title: Human podocytes TF knockout and IFNAR1 Knockown 3

city: Braunschweig

first: Robert

last: Geffers

address: Inhoffenstr. 7

department: Dep. Molecular Bacteriology

institute: HCI - Helmholtz Centre for Infection Research

description:
IFN3
RawCountsAnnotated_21-0007.txt

data_processing:
Before alignment to reference genome each sequence in the raw FASTQ files were trimmed on base call quality and sequencing adapter contamination using fastq-mcf. Reads shorter than 15 bp were removed from FASTQ file. Trimmed reads were aligned to the reference genome using open source short read aligner STAR (https://code.google.com/p/rna-star/)) with settings according to log file.
Feature counts were performed using R packkage Rsubread:FeatureCount with default settings for paired end reads.
Assembly: mm10 or GRCh38
Supplementary files format and content: tab-delimited text file includes raw counts for each sample

source_name: Immortalized human podocytes

extract_protocol:
RNA was extracted using RNeasy micro plus kit from Qiagen
Quality and integrity of total RNA was controlled on Agilent Technologies 2100 Bioanalyzer (Agilent Technologies; Waldbronn, Germany). The RNA sequencing library was generated from 500ng total RNA using Dynabeads® mRNA DIRECT™ Micro Purification Kit (Thermo Fisher) for mRNA purification followed by NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England BioLabs) according to manufacture´s protocols. The libraries were sequenced on Illumina NovaSeq 6000 using NovaSeq 6000 S1 Reagent Kit (100 cycles, paired end run) with an average of 5 x107 reads per RNA sample.

value: Immortalized human podocytes

value: TF KO IFNAR1 KD


title: 097 RNA

status: Public on Dec 31 2024

type: SRA

city: Tampa

first: Chia-Ho

last: Cheng

address: 12902 USF Magnolia Drive

country: USA

institute: Moffitt Cancer Center

library_source: transcriptomic

data_processing:
Sequencing reads were subjected to a variety of pre- and post-alignment QC measures before mapping against human reference genome hs37d5 using STAR-2.5.3a
Gene-level quantification was determined using HTSeq 0.6.1 by summation of raw counts of reads aligned to the region (Gencode29) associated with each gene.
To account for differences in library size between samples, Trimmed Mean of M values (TMM) normalization using calcNormFactors(), also from edgeR, was applied.
All data were then transformed using voom.
Assembly: hs37d5
Supplementary files format and content: tab-delimited text files include raw gene count for each sample

molecule: total RNA

organism: Homo sapiens

source_name: ovarian tumors

extract_protocol:
RNA was harvested using the Qiagen All-Prep RNA isolation kit
RNA libraries were prepared by using Illumina TruSeqTM RNA Exome Library Preparation Kit (Illumina Inc., San Diego, CA) , following the manufacturer’s protocol

tag: yob decade

tag: pre/post menopause

value: pre


title: dinBT_CIP_replicate_1

city: Hangzhou

first: min

last: yue

address: No.1, Sub-Lane Xiangshan, Xihu District, Hangzhou, China

country: China

institute: Hangzhou Institute for Advanced Study, University of Chinese Academy of Science

data_processing:
Raw data were processed by Trimmomatic (v.0.36).[Bolger, et al. Bioinformatics, 2014, PMID: 24695404]
Clean reads were mapped to NCBI Rfam databases, to remove the rRNA sequences by Bowtie2 (v2.33).[Langmead, et al. Nat Methods, 2012, PMID: 22388286]
The remaining mRNA sequences were mapped to the SL1344 genome (NC_016810.1, NC_017718.1, NC_017719.1, NC_017720.1) by Hisat2 (2.1.0).[Kim, et al. Nature Biotechnology, 2019, PMID]
The read numbers mapped to each gene were calculated using HTSeq-count (v0.9.1).[Anders, et al. Bioinformatics, 2015, PMID: 25260700]
For gene expression analysis, the read count number was calculated and then normalized to FPKM (fragments per kilobase per million mapped reads) value using RSEM software (v1.3.1).[Li, et al. BMC Bioinformatics, 2011, PMID: 21816040]
Assembly: SL1344 genome (NC_016810.1, NC_017718.1, NC_017719.1, NC_017720.1)
Supplementary files format and content: Comma-separated values file includes raw counts for each Sample
Supplementary files format and content: Comma-separated values files include FPKM values for each Sample

organism: Salmonella enterica subsp. enterica serovar Typhimurium

source_name: strain

growth_protocol: Single colonies were seeded into LB medium and cultured to ~0.1 of OD600.

extract_protocol:
Total mRNA was extracted with the HiPure RNA Kit (R4130-02, Magen, China)
cDNA libraries were generated using a NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (E7420; New England Biolabs; Ipswich, USA) following the manufacturer’s recommendation

treatment_protocol: The bacterial solution containing 0.03125μg/ml CIP was subsequently incubated without shaking at room temperature for 10 min, and a liquid nitrogen flash freezer was used to stop the reaction.

tag: tissue

value: strain

tag: cell line

tag: cell type

tag: genotype

value: frameshift dinB (GeneID:1251832)

tag: treatment

value: LB medium containing 0.03125μg/ml CIP, without shaking at room temperature for 10 min, and a liquid nitrogen flash freezer was used to stop the reaction.


title: Col_2C

city: Praha 6

first: Karel

last: Muller

address: Rozvojová 263

department: Lab. of hormonal regulations in plants

country: Czech Republic

institute: Institute of Experimenal Botany of the Czech Academy of Sciences

data_processing:
reads were quality filterred by rcorrector and Trimmomatic
transcript quantification and normalization done by Salmon v1.3.0 with parameters--seqBias, --gcBias, --PosBias, --numBootstraps 30
Assembly: Arabidopsis thaliana, TAIR10 cds library, version 20101214
Supplementary files format and content: sf files generated by Salmon software, include TPM values

organism: Arabidopsis thaliana

source_name: root

growth_protocol: Chlorine gas was used for surface-sterilization. After this, seeds were stratified for 2 d at 4°C. Seedlings were grown vertically on plates containing 1% (w/v) agar (Duchefa) with ½ Murashige and Skoog (MS, Duchefa, 0,5g/l MES, 1 % (w/v) sucrose, pH 5.8 adjusted with 1M KOH). Plants grown in growth chamber with following conditions: 60 % humidity, 22°C by day (16 h), 18°C by night (8 h), light intensity of 120 μmol photons m-2 s-1.

extract_protocol:
protocol from Plant Total RNA Mini Kit, Favorgen
KAPA HyperPrep kit

treatment_protocol: To treat the plants, 5d old plants were transferred to treatment-containing medium. Estradiol (Sigma, 20mM stock in DMSO, 2.5 μM working concentration) diluted in DMSO was used for treatments.

value: root

tag: biological replicates

value: EST

tag: treatment time


title: Human podocytes TF knockout and IFNAR1 Knockown 2

country: Germany

description:
IFN2
RawCountsAnnotated_21-0007.txt


title: 098 RNA


title: dinBT_CIP_replicate_3


title: 096 RNA


title: RA1 shCONTROL

city: Guangzhou

first: Yu

last: Kuang

address: No.58 Zhongshan Er Road Guangzhou

institute: The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China)

description:
R1CTRL
lncRNA-shFTOVSCONTROL.log2FC1.Pvalue0.05.fpkm.anno
mRNA-shFTOVSCONTROL.All.fpkm.anno

data_processing:
Reads were aligned to the human Ensemble genome GRCh38 using Hisat2 aligner (v2.1.0) under parameters: “--rna-strandness RF”.
The reads mapped the genome were calculated using featureCounts (v1.6.3).
Differential gene expression analysis was performed using the DESeq2 R-package.
Assembly: human Ensemble genome GRCh38
Supplementary files format and content: shFTOVSCONTROL.processed data file.txt, All.HTSeq.counts.txt

source_name: synovial tissues

extract_protocol:
Total RNA was extracted using TRIzol™ Reagent (Invitrogen, 15596018). Then, RNA samples undergo the following process. (1) DNase I digestion: Add DNase I to the RNA sample, digest at 37 ℃ for 30 minutes to remove residual DNA from the sample, and purify and recover RNA using magnetic beads. (2) Remove ribosomal RNA and RNA fragmentation: Add 5XRT Buffer and ribosomal RNA probe to the sample RNA, perform temperature gradient reaction on RNA sample fragmentation and remove rRNA. (3) Synthesis of first strand cDNA: Add EpiScriptTM IV, RNase inhibitor, DTT, TSO, random primers to the RNA sample in step 2. Mix gently with the tube wall and centrifuge rapidly. React in a PCR machine according to the following procedure: 37 ° C, 90 minutes; 70 ° C for 15 minutes. (4) First round of PCR amplification: Add 2XpfuMax HiFi PCR ProMix and sequencing primers to the first strand cDNA sample, mix well, and perform amplification reaction in the PCR instrument. (5) Purify the PCR amplification product using EpiTM DNA Clean Beads at a ratio of 1X, and recover 300-400bp of DNA fragments using magnetic beads at a ratio of 0.65X/0.2X for the purified product. (6) Perform a second round of PCR amplification on the purified product, enriching 300-400bp of DNA fragments. (7) Quality inspection: Use Biotic Qsep100 Analyzer to inspect the library and check if the distribution of library size meets the theoretical size.
EpiTM mini longRNA-seq kit (Epibiotek, E1802) was used for library preparation according to the instructions.

