: whole blood, preHD B 13, year 1

value: vancouver


title: NHP22.D006.FRZ-MDCK.a1

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: NHP22; Day post infection: D006; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a1
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.

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

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: FRZ

value: TRUE

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

value: std

tag: tissue


title: NHP22.D006.FRZ-MDCK.a2

description:
Animal: NHP22; Day post infection: D006; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a2
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.

value: Peripheral blood mononuclear cells (PBMC)


title: whole blood, preHD B 15, year 1

value: Female


title: whole blood, preHD B 14, year 1


title: NHP19.D004.FRZ-MDCK.a2

description:
Animal: NHP19; Day post infection: D004; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a2
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.


title: NHP19.D004.FRZ-MDCK.a1

description:
Animal: NHP19; Day post infection: D004; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a1
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.


title: NHP16.D005.FRZ.a2

description:
Animal: NHP16; Day post infection: D005; 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: FALSE


title: CEMBA181023_6B_1_CEMBA181023_6B_2_A12_ad001

city: La Jolla

first: Joseph

middle: R

last: Ecker

state: CA

address: 10010 North Torrey Pines Road

department: Genomic Analysis Laboratory

institute: HHMI-Salk-Institute

library_source: genomic

data_processing:
Library strategy: snmC-seq2
Reads trimed with Cutadapt for adapters and low quality bases (Phred score >= 20)
The resulting files are mapped to in-silico bisulfite converted mm10 reference genome using Bismark.
The raw BAM file will be filtered using samtools to filter reads by MAPQ > 10 and use Picard to remove PCR duplicates
Tab-delimited files (ALLC files) describing the quantity of unmethylated and methylated cytosine base calls will be generated with in-house developing package using the filtered BAM files
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited files (ALLC files) describing the quantity of unmethylated and methylated cytosine base calls. Columns are: 1. Chromosome, 2. Position, 3. Strand, 4. Cytosin Context, 5. methylated base call, 6. total mapped base call, 7. not used, place holder column

molecule: genomic DNA

organism: Mus musculus

source_name: C57BL/6J Male P56 Mouse Brain

growth_protocol: All mice are C57BL/6J Male, brain dissected in P56

extract_protocol:
the brain of P56 male C57BL/6J mouse will be removed and cut into 600 microns thick coronal sections using a tissue matrix. Then each region will be microdissected from the coronal tissue sections based on identified landmarks and brain atlas coordinates, as defined in Allen brain atlas CCF3 (Avaliable at: http://atlas.brain-map.org). For each dissection region, the tissue from several littermate mice will be combined together into to separate replicate groups. The number of animals in a replicate depends on the size of the tissue. Nuclei preparation will be stained with the fluorescently labeled anti-NeuN body and nucleic acid stain Hoechst 33342.
Library prepared following protocol discribed in Luo, Chongyuan, Angeline Rivkin, Jingtian Zhou, Justin P. Sandoval, Laurie Kurihara, Jacinta Lucero, Rosa Castanon, et al. 2018. “Robust Single-Cell DNA Methylome Profiling with snmC-seq2.” Nature Communications 9 (1): 3824.

tag: brain region

value: SSp

value: male

tag: age

tag: strain


title: NHP16.D005.FRZ.a1

description:
Animal: NHP16; Day post infection: D005; Fresh or freeze-thawed: FRZ; 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: NHP16.D005.FRZ-MDCK.a6

description:
Animal: NHP16; Day post infection: D005; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a6
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.


title: NHP16.D005.FRZ-MDCK.a5

description:
Animal: NHP16; Day post infection: D005; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a5
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.


title: whole blood, preHD B 10, year 3


title: whole blood, preHD B 11, year 1


title: whole blood, preHD B 11, year 3


title: NHP16.D005.FRZ-MDCK.a4

description:
Animal: NHP16; Day post infection: D005; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a4
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.


title: whole blood, preHD B 09, year 1


title: whole blood, preHD B 10, year 1


title: whole blood, preHD B 08, year 3


title: whole blood, preHD B 08, year 1


title: whole blood, preHD B 07, year 3


title: whole blood, preHD B 09, year 3


title: NHP14.D003.FRZ.a3

description:
Animal: NHP14; Day post infection: D003; 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: whole blood, preHD B 07, year 1


title: NHP14.D003.FRZ.a2

description:
Animal: NHP14; Day post infection: D003; 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: whole blood, preHD B 06, year 3


title: whole blood, preHD B 06, year 1


title: NHP14.D003.FRZ.a1

description:
Animal: NHP14; Day post infection: D003; Fresh or freeze-thawed: FRZ; 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: AtRH17 OX_14DAG_rep2

city: Busan

first: Yong-Hwan

last: Moon

address: Busandaehak-ro 63Beon-gil 2, Geumjeong-gu

department: Molecular Biology

country: South Korea

institute: Pusan National University

description: Gene expression in 14-day-old seedlings of AtRH17 OX grown under SD conditions (8-h-light and 16-h-dark cycle). Whole seedlings were sampled and total RNA was analyzed.