tag: disease state

value: rheumatoid arthritis

value: synovial tissues

value: primary cell

value: rheumatoid arthritis fibroblast synoviocytes

value: WT

value: control lentivirus infection


title: WT_CIP_replicate_3

value: wild type


title: Col_2B


title: 103 RNA


title: Human podocytes TF knockout and IFNAR1 Knockown 1

description:
IFN1
RawCountsAnnotated_21-0007.txt


title: RA3 shFTO

description:
R3F-10L
lncRNA-shFTOVSCONTROL.log2FC1.Pvalue0.05.fpkm.anno
mRNA-shFTOVSCONTROL.All.fpkm.anno

value: FTO knockdown

value: shFTO-lentivirus infection


title: Col_2A


title: 095 RNA


title: Human podocytes TF knockout 3

description:
TKO3
RawCountsAnnotated_21-0007.txt

value: Immortalized human podocytes

value: TF KO


title: △dinB_CIP_replicate_1


title: Human podocytes TF knockout 2

description:
TKO2
RawCountsAnnotated_21-0007.txt


title: Col_1C


title: 099 RNA

value: pre


title: 094 RNA


title: 093 RNA


title: WT_CIP_replicate_1

value: strain


title: Col_1B


title: △dinB_LB_replicate_3

value: LB medium, without shaking at room temperature for 10 min, and a liquid nitrogen flash freezer was used to stop the reaction.


title: Human podocytes TF knockout 1

description:
TKO1
RawCountsAnnotated_21-0007.txt


title: △dinB_LB_replicate_2


title: 092 RNA


title: Col_1A


title: Human podocytes wild type 2

description:
CONT2
RawCountsAnnotated_21-0007.txt


title: △dinB_LB_replicate_1


title: AXR_3C

value: DMSO


title: 089 RNA


title: Human podocytes wild type 1

description:
CONT1
RawCountsAnnotated_21-0007.txt


title: AXR_3B

value: root


title: WT_CIP_replicate_2

value: LB medium containing 0.03125μg/ml CIP, without shaking at room temperature for 10 min, and a liquid nitrogen flash freezer was used to stop the reaction.


title: dinBT_LB_replicate_2

value: frameshift dinB (GeneID:1251832)


title: AXR_3A


title: dinBT_LB_replicate_3


title: 087 RNA


title: sample4_endoderm_day3

city: Ann Arbor

first: Shiyu

last: Sun

state: Michigan

address: G.G. Brown Building, 2350 Hayward St

department: Mechanical Engineering

institute: University of Michigan

data_processing:
Illumina NovaSeq-6000 for basecalling
10x Genomics Cell Ranger pipeline (version 3.1.0) for barcoding, alignment, filtering
Assembly: GRCh38
Supplementary files format and content: TSV and MTX files are generated by Cell Ranger pipeline, including barcodes, features and matrix. RD file is generated using Seurat.

source_name: H9 hESC

growth_protocol: The samples were generated according to published manuscript.

extract_protocol:
The embryoids were dissociated into single cells. Dissociated cells were then lysed and barcoded with 10x Chromium Controller Instrument (10x Genomics).
cDNA library was constructed according to the standard 10x Genomics scRNA-seq library construction protocol (Single Cell 3' v2). The libraries were sequenced on Illumina NovaSeq-6000 with paired-end sequencing (One sample per flow cell).

value: H9 hESC

value: human embryonic stem cell (hESC)

value: hESC-derived PTED embryoids with promoted endoderm differentiation


title: AXR_2C

value: EST


title: 085 RNA


title: WT_LB_replicate_3


title: WT_LB_replicate_2


title: AXR_2A


title: 084 RNA


title: sample2_day5

source_name: H9 ESC

value: hESC-derived PTED embryoids


title: sample3_day8


title: Human podocytes wild type 3

description:
CONT3
RawCountsAnnotated_21-0007.txt

value: WT


title: 091 RNA


title: 083 RNA


title: WT_LB_replicate_1

value: wild type


title: 088 RNA


title: Mouse_U2_Unmarked

city: New York City

first: Benjamin

last: Chen

state: New York

address: 1468 Madison Avenue

department: Infectious Disease

institute: Icahn School of Medicine at Mount Sinai

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
10xGenomics CellRanger (v.6.21.) https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/tutorial_in
Seurat v4.3 (https://satijalab.org/seurat/)
Analysis was conducted using Seurat (v4.3.0) as well as scCustomize (v1.1.1) in R (v4.2.0). Standard QC based on individual mitochondrial gene cutoffs (5%,10%, or 15%) and overall minimum of 200 and individual maximum of unique feature counts ranges were applied. Furthermore, cells with more than 5% of mouse gene UMIs were removed, likewise keeping cells with >95% human gene transcripts. DoubletFinder (v2.0.3) was used to identify potential technical doublets in the datasets, however due to the homogeneity of the cells in the datasets we chose not to remove those doublets but keep the distinction within the metadata.  To reduce batch and donor effects we avoided merging and integrating datasets from different mice or sequencing runs. We utilized the multimodal reference mapping workflow from Satija Lab which uses a K Nearest Neighbor approach to predict a likely CD4 T cell type that we used to annotate the cells in our dataset in relation to a comprehensive Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) derived reference map of 162,000 PBMCs using 228 antibodies in conjunction with scRNAseq expression data and added the predicted cell type data as meta data to the original datasets.  Datasets from the same mouse were merged and normalized using SCTransform, which utilizes regularized negative binomial regression approach. Principal component analysis (PCA) was performed identifying variable genes, Lenti-RG and HIV specific genes were manually removed. The number of principal components was chosen using the JackStraw and ElbowPlot function within Seurat (v4.3.0) and was individual for each dataset between 15 to 30. Non-linear dimensionality reduction analysis was used to produce unsupervised clustering using individual cluster resolutions based on clustree results (v0.5.0). Samples were visualized via Uniform Manifold Approximation and Projection (UMAP).  Differential gene expression analysis was conducted independent of cluster identity using the FindMarkers function of Seurat (v4.1.0) and Model-based analysis of single cell transcriptomics (MAST). Multiplicity correction was performed with the Benjamini-Hochberg method. These results were displayed with an false discovery rate cutoff (FDR) < 0.05 using EnhancedVolcano (v1.16.0). Lastly, we chose to merge, SCTransform, and integrate based on mutual-nearest-neighbor (MNN) using fastMNN within SeuratWrappers (v.0.3.1).

source_name: Spleen

extract_protocol:
Spleen from infected mice were harvested and processed by homogenizing the spleen through 40um sterile filters. The cells were washed with 2mM EDTA containing PBS and treated with lysing buffer ACK (Life Technologies) to remove RBC. The remaining cells were stained with antibodies described in engraftment panel and flow sorted. Cells were sorted to enrich for GFP+ infected cells as described in results section. In 15 acutely infected cells, GFP+ HIV infected cells were pooled with dsRed+ and unmarked cells for 10x sample processing. 10d and 29d ART treated GFP+ latently infected cells were sorted into carrier wild type mouse B cells and processed for 10x sample processing. Unmarked and dsRed+ bystander cells were sorted separately and sequenced separately.
FACS sorting followed by Genomics Chromium Next GEM Single Cell 3’ Reagent Kits v.3.1 (CG000204 Rev D) protocol

value: Spleen

value: UCB CD34+

value: CD4+ T-cells

value: NOD.Cg-Prkdcscid ||2rgtm1Wjl/SzJ, NSG


title: AXR_1C


title: 082 RNA

value: pre


title: 081 RNA


title: Yak ovary, December 2, scRNAseq

city: Lanzhou

first: Jie

last: Pei

state: Gansu

address: No. 335 Jiangouyan ST, Qilihe District

institute: Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences

description: 10x Genomics

data_processing:
Raw scRNA-seq data was converted using the Cell Ranger analysis pipeline provided by 10x Genomics (v2.2.0) and reads were aligned to the yak genome version BosGru3.0 using the STAR aligner.
Assembly: BosGru3.0
Supplementary files format and content: Tab-separated values files and matrix files

organism: Bos grunniens

source_name: ovary

extract_protocol:
The pieces of ovarian tissue were washed in cooled RPMI 1640 medium containing 0.04% bovine serum albumin twice. The ovarian samples were further minced into approximate 0.5 mm3 pieces with a sterile scalpel blade in RPMI 1640 medium containing 0.04% BSA. The pieces were incubated with 1 mg/ml collagenase Type II in 0.25% trypsin-EDTA on ice overnight. The digestion was stopped by adding 10% of fetal calf serum. Next, the dissociated cells were collected by centrifugation at 160 × g for 3 min and incubated with advanced DMEM/F12 Glutamax containing 1% insulin-transferrin-selenium , 1% penicillin-streptomycin, and 27 IU/ml RNase-free DNase I at 37 °C for 1 h. The ovarian cells were resuspended in DPBS containing 2% FBS and passed through a 40 μm cell strainer.
The cell suspensions containing high viability cells were prepared immediately. Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 (Chromium, PN-1000128) was used for gel beads in emulsion (GEM) formation. The cell suspensions, gel beads, and partitioning oil were loaded into a Chromium Single Cell Controller to produce single-cell GEM. Then, a primer containing Illumina TruSeq Read 1, 10X Barcode, and unique molecular identifier (UMI) was applied to produce barcoded full-length cDNA by reverse transcriptase reaction. The GEMs were broken and the pooled fractions were recovered. The first-strand cDNAs were purified with silane magnetic beads and amplified to generate sufficient mass for library construction. Enzymatic fragmention and size selection were used to optimize cDNA amplicon size. P5, P7, a sample index, and a TruSeq Read 2 were added to the amplicon via end repair, A-tailing, adaptor ligation, and PCR.

value: ovary

tag: age

value: 4 years old

tag: reproductive state

value: pregnant

tag: physical condition

value: healthy

tag: collection time

value: December


title: AXR_1B

value: root


title: 080 RNA


title: dinBT_LB_replicate_1

value: strain

value: frameshift dinB (GeneID:1251832)

value: LB medium, without shaking at room temperature for 10 min, and a liquid nitrogen flash freezer was used to stop the reaction.


title: Yak ovary, April 1, scRNAseq

description: 10x Genomics

value: anestrus

value: April


title: Mouse_U1_Unmarked

description: 10x Genomics


title: AXR_1A

value: EST


title: Yak ovary, December 1, scRNAseq

description: 10x Genomics


title: AXR_2B


title: Mouse_U1_dsRed

description: 10x Genomics

value: Spleen


title: AF_STAGE4_R3

city: East Lansing

first: Frances

last: Trail

state: MI

address: 612 Wilson Road, Room 342

department: Plant Biology

institute: Michigan State University

data_processing:
Base calling was done by Illumina Real Time Analysis (RTA) v2.7.7 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.20.0. The low-quality reads were removed using the read filtering tool, Trimmomatic, Version 0.39. The 3’ ends of NovaSeq6000 single-end 100 bp reads were trimmed to 50 bp reads (keeping the first 50 bp) using Trimmomatic Version 0.39.
The trimmed reads were mapped to the indexed reference sequences using HISAT 2.2.1.
The HISAT alignments in SAM format were then sorted and converted to BAM format using SAM tools.
Count files were then generated using HTSeq Version 0.11.1.
The DESeq2 package Version 1.30.1 in R was used to normalize the read counts and to perform differential expression analysis.
Assembly: The reference sequence used for the alignment are A. fumigatus AF293 (GCA_000002655.1), A. nidulans A4 (GCA_000149205.2), and F. oxysporum NRRL 32931 (GCA_000271745.2)
Supplementary files format and content: Tab-demilited text file includes raw counts for each sample

organism: Aspergillus fumigatus

source_name: Germinated conidia

growth_protocol: For total RNA extraction of each germination stage on standard medium (Bird agar medium), a 200 µL aliquot of the conidial suspension (1 x 106 conidia/mL) of each species was inoculated onto a sterile cellophane sheet overlaid on Bird medium in a Petri dish (60 x 15 mm). Petri dishes containing inoculated sheets were then incubated at RT till the conidia reached respective germination stages.

extract_protocol:
Total RNA  from cellophane membranes was isolated using Trizol reagent. RNA was treated with RNase-Free DNase (Qiagen), purified further using RNA Clean and ConcentratorTM - 5 (ZymoResearch)
Libraries were prepared using the Illumina Stranded mRNA Prep - Ligation kit with IDT for Illumina RNA UD Indexes.

value: Germinated conidia

tag: Stage

value: WT

value: Bird medium


title: AF_STAGE4_R2


title: Yak ovary, August 1, scRNAseq

description: 10x Genomics

value: estrus

value: August


title: Mouse_T3_Mixed

description: 10x Genomics

value: UCB CD34+

value: CD4+ T-cells

value: NOD.Cg-Prkdcscid ||2rgtm1Wjl/SzJ, NSG

value: 10 day ART


title: sample1_day2

value: H9 hESC

value: human embryonic stem cell (hESC)

value: hESC-derived PTED embryoids


title: AF_STAGE4_R1


title: 063V03D1

city: Siena

first: Maria

last: Novedrati

address: Viale Bracci, 16

country: Italy

institute: University of Siena

data_processing:
Base-calling, alignment and filtering were performed by the Ion Proton Server
Assembly: GRCh37
Supplementary files format and content: Counts_Leish2b_20230615.csv contains read counts for all the samples
Supplementary files format and content: vstNormCount.csv containing normalized counts (log2-like)

source_name: Whole blood

extract_protocol:
PAXgene Blood RNA kit
Ion AmpliSeq™ Library Kit Plus
Ion S5 Sequencing Kit

value: Whole blood

value: 7.5x10^10 viral particles

value: youth

tag: timepoint


title: 078 RNA

value: pre


title: Mouse_T1_dsRed

description: 10x Genomics

value: Spleen


title: AF_STAGE3_R3

value: Germinated conidia


title: 077 RNA


title: Mouse_U2_dsRed

description: 10x Genomics


title: 063V02D0


title: 076 RNA


title: AF_STAGE3_R2

value: WT


title: Mouse_A3_dsRed2

description: 10x Genomics

value: NOD.Cg-Prkdcsci ||2rgtm1Wjl/SzJ, NSG


title: 075 RNA


title: 062V02D0


title: AF_STAGE2_R3

value: Bird medium


title: AF_STAGE3_R1


title: 079 RNA


title: 061V02D1


title: Mouse_A3_GFP

description: 10x Genomics


title: Mouse_A3_dsRed1

description: 10x Genomics


title: AF_STAGE2_R2


title: 073 RNA


title: 061V02D0


title: 070 RNA


title: 074 RNA


title: AF_STAGE2_R1


title: 060V02D1


title: 069 RNA


title: AF_STAGE1_R3

source_name: Conidia

value: Conidia


title: Mouse_A1_Mixed

description: 10x Genomics

value: UCB CD34+


title: SE_Met_3

city: Utrecht

first: Frank

middle: van

last: Leeuwen

address: Heidelberglaan 25

country: Netherlands

institute: Princess Maxima Center

data_processing:
Raw data (raw reads) of FASTQ format were processed through fastp
alignment to reference genome (hg38) using the HISAT2 v2.0.5 algorithm program
FeatureCounts v1.5.0-p3 was used to count the read numbers mapped of each gene.
FPKM of each gene was calculated
Assembly: Homo Sapiens(GRCh38/hg38)
Supplementary files format and content: text file of FPKM values for each Sample
Supplementary files format and content: text file of raw read counts for each Sample

source_name: SEM

growth_protocol: Cells were grown in RPMI1640 (Invitrogen), supplemented with 10% fetal bovine serum (Greiner Bio-One) and 1% penicillin/streptomycin (Invitrogen) at a cell density between 0.2E6 and 2.5E6 cells per milliliter.