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence(using fastx_trimmer), then mapped to TAIR10 whole genome using Bowtie2
Read count extraction and nomalization were performed using edgeR.
Genome_build: TAIR10
Supplementary_files_format_and_content: tab-delimited text files include read count values for each Sample

organism: Arabidopsis thaliana

source_name: AtRH17 OX, whole seedlings, 14 DAG, SD

growth_protocol: Arabidopsis plants were of the Columbia (Col-0) ecotype background. Arabidopsis seeds were surface-sterilized and water-imbibed in the dark for 2 d at 4℃. The seeds were sown on 0.7% (w/v) agar plates containing on-half-strength Murashige and Skoog (MS) media supplemented with B5 vitamins and 1.5% (w/v) sucrose. They were grown at 22℃ under SD conditions (8-h-light/16-h-dark cycle).

extract_protocol:
RNA was harvested using Trizol reagent (Invitrogen). 500 ng of total RNA was used for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using LEXOGEN Quant-Seq Library Prep Kit (Cat#001.24) standard protocols.

treatment_protocol: No treatment

tag: ecotype

value: 14 days after germination

tag: genotype

value: whole seedlings


title: NHP14.D003.FRZ-MDCK.a6

description:
Animal: NHP14; Day post infection: D003; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a6
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.


title: AtRH17 OX_14DAG_rep1


title: NHP14.D003.FRZ-MDCK.a5

description:
Animal: NHP14; Day post infection: D003; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a5
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.


title: HRE1β OX_14DAG_rep2

description: Gene expression in 14-day-old seedlings of HRE1β grown under SD conditions (8-h-light and 16-h-dark cycle). Whole seedlings were sampled and total RNA was analyzed.

source_name: HRE1β OX, whole seedlings, 14 DAG, SD


title: whole blood, preHD B 05, year 3

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


title: HRE1β OX_14DAG_rep1


title: NHP23.D006.FRZ-MDCK.a4

description:
Animal: NHP23; Day post infection: D006; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a4
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.


title: NHP14.D003.FRZ-MDCK.a4

description:
Animal: NHP14; Day post infection: D003; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a4
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.


title: HRE1α OX_14DAG_rep2

description: Gene expression in 14-day-old seedlings of HRE1α grown under SD conditions (8-h-light and 16-h-dark cycle). Whole seedlings were sampled and total RNA was analyzed.

source_name: HRE1α OX, whole seedlings, 14 DAG, SD


title: whole blood, preHD B 05, year 1


title: whole blood, preHD B 04, year 3


title: NHP24.D006.FRZ.a2

description:
Animal: NHP24; Day post infection: D006; 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: HRE1α OX_14DAG_rep1


title: whole blood, preHD B 03, year 3


title: NHP24.D006.FRZ.a1

description:
Animal: NHP24; Day post infection: D006; Fresh or freeze-thawed: FRZ; MDCK Spike in: False; Array number: a1
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.


title: NHP24.D006.FRZ-MDCK.a6

description:
Animal: NHP24; Day post infection: D006; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a6
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.


title: WT_14DAG_control_rep1

description: Gene expression in 14-day-old seedlings of WT grown under SD conditions (8-h-light and 16-h-dark cycle). Whole seedlings were sampled and total RNA was analyzed.

source_name: WT, whole seedlings, 14 DAG, SD

value: WT


title: NHP24.D006.FRZ-MDCK.a5

description:
Animal: NHP24; Day post infection: D006; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a5
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.


title: Ulk1 dominant negative replicate 3

city: Goettingen

first: Gabriela

last: Salinas

state: Lower-Saxony

address: Kreuzbergring 57

department: Department of Pathology

country: Germany

institute: Universitaetsmedizin Goettingen

data_processing:
Demultiplexed to fastq files with bcl2fastq v2.17.1.14
Quality control of FastQ files with FastQC version 0.11.5
FastQ files aligned to rat genome using STAR version 2.5.2a using paired-end option and allowing for 2 mismatches within 50 bases
Reads counted for exons using dexseq (version 1.22.0) python script
Differential exon analysis was run using DEXSeq (version 1.22.0) with differential exons having FDR<0.05
Genome_build: rn6 version 93
Supplementary_files_format_and_content: gene and exon Counts

organism: Rattus norvegicus

source_name: Ulk

growth_protocol: Primary rat cortical neurons were prepared from embryonic day 18 (E18) rats according to standard protocols and cultured in cortex medium (serum-free neurobasal medium supplemented with B-27, penicillin/streptomycin/neomycin, L-glutamine and transferrin) in 24-well-plates after poly-L-ornithine and laminin coating in a density of 4x10E5 neurons per well for 8 DIV.

extract_protocol:
Lysates were obtained with TRI Reagent (Sigma ®), and RNA was extracted by phenol-chloroform precipitation
TruSeq RNA Library Preparation Kit v2, Set A

treatment_protocol: On DIV 1, AAV.mCherry (control, Genbank ID: KT345943, 5x10E6 TU/well) or AAV.ULK1.DN (9x10E6 TU/well) were added to each well.