extract_protocol:
mRNA was purified from cell cultures using a NucleoSpin RNA isolation kit (740955, Machery-Nagel).
Library prep, RNA sequencing, and data analysis was performed by NovoGene (Cambridge, UK) on triplicate samples.

treatment_protocol: NALM-6 and SEM cells were incubated for 24-hours in complete methionine depleted RPMI1640 medium or normal, control RPMI1640 medium.

value: SEM

value: BCP-ALL

value: Methionine Restricted


title: Mouse_A2_GFP

description: 10x Genomics

value: CD4+ T-cells

value: NOD.Cg-Prkdcscid ||2rgtm1Wjl/SzJ, NSG


title: Mouse_A2_dsRed

description: 10x Genomics


title: 060V02D0


title: AF_STAGE1_R2


title: 062V03D1


title: 068 RNA


title: 059V02D1


title: 067 RNA


title: AF_STAGE1_R1


title: 059V02D0


title: SE_Met_1


title: 066 RNA


title: IL4_IL13_72h_rep4

type: genomic

city: Tampere

first: Dario

last: Greco

address: Arvo ylpön Katu 34

department: Faculty of Medicine and Health Technology

country: Finland

institute: Tampere University

hyb_protocol: Genomic DNA was treated with sodium bisulfite using EZ-96 DNA methylation kit (Zymo Research, cat no.: D5004), following the manufacturer’s standard protocol

scan_protocol: Illumina iScan and Illumina GenomeStudio Software

data_processing:
minfi package v1.46 and Maksimovic et al 2017 pipeline
Average Beta

label: Cy3 and Cy5

molecule: genomic DNA

label_protocol: Standard Illumina protocol

growth_protocol: THP-1 cells (ATCC TIB-202, USA) were cultured in RPMI-1640 (Gibco, USA) supplemented with 10% FBS (Gibco, USA) (culture media). Cells were cultured in 75 cm2 flasks at a density < 1 × 106 cells/ml. Cells were differentiated in 12 well plates, with 500,000 cells/ well (127,000 cells/cm2), in the culture media supplemented with 50 nM of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, USA) for 48h

extract_protocol: Cells were lysed on the plate and the DNeasy blood and tissue kit (Qiagen, Germany) was used as per the vendor’s instructions.

treatment_protocol: After PMA differentiation, cells are exposed to two different cytokines cocktails and fresh media for 24h, 48h, and 72h. In particular, to simulate a PI environment the cells are exposed to LPS (Merck) 10 pg/ml and IFNγ (Sigma-Aldrich, USA) 20 ng/ml; to induce an AI phenotype instead, the cells are exposed to IL-13 (Sigma-Aldrich, USA) 20 ng/ml and IL-4 20 (Sigma-Aldrich, USA) ng/ml.

tag: time

tag: replicate

value: monocyte

tag: other treatment

value: before the exposure 30.9 ng/ml PMA for 48h

tag: well-plate

tag: cell density

value: 0.5 106 cells/ml

tag: pma concentration

tag: pma exposure

tag: il-4 conc

value: 20 ng/ml

tag: il-13 conc

tag: cytokines exposure


title: SE_Ctr_3

tag: cell line

tag: cell type

tag: genotype

tag: treatment

value: Untreated


title: 058V02D1

tag: tissue

value: Whole blood

value: 7.5x10^10 viral particles

tag: age

value: youth


title: 058V02D0

tag: timepoint


title: IL4_IL13_72h_rep3

tag: time


title: 065 RNA

tag: yob decade

tag: pre/post menopause

value: pre


title: SE_Ctr_2

value: WT


title: 064 RNA


title: SE_Ctr_1


title: 064V02D0


title: IL4_IL13_72h_rep1

tag: replicate

tag: other treatment

tag: well-plate


title: 064V03D1


title: IL4_IL13_72h_rep2

tag: cell density

tag: pma concentration

tag: pma exposure


title: 063 RNA


title: N6_Met_3


title: 062 RNA

value: post


title: LPS_INFgamma_72h_rep4

value: LPS-INFgamma

tag: infgamma conc

tag: lps conc

tag: cytokines exposure


title: 057V02D1


title: N6_Met_2

tag: cell line

tag: cell type

tag: genotype


title: 061 RNA


title: LPS_INFgamma_72h_rep3

tag: treatment


title: 057V02D0

tag: tissue

tag: age

tag: timepoint


title: 060 RNA

tag: yob decade

tag: pre/post menopause


title: SE_Met_2

value: SEM


title: N6_Met_1

value: BCP-ALL


title: LPS_INFgamma_72h_rep2

tag: time

tag: replicate

tag: other treatment


title: 056V02D1


title: N6_Ctr_3


title: 059 RNA


title: LPS_INFgamma_72h_rep1

tag: well-plate

tag: cell density

tag: pma concentration


title: 056V02D0


title: N6_Ctr_2


title: 058 RNA


title: no_treatment_72h_rep4

value: C

tag: pma exposure


title: 055V02D1


title: 057 RNA


title: no_treatment_72h_rep3

tag: cytokines exposure


title: 055V02D0


title: no_treatment_72h_rep2

tag: cell line


title: ETP-ALL3 [CITE-Seq]

city: Toronto

first: Soheil

last: Jahangiri

state: Ontario

address: 610 University Ave

department: Princess Margaret Cancer Centre

country: Canada

institute: University Health Network

data_processing:
Raw FASTQ sequencing data was processed for downstream analysis using CellRanger version 6.0.0 (10X Genomics).
ADT and RNA counts were generated from raw sequencing data using the SC5P-R2 mode for 5 prime paired end read analysis and expected cells were estimated at 26000.
The single-cell analyses were carried out using the Seurat v4.3.022 in R. CellRanger h5 files were imported directly into Seurat.
Antibody derived tags (ADTs) were normalized to spike-in murine IgG.
Cells with less than 200 genes and more than 20% mitochondrial gene counts were removed
Hashtag oligos (HTOs) were demultiplexed using the HTODemux function at 0.99 percentile.
TCR clonotypes were reviewed manually to assign sample-specific clonotypes
Cells of samples expressing clonotypes from other samples were marked as doublets
Cells that were negative by HTO-based assignment or marked as doublet by HTO or clonotypes were filtered out.
Assembly: GENCODE human reference genome v38
Supplementary files format and content: Gene expression – Raw counts
Library strategy: CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by sequencing)

source_name: Bone Marrow

extract_protocol:
Cells aliquoted for CITE-Seq were washed 3X with CITE-Seq staining buffer (CSM; 2% BSA, 0.01% Tween in PBS) in conical bottom polypropylene FACS tubes. After washing, cells were resuspended in 50μl of CITE-Seq antibody mix containing 0.1μg CITE-Seq antibody (BioLegend, see Table X) per sample. Next, 0.1μg Hashtag oligos (HTO, Biolegend, table X) were added to each sample individually, and samples were incubated for 30 minutes in the dark at RT. Each library was made up of 4 hashed samples, including 3 malignant and 1 healthy bone marrow specimen. After staining, cells were washed 3X with CSM, counted, pooled evenly and delivered to the Princess Margaret Genomics Center (PMGC) for single cell sequencing preparation using the 10X platform.
Library construction was carried out with the Chromium Next GEM Single Cell 5ʹ Kit v2 (cat#1000263) for Whole transcriptome gene expression library, the 5' Feature barcoding kit (cat#1000541) for Antibody Derived Tag library and the Chromium Single Cell Human TCR Amplification Kit (cat#1000252) for the VDJ library all following manufacturor's recommendations

value: Bone Marrow

tag: disease

value: primary TALL

tag: cell type

value: primary human BM mononuclear cells


title: 056 RNA


title: 054V02D1

tag: treatment


title: 055 RNA


title: no_treatment_72h_rep1

value: monocyte

value: before the exposure 30.9 ng/ml PMA for 48h


title: 054V02D0

tag: tissue


title: 054 RNA


title: IL4_IL13_48h_rep4

value: 0.5 106 cells/ml

tag: il-4 conc

value: 20 ng/ml

tag: il-13 conc


title: ETP-ALL4 [CITE-Seq]

tag: genotype


title: 053V02D1

value: Whole blood

tag: age

tag: timepoint


title: IL4_IL13_48h_rep2

tag: time

tag: replicate


title: 053V02D0

value: 7.5x10^10 viral particles


title: hBM6 [CITE-Seq]

value: Healthy


title: 053 RNA

tag: yob decade

tag: pre/post menopause

value: pre


title: 052 RNA


title: IL4_IL13_48h_rep1

tag: other treatment

tag: well-plate

tag: cell density

tag: pma concentration


title: T-ALL13 [CITE-Seq]

tag: disease

value: WT


title: N6_Ctr_1

tag: cell line

tag: cell type

value: Untreated


title: 052V02D1

tag: tissue

tag: treatment

value: youth


title: 051 RNA

value: post


title: LPS_INFgamma_48h_rep4

value: LPS-INFgamma

tag: pma exposure

tag: infgamma conc

tag: lps conc

tag: cytokines exposure


title: MPAL2 [CITE-Seq]

tag: genotype


title: LPS_INFgamma_48h_rep3

tag: time

tag: replicate


title: 052V02D0

tag: age

tag: timepoint


title: MPAL3 [CITE-Seq]


title: 051V02D1


title: 050 RNA

tag: yob decade

tag: pre/post menopause


title: T-ALL14 [CITE-Seq]

value: primary human PB mononuclear cells


title: hBM7 [CITE-Seq]

value: Bone Marrow


title: hBM5 [CITE-Seq]


title: LPS_INFgamma_48h_rep1

tag: other treatment

tag: well-plate

tag: cell density

tag: pma concentration


title: 047 RNA


title: LPS_INFgamma_48h_rep2

tag: cell line

tag: cell type


title: 050V02D1

tag: tissue

tag: treatment


title: 051V02D0


title: 046 RNA


title: 050V02D0


title: no_treatment_48h_rep4

value: C

tag: pma exposure


title: T-ALL10 [CITE-Seq]

tag: disease

value: primary TALL

value: primary human BM mononuclear cells


title: T-ALL11 [CITE-Seq]

tag: genotype


title: 045 RNA


title: no_treatment_48h_rep3

tag: time

value: monocyte

value: before the exposure 30.9 ng/ml PMA for 48h

tag: cytokines exposure


title: T-ALL12 [CITE-Seq]


title: 049V02D1

tag: age


title: hBM4 [CITE-Seq]


title: 049V02D0

tag: timepoint


title: no_treatment_48h_rep1

tag: replicate

value: 0.5 106 cells/ml


title: 047V02D1

value: Whole blood


title: T-ALL9 [CITE-Seq]


title: 044 RNA

tag: yob decade

tag: pre/post menopause


title: IL4_IL13_24h_rep4

tag: other treatment

tag: well-plate

tag: il-4 conc

value: 20 ng/ml

tag: il-13 conc


title: 048 RNA


title: 043 RNA


title: T-ALL3 [CITE-Seq]

tag: cell type


title: 047V02D0

tag: tissue

value: 7.5x10^10 viral particles


title: 040 RNA

value: pre


title: IL4_IL13_24h_rep3

tag: treatment

tag: cell line

tag: cell density

tag: pma concentration


title: T-ALL4 [CITE-Seq]

tag: disease

tag: genotype

value: WT


title: IL4_IL13_24h_rep2

tag: time

tag: pma exposure

tag: cytokines exposure


title: 039 RNA

tag: yob decade


title: 046V02D0

tag: age

tag: timepoint


title: hBM3 [CITE-Seq]

value: Healthy


title: IL4_IL13_24h_rep1

tag: replicate

tag: other treatment

tag: well-plate


title: T-ALL6 [CITE-Seq]

tag: cell type


title: 037 RNA

tag: pre/post menopause


title: 046V02D1

tag: tissue

tag: treatment

value: youth


title: LPS_INFgamma_24h_rep4

tag: cell line

tag: cell density

tag: infgamma conc

tag: lps conc


title: no_treatment_48h_rep2

tag: time

tag: pma concentration


title: 045V02D1


title: T-ALL7 [CITE-Seq]

tag: disease

tag: genotype


title: 045V02D0

tag: age


title: 036 RNA

tag: yob decade


title: LPS_INFgamma_24h_rep3

tag: replicate

tag: pma exposure

tag: cytokines exposure


title: T-ALL5 [CITE-Seq]

tag: cell type


title: 044V02D1

tag: timepoint


title: 042 RNA

tag: pre/post menopause


title: 035 RNA


title: LPS_INFgamma_24h_rep2

tag: treatment

tag: other treatment

tag: well-plate


title: hBM2 [CITE-Seq]

tag: tissue


title: no_treatment_24h_rep4

tag: cell line

tag: cell density


title: 034 RNA


title: T-ALL8 [CITE-Seq]


title: no_treatment_24h_rep2

tag: time

tag: pma concentration


title: 033 RNA


title: ETP-ALL1 [CITE-Seq]

tag: disease

tag: genotype


title: no_treatment_24h_rep1


title: 043V02D0

tag: age


title: 038 RNA

tag: yob decade


title: hBM1 [CITE-Seq]


title: 044V02D0


title: 030 RNA


title: 041V02D1


title: T-ALL2 [CITE-Seq]


title: ETP-ALL2 [CITE-Seq]


title: 041V02D0


title: 042V02D0


title: embryos, 72 hpf, rhpn2 mutant-c

city: NANTONG

first: DAI

middle: YU

last: BEI

address: seyuan street num 9

institute: nantong university

description: Library name: sample6

data_processing:
Raw sequencing BCL data base-calling using bcl2fastq v2.20.0.422
Data filtering using fastp v0.23.2 (--cut_front --cut_tail --cut_mean_quality 20 --length_required 35) followed by rRNA filtering using SortMeRNA v4.3.2 with default parameters
Read alignment using STAR v2.7.10a
Quantify using Salmon v1.9.0 with default parameters
Assembly: ZDB-GENE-030131-9927
Supplementary files format and content: Tab-separated values files

organism: Danio rerio

source_name: whole embryo

growth_protocol: zebrafish embryos were raised in E3 solution at 28.5℃ to the indicated developmental stage.

extract_protocol:
For total mRNA extraction from  zebrafish embryos, TRIzol Reagent (Catalogue Number: C2432, Invitrogen) was used according to the manufacturer's instructions.
For library construction, NEBNext Ultra RNA Library Prep Kit for Illumina was used according to the manufacturer's instructions.

treatment_protocol: rhpn2 gene were knockouted in embryos

value: whole embryo

tag: developmental stage

value: 72 hpf

value: rhpn2 mutant


title: 028 RNA


title: embryos, 72 hpf, rhpn2 mutant-b

description: Library name: sample5


title: 031 RNA


title: 040V02D1


title: 043V02D1


title: embryos, 72 hpf, wild type-c

description: Library name: sample3

value: wild type


title: 032 RNA


title: T-ALL1 [CITE-Seq]

value: Bone Marrow


title: embryos, 72 hpf, wild type-b

description: Library name: sample2


title: 027 RNA


title: 029 RNA


title: T-ALL6

data_processing:
Short read quality control was performed using FastQC v0.11.5
The sequencing adapters and low-quality end reads were trimmed using Trim Galore v0.6.6
STAR v.2.7.9a was used to align the raw reads to GENCODE human reference genome v38. After removing low quality alignments, (mapq < 15)
featureCounts v2.0.1 was used for gene expression quantification
DESeq2 v1.36.0  was used to identify differentially expressed genes between IRF5+ and IRF5- groups
Gene set enrichment analysis (GSEA) was performed using fgsea v1.22.0 and Bader lab gene sets, updated November 2022 (http://baderlab.org/GeneSets)
Assembly: GENCODE human reference genome v38
Supplementary files format and content: Gene expression – Raw counts

extract_protocol:
RNA was extracted using RNeasy Plus Micro Kit (QIAGEN, Cat. #74034) following manufacturer’s protocol.
The cDNA libraries were generated using SMART-seq v4 PLUS Kit (Takara, cat. #R400752) following manufacturer’s protocol.
paired end, 150 bp, ~ 50 million reads per sample