tag: cell type

value: primary cortical neurons

value: wild type

tag: transduction


title: whole blood, preHD B 03, year 1

tag: year

tag: Sex

value: Male


title: WT_14DAG_control_rep2


title: NHP24.D006.FRZ-MDCK.a4

description:
Animal: NHP24; Day post infection: D006; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a4
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.


title: Control replicate 3

source_name: ctrl

value: control


title: whole blood, preHD B 04, year 1

tag: age at year 1

tag: location

value: preHD B


title: whole blood, preHD B 01, year 1

value: london

tag: disease stage

tag: expanded allele cag repeat count


title: whole blood, preHD B 02, year 1

tag: uhdrs tms

tag: uhdrs tfc


title: whole blood, preHD A 18, year 1

value: preHD A


title: whole blood, preHD A 17, year 3

value: vancouver


title: whole blood, preHD A 18, year 3


title: Ulk1 dominant negative replicate 2


title: NHP13.D003.FRZ.a2

description:
Animal: NHP13; Day post infection: D003; 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.

tag: fresh_vs_freezethawed (frz)

value: FRZ

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

value: std


title: NHP13.D003.FRZ.a1

description:
Animal: NHP13; Day post infection: D003; Fresh or freeze-thawed: FRZ; 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: tissue

value: Peripheral blood mononuclear cells (PBMC)


title: whole blood, preHD A 17, year 1

value: Female


title: Ulk1 dominant negative replicate 1

tag: age


title: NHP16.D005.FRZ.a3

description:
Animal: NHP16; Day post infection: D005; 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.

value: FALSE


title: Control replicate 2


title: KEN3: U2OS_KEN_3

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-KEN/AAA overexpression


title: KEN2: U2OS_KEN_2


title: whole blood, preHD A 16, year 1


title: whole blood, preHD A 15, year 3


title: whole blood, preHD A 15, year 1


title: NHP13.D003.FRZ-MDCK.a5

description:
Animal: NHP13; Day post infection: D003; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a5
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.

value: TRUE


title: Control replicate 1


title: NHP13.D003.FRZ-MDCK.a4

description:
Animal: NHP13; Day post infection: D003; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a4
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.


title: NHP13.D003.FRZ.a3

description:
Animal: NHP13; Day post infection: D003; 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: WT1: U2OS_WT_1

value: GFP-UHRF1-WT overexpression


title: WT3: U2OS_WT_3


title: NHP13.D003.FRZ-MDCK.a6

description:
Animal: NHP13; Day post infection: D003; Fresh or freeze-thawed: FRZ-MDCK; MDCK Spike in: True; Array number: a6
Data for a given Seq-Well array for a specific biological sample
PBMC purification: We centrifuged whole blood in K3EDTA tubes at 1800 x g for 10 min at room temperature, removed EDTA plasma, and added phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to the pelleted cells to double the original whole blood volume. We gently poured the PBS-blood cell mixture into an Accuspin tube containing Histopaque (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 1000 x g for 10 min at room temperature with the brake set to 1. Following centrifugation, we removed the top, clear supernatant layer to within 0.5 cm of the cloudy white layer containing PBMCs. We transferred the cloudy PBMC layer to a clean 15 mL conical tube and increased the volume to 10 mL using PBS supplemented with 2% heat-inactivated fetal bovine serum (PBS/2%HI-FBS) and mixed by inversion. We then centrifuged at 300 x g for 10 min at 4 °C with the brake set to 1. Following centrifugation, we removed the supernatant, resuspended the cell pellet with PBS/2%HI-FBS to a final volume of 10 mL, and mixed using gentle raking to wash the cells. We repeated the wash step 2 more times with the centrifuge set to 200 x g for 10 min at 4 °C with the brake set to 1. We then resuspended the cell pellet in 9.5mL PBS/2%HI-FBS for counting using the Countess Cell Counting system (Thermo Fisher Scientific). We aliquoted 0.5 mL for Seq-Well, and used the remaining 9 mL volume for CyTOF.
Seq-Well: We performed Seq-Well as described previously (Gierahn et al., 2017), with the S3 protocol (Hughes et al., 2019) and some controls and modifications to adhere to the BSL-4 environment. After loading and sealing beads and cells in Seq-Well arrays, we placed them in a -80 °C freezer until further processing – this step was required due to time constraints in the BSL-4. Later, we removed sealed Seq-Well arrays from the -80 °C freezer, placed them in 4-well dishes, and allowed them to equilibrate to room temperature for at least 30 min. We then covered arrays in 5 mL Seq-Well Lysis Buffer per protocol. We performed RNA hybridization and RT as specified in the protocol (Hughes et al., 2019). After RT, we collected beads by centrifugation at 1000 x g for 1 min at room temperature. We resuspended beads with GeneXpert Lysis Buffer (Cepheid, Sunnyvale, CA, USA) for inactivation, which was required prior to removal from the BSL-4 laboratory according to standard operating procedures. After removal, we washed beads thrice with TE buffer containing 0.01% Tween 20 and shipped at 4 °C for further library construction and sequencing.