value: primary TALL


title: 042V02D1


title: embryos, 72 hpf, wild type-a

description: Library name: sample1


title: T-ALL13

value: primary human BM mononuclear cells


title: 026 RNA


title: 039V02D1


title: 025 RNA


title: ETP-ALL4


title: PWD_Untreated_rep4

city: Burlington

first: Seth

last: Frietze

state: VT

address: 149 Beaumont Avenue

department: Biomedical and Health Sciences

institute: University of Vermont

data_processing:
RNA-seq reads were aligned to the mm10 genome using STAR version v2.7.9a using  default parameters.
Raw RNA-seq count data were processed using STAR aligner with --quantMode GeneCounts and loaded into edgeR to generate a table of Transcripts Per Million (TPM) values.
Assembly: mm10
Supplementary files format and content: comma-separated text file including TPM values for each sample

organism: Mus musculus

source_name: BDMC

growth_protocol: Bone marrow-derived dendritic cells (BMDCs) were generated from mouse bone marrow using a standard 7-day granulocyte-macrophage colony-stimulating factor (GM-CSF, 20ng/ml, Shenandoah #200-15) differentiation protocol, as previously described(15). BMDCs were cultured in complete medium (RPMI 1640 containing 10% FBS, 2 mM L-glutamine, 100 Units/ml penicillin, 100 μg/ml streptomycin, and 55 μM beta-mercaptoethanol, all from Gibco).

extract_protocol:
RNA was extracted according to the manufacturer’s instructions using the RNeasy plus Mini Kit (cat # 74104) by Qiagen (Hilden, Germany). 1ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using NEB Ultra2 stranded RNA-seq library Prep Kit following manufacturer's protocols.

treatment_protocol: BMDCs were seeded in 1 million per well, and were stimulated with lipopolysaccharide (LPS) from E. coli 0111:B4 for 6 hours at 10ng/ml concentration.

value: BDMC

value: PWD

value: Untreated

tag: replicate


title: embryos, 72 hpf, rhpn2 mutant-a

description: Library name: sample4


title: MPAL1 [CITE-Seq]

tag: cell type


title: PWD_LPS_rep4

value: LPS


title: ETP-ALL3


title: 023 RNA


title: 038V02D0

value: Whole blood

tag: timepoint


title: 022 RNA

tag: pre/post menopause


title: 038V02D1

tag: treatment

value: 7.5x10^10 viral particles


title: T-ALL14


title: PWD_LPS_rep3


title: PWD_Untreated_rep3


title: ETP-ALL5


title: 037V02D1


title: PWD_Untreated_rep2


title: 040V02D0


title: MPAL2


title: PWD_LPS_rep2


title: 037V02D0


title: 019 RNA

value: pre


title: T-ALL18

value: WT


title: 036V02D1


title: 018 RNA


title: 039V02D0


title: 036V02D0


title: PWD_Untreated_rep1


title: ETP-ALL6


title: 017 RNA


title: 035V02D1


title: PWD_LPS_rep1


title: T-ALL12


title: 016 RNA


title: C57_Untreated_rep4

tag: strain


title: 035V02D0


title: ETP-ALL8


title: T-ALL17


title: 015 RNA


title: 020 RNA


title: 034V02D1


title: C57_LPS_rep4


title: 024 RNA


title: MPAL4


title: 034V02D0


title: C57_Untreated_rep3


title: ETP-ALL7


title: 065V03D1


title: 011 RNA


title: T-ALL15


title: 012 RNA

value: post


title: 065V02D0

value: youth


title: 010 RNA


title: 066V03D1


title: T-ALL16


title: 009 RNA


title: C57_Untreated_rep1


title: 066V02D0


title: T-ALL11


title: ETP-ALL2


title: C57_LPS_rep1


title: 006 RNA


title: 007 RNA


title: C57_LPS_rep3


title: 033V02D0


title: 013 RNA


title: B6_Untreated_rep4


title: B6_LPS_rep4


title: C57_Untreated_rep2


title: ETP-ALL1


title: 020V02D1

value: adults


title: 008 RNA


title: 020V02D0


title: C57_LPS_rep2


title: T-ALL8


title: B6_LPS_rep3


title: 004 RNA


title: 019V02D1


title: T-ALL1


title: T-ALL2


title: B6_Untreated_rep2


title: 003 RNA


title: B6_Untreated_rep3


title: 019V02D0


title: MPAL1


title: B6_LPS_rep2


title: 033V02D1


title: 018V02D1


title: T-ALL9


title: 018V02D0


title: T-ALL7


title: NPH_23, Infected, scRNA-seq, 5'

city: Pittsburgh

first: Kathy

last: Shair

state: PA

address: 5117 Centre Avenue

department: UPMC Hillman Cancer Center

institute: University of Pittsburgh

description: 10x Genomics, 5' V2

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v7.1.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: GRCh38 + Custom Akata EBV Genome (KC207813.1)
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Nasopharynx

extract_protocol:
Nasopharyngeal organotypic rafts were dissociated with collagenase, dispase, and trypsin (no EDTA). Rafts were filtered sequentially through a 0.77 micron and a 0.4 micron Belart filter. Viability was assessed by an Acridine Orange Propidium Iodide assay.
10X Genomics 5' V2 dual indexed chemistry. Samples are loaded in the 10X Genomics Chromium Controller.

value: Nasopharynx

value: Epithelial cells


title: 005 RNA


title: NPH_23, Mock, scRNA-seq, 5'

description: 10x Genomics, 5' V2


title: 017V02D1


title: B6_LPS_rep1


title: Normal saline treated mice 3

city: Ipoh

first: Muhammad

middle: Asyaari

last: Zakaria

state: Perak

address: 3, Jalan Greentown

department: Faculty of Pharmacy and Health Sciences

country: Malaysia

institute: Universiti Kuala Lumpur-Royal College of Medicine Perak

data_processing:
Raw reads were filtered to remove unwanted reads: adaptors, undeterminable bases (N > 10% ), and reads with > 50% bases (quality scores ≤ 5)
Clean reads were aligned to the Mus musculus genome (GRCm38/mm10) using STAR version 2.5
Gene-level read counts were computed using HTSeq version 0.6.1
Processed data was obtained using DESeq2
Assembly: mm10
Supplementary files format and content: tab-delimited text file includes raw counts for each sample

source_name: lung

growth_protocol: All BALB/c mice were placed in animal house, with ad libitum access to water and normal mice diet. All mice were housed under the same laboratory conditions of ambient room temperature and lighting (12 h light–dark cycle).

extract_protocol:
The total RNA was extracted using innuPREP RNA Mini Kit (Analytik Jena, Germany) according to the manufacturer’s guidelines. Then, contaminating genomic DNA in the samples was removed using the RapidOut DNA Removal Kit (Thermo Scientifc Inc., USA). All RNA were ensured to have good purity, exhibited discrete 28s and 18s bands and passed the RIN value of > 6.8.
RNA libraries for RNA-seq were prepared using NEBNext® Ultra TM RNA Library Prep Kit

treatment_protocol: LUSC in BALB/c mice was induced using 0.04M NTCU for 30 weeks. Whereas, the control group received 0.9% normal saline only. As much as 25 uL of treatment was given twice per week with an interval of 3.5 days via skin painting.

value: lung

tag: Sex

value: female

value: 30 weeks

value: 0.9% normal saline


title: NPH_22, Infected, scRNA-seq, 5'

description: 10x Genomics, 5' V2

tag: tissue


title: T-ALL4

value: Bone Marrow

value: primary TALL

value: primary human BM mononuclear cells


title: T-ALL5


title: NPH_22, Mock, scRNA-seq, 5'

description: 10x Genomics, 5' V2


title: Normal saline treated mice 2


title: 015V02D1


title: HK1 EBV pBabe HA IRES, HTO, scRNA-seq, 5'

description: 10x Genomics, 5' V2

extract_protocol:
Freshly collected HK1 EBV China 1 and IRES cells were washed with PBS. The cells were then trypsinized (trypsin-EDTA) and collected using RPMI media (supplemented with heat inactivated FBS). Cells were then spun down and resuspended in RPMI media (supplemented with heat inactivated FBS) and counted and aliquoted to ~200,000 cells. Next they were filtered through a 70uM filter and resuspended in PBS + 1%BSA + hashtagging antibody. Following this, cells were incubated for 30min at 4C. Cells were then washed by adding PBS + 1%BSA, gently mixing, spinning for 150rcf for 10min at 4C, removing supernatant, adding PBS +1% BSA, gently mixing, spinning down and repeating the wash step for a third time. Cells were then resuspended in PBS + 1%BSA.
10X Genomics 5' V2 dual indexed chemistry with Feature Barcoding using BioLegend's TotalSeq-C0259, C0260, C0262, C0263, C0264, C0265 anti-human Hashtag 9, 10, 12, 13, 14, 15 antibodies.  Samples are loaded in the 10X Genomics Chromium Controller.

tag: cell line


title: 015V02D0


title: Normal saline treated mice 1


title: 001 RNA

data_processing:
Sequencing reads were subjected to a variety of pre- and post-alignment QC measures before mapping against human reference genome hs37d5 using STAR-2.5.3a
Gene-level quantification was determined using HTSeq 0.6.1 by summation of raw counts of reads aligned to the region (Gencode29) associated with each gene.
To account for differences in library size between samples, Trimmed Mean of M values (TMM) normalization using calcNormFactors(), also from edgeR, was applied.
All data were then transformed using voom.
Assembly: hs37d5
Supplementary files format and content: tab-delimited text files include raw gene count for each sample

source_name: ovarian tumors

extract_protocol:
RNA was harvested using the Qiagen All-Prep RNA isolation kit
RNA libraries were prepared by using Illumina TruSeqTM RNA Exome Library Preparation Kit (Illumina Inc., San Diego, CA) , following the manufacturer’s protocol


title: LX-2 cells, CA_3

city: chongqin

first: wu

last: kang

address: chongqin

institute: CQMU

data_processing:
The raw paired end reads were trimmed and quality controlled by SeqPrep (https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle) with default parameters.
Then clean reads were separately aligned to reference genome with orientation mode using HISAT2 (http://ccb.jhu.edu/software/hisat2/index.shtml)[1] software.
The mapped reads of each sample were assembled by StringTie (https://ccb.jhu.edu/software/stringtie/index.shtml? t=example) in a reference-based approach.
To identify DEGs (differential expression genes) between two different samples, the expression level of each transcript was calculated according to the transcripts per million reads (TPM) method.
RSEM (http://deweylab.biostat.wisc.edu/rsem/) was used to quantify gene abundances.
Assembly: mm10
Supplementary files format and content: CA_vs_Control.deseq2.annot2.xlsx: Excel file includes raw counts and TPM values for each Sample.

extract_protocol:
Total RNA was extracted from the tissue using TRIzol® Reagent according to the manufacturer’s instructions (Invitrogen) and genomic DNA was removed using DNase I (TaKara). Then RNA quality was determined by 2100 Bioanalyser (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). Only high-quality RNA sample (OD260/280=1.8~2.2, OD260/230≥2.0, RIN≥6.5, 28S:18S≥1.0, >1μg) was used to construct sequencing library.
RNA-seq transcriptome library was prepared following TruSeqTM RNA sample preparation Kit from Illumina (San Diego, CA) using 1μg of total RNA. Shortly, messenger RNA was isolated according to polyA selection method by oligo(dT) beads and then fragmented by fragmentation buffer firstly. Secondly double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) with random hexamer primers (Illumina). Then the synthesized cDNA was subjected to end-repair, phosphorylation and ‘A’ base addition according to Illumina’s library construction protocol.

value: Human hepatic stellate cells

value: cholic acid (CA)


title: NTCU treated mice 3

value: 0.04 NTCU


title: 032V02D1

value: Whole blood

value: 7.5x10^10 viral particles


title: LX-2 cells, CA_2


title: 032V02D0


title: NTCU treated mice 2


title: B6_Untreated_rep1

value: BDMC

value: Untreated


title: 017V02D0

value: adults


title: LX-2 cells, ctr_3

value: vehicle


title: NTCU treated mice 1


title: 031V02D1

value: youth


title: HK1 EBV pBabe HA Ch1, GEX, scRNA-seq, 5'

description: 10x Genomics, 5' V2

value: Nasopharynx

value: Epithelial cells


title: HK1-EBV pBabe HA IRES, Day 4

data_processing:
STAR Aligned + RSEM quantifier
Sequences were aligned to the GRCh38 + Custom Akata EBV Genome using STAR aligner through the RSEM wrapper (--star flag)
Assembly: GRCh38 + Custom Akata EBV Genome (KC207813.1)
Supplementary files format and content: tab-delimited text files include TPM, FPKM values for each Sample

extract_protocol:
Cell were collected in ~350uL of TRIzol reagent. Total RNA was extracted from fresh frozen cell pellet samples using Qiagen RNeasy Plus Universal mini kit. RNA samples were quantified using Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and RNA integrity was checked with 4200 TapeStation (Agilent Technologies). RNA purification, quantification and QA were performed by Genewiz.
Trizol-purified total RNA samples were treated with TURBO DNase (Thermo Fisher Scientific) and subjected to rRNA depletion using QIAseq FastSelectTM−rRNA HMR kit (Qiagen). Strand-specific RNA sequencing libraries were prepared with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) using the dUTP incorporation method. Sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies) and quantified on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and by quantitative PCR (KAPA Biosystems). Sequencing libraries were multiplexed and clustered on one lane of a flowcell and run on an Illumina HiSeq instrument using a 2x150bp Paired-End (PE) protocol. Library construction, QC/QA, and sequencing were performed by Genewiz.

treatment_protocol: Cells were reactivated by ALI culture following the published protocol in PMID: 30021875.

value: ALI Day 4


title: 031V02D0


title: LX-2 cells, ctr_2


title: T-ALL10

value: WT


title: 030V02D1


title: LX-2 cells, ctr_1


title: HK1-EBV pBabe HA IRES, Day 0

value: ALI Day 0


title: HK1-EBV pBabe HA IRES, Day 2

value: ALI Day 2


title: 030V02D0


title: HK1 EBV pBabe HA IRES, GEX, scRNA-seq, 5'

description: 10x Genomics, 5' V2


title: Multiplexed scRNA, 5 individuals-3

city: Nashville

first: Lindsey

middle: "Joey"

last: Stolze

state: TN

address: 1211 Medical Center Dr

institute: Vanderbilt University Medical Center

description: Multiplexed scRNA-seq with 5 individuals blood derived PBMCs, 4 individuals shared with Sample 2

data_processing:
We aligned the raw sequencing reads to the human genome (hg38) using STAR v.2.7.2a. We identified cells using cell ranger (version 7.1)
Downstream analysis was performed using the Seurat V4. We filtered out cells with ≤ 200 genes, umi ≤ 400 or > 4000, and ≤ 10% mitochondrial genes expressed.
We used DoubletFinder with default settings to identify heterotypic doublets.
We then performed downstream analyses on the RNA count matrix including feature selection, scaling and centering counts, principal component analysis, Uniform Manifold Approximation and Projection (UMAP) reduction, shared nearest neighbor graph construction, and finally cluster determination.
Assembly: hg38

source_name: Blood PBMCs

extract_protocol:
PBMCs were thawed and washed with PBS (no calcium or magnesium), and the pellet resuspended in 100µL of cell staining buffer. The cells were incubated with Fc receptor blocking solution (5 µL, TruStain). Cells were incubated on ice for 10 minutes. A Total Seq C master mix containing surface antibodies (CD3 clone UCHT1 #300479, CD4 clone SK3 #344651, CD8a clone SK1 #344753, CD14 clone 63D3 #367137, CD16 clone 3G8 #302065, CD69 clone FN50 #310951) was prepared. 3µL of the master mix was added each sample, which were incubated on ice for 30 minutes. The cells were washed three times and resuspended in 0.04% BSA in PBS.
We submitted twenty thousand cells that were loaded onto the Chromium Single Cell 5’ ssay (10x Genomics). Cells were encapsulated and feature barcoded, with library preparation, cDNA amplification and sequencing (50bp paired-end reads, on NovaSeq 6000 S2 platform).

value: Blood PBMCs


title: 029V02D1


title: HK1-EBV pBabe HA Ch1, Day 2


title: Multiplexed scRNA, 5 individuals-2

description: Multiplexed scRNA-seq with 5 individuals blood derived PBMCs, 4 individuals shared with Sample 3


title: HK1 EBV pBabe HA Ch1, HTO, scRNA-seq, 5'

description: 10x Genomics, 5' V2


title: LX-2 cells, CA_1

value: Human hepatic stellate cells


title: S3_PI-_28_664

type: RNA

city: Giessen

first: Gerald

middle: Herbert

last: Reiner

address: Frankfurter Strasse 112

department: Veterinary Medicine

institute: Justus-Liebig-University Giessen

hyb_protocol: After checking the quality and quantity of the labeled cRNA, cRNA was fractionated and hybridized with the Affymetrix GeneChips. GeneChips were washed and stained with the Affymetrix GeneChip Fluidics station 450. The GeneChips were then scanned with an Affymetrix GeneChip scanner 3000. All procedures were performed according to Affymetrix protocols (GeneChip expression analysis, Technical manual from Affymetrix). The quality of hybridization was assessed in all samples following the manufacturer´s recommendations.

scan_protocol: After scanning the arrays, cell intensity files containing a single intensity value for each probe cell were computed from the image data with the Affymetrix GeneChip Command Console Software.

data_processing: Probe cell intensity data were further analyzed in the Affymetrix Expression Console software using the Robust Multichip Analysis (RMA) algorithm.

label: Biotin

organism: Sus scrofa

source_name: Liver SINS high, breed Pietrain susceptible (PI-)

label_protocol: The cRNA was labeled with biotin using the Affymetrix GeneChip labeling kit.

growth_protocol: The study was approved by the responsible animal welfare office of the Justus-Liebig-University in Giessen, Germany (file number 708_M). The animal experiment was carried out in the conventional pig breeding stables of the Oberer Hardthof teaching and research station at Justus-Liebig University Giessen. 21 out of 236 piglets from 37 sows of a herd of Topigs hybrid sows artificially inseminated with Duroc (DU), vafourable Pietrain (PI+) and unfavourable Pietrain (PI-) (with regard to susceptibility to SINS) semen were used for the experiment. From all piglets scored for SINS in this experiment, eleven piglets with least (SINS low) and ten piglets with highest (SINS high) SINS-Scores were selected for transcriptomics.

extract_protocol: Total RNA from liver aliquots (20 mg) of 11 and 10 suckling piglets of the SINS low group (either no or only low-grade signs of SINS) and the SINS high group (high-grade signs of SINS), respectively was extracted using TRIzol reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s protocol. RNA quantity and quality were determined spectrophotometrically and by electrophoresis. The average RNA concentration and RNA integrity number (RIN) of all total RNA samples (n = 21, means ± SD) was 454 ± 34 ng/µL and 8.0 ± 0.6, respectively. For microarray hybridization, RNA samples were sent on dry-ice to the Center of Excellence for Fluorescent Bioanalytics (Regensburg, Germany) and, following a further RNA quality check using an Agilent 2100 Bioanalyzer (RIN of all samples: 7.7 ± 0.5), processed using an Affymetrix GeneChip Porcine Gene 1.0 Sense Target array.

treatment_protocol: From all piglets scored for SINS in this experiment, six piglets with least (SINS low) and six piglets with highest (SINS high) SINS-Scores were selected for transcriptomics.

value: mixed

tag: condittion

value: SINS high

value: liver

tag: breed

value: Pietrain


title: 029V02D0

value: Whole blood

value: 7.5x10^10 viral particles


title: Multiplexed scRNA, 5 individuals-1

description: Multiplexed scRNA-seq with 5 individuals blood derived PBMCs


title: HK1-EBV pBabe HA Ch1, Day 0


title: S4_PI-_29_664


title: A549 cells, SAMD9-Myc-DDK, 24 hr, replicate 2

city: St. Louis

first: Gaopeng

last: Hou

state: MO

address: McDonnell Pediatric Research Building, Rm 9220

department: molecular microbiology

institute: Washinton University inSt. Louis

data_processing:
Basecalling with Illumina RTA 1.9
Demultiplexing with Illumina bcl2fastq2
Fastq files were combined and aligned with STAR 2.5.1a
Gene counts quantitated with Subread:featureCount 1.4.6-p5 with CPM and RPKM transformations added with custom Rscripting.
Assembly: Ensembl GRCh38.76
Supplementary files format and content: Gene counts with CPM and RPKM in tab-delimitted text format

extract_protocol:
Trizol extraction and RNA quality control by Agilent Bioanalyzer or 4200 Tapestation
KAPA RiboErase

treatment_protocol: Wild type A549 cells were transfected with 1 μg of pCMV6-Entry or SAMD9-Myc-DDK plasmids for 24 hr.

value: Lung carcinoma epithelial cell

value: SAMD9-Myc-DDK transfected

tag: time

value: 24 hr

value: SAMD9 overexpression


title: 028V02D1

value: youth


title: BT183 cells

city: Boston

first: Lisa

last: Gabler

address: Brookline Avenue 360

institute: Dana Farber Cancer Institute

data_processing: Raw sequencing reads were aligned to the hg19 genome and normalized gene counts were quantified using RSEM (v1.3.0) as transcript-per-million (TPM) (B. Li & Dewey, 2011). Expression levels were quantified as previously described (Hovestadt et al., 2019). In brief, expression levels were calculated by log transformation of the transcripts-per-million (TPMi,j) for each gene (i) per sample (j): Ei,j = log2(TPMi,j/10 + 1). To filter out low-quality cells in fresh samples, cells with <2,000 genes or an average housekeeping gene expression of <2.5 were removed. For frozen tumors, a filtering threshold of <1,000 genes and an alignment rate of <0.4 were employed. In sum, 3,406 high-quality cells (2,520 derived from fresh and 886 from frozen tissue samples) were retained. Additionally, we removed genes with a mean expression <4, obtaining a total number of 7,795 and 10,809 genes for fresh and frozen tissues, respectively. For the remaining cells and genes, we computed the aggregate expression of each gene as Ea(i) = log2(average(TPMi,1…n) + 1).

molecule: polyA RNA

source_name: cell model

extract_protocol:
Tumor tissues were collected at the time of surgery and processed directly for scRNAseq or frozen and processed later for snRNAseq.BT183 (100,000 cells) , CBTP03, or NCH3602 (1,000,000) ETMR cells were injected into the cerebral cortex of NSG mice. Tumor tissues were collected fresh and processed directly or frozen and processed at a later time point.
The whole transcriptomes of single cells were amplified, libraries were constructed, and sequencing was performed following the modified Smart-seq2 protocol (Filbin et al., 2018; Gojo et al., 2020; Hovestadt et al., 2019; Liu et al., 2022; Neftel et al., 2019; Tirosh, Venteicher, et al., 2016; Venteicher et al., 2017). RNA was purified using Agencourt RNAClean XP beads (Beckman Coulter). Oligo-dT-primed reverse transcription was performed with Maxima H Minus reverse transcriptase (Life Technologies) and a template-switching oligonucleotide (TSO, Qiagen). cDNA was amplified using KAPA HiFi HotStart ReadyMix (KAPA Biosystems) and purified using Agencourt AMPure XP beads (Beckman Coulter). Libraries were generated using the Nextera XT Library Prep kit (Illumina). Multiplexed libraries were sequenced on a NextSeq 500/550 High Output Kit v2.5 (Illumina).

value: cell model

value: Embryonal tumor with multilayered rosettes (ETMR) cells

tag: gender

value: male

value: treatment naïve


title: 028V02D0

extract_protocol:
PAXgene Blood RNA kit
Ion AmpliSeq™ Library Kit Plus
Ion S5 Sequencing Kit


title: NCH3602 PDX #2

source_name: PDX tissue

value: PDX tissue

value: female


title: A549 cells, SAMD9-Myc-DDK, 24 hr, replicate 1

tag: genotype


title: S2_PI-_32_702

value: mixed


title: C3_PI-_25_544

source_name: Liver SINS low, breed Pietrain susceptible (PI-)

value: SINS low


title: S1_PI-_41_736

value: SINS high

value: liver


title: HK1-EBV pBabe HA Ch1, Day 4

value: Nasopharynx

value: Epithelial cells

value: ALI Day 4


title: 027V02D1

value: Whole blood

value: 7.5x10^10 viral particles

tag: age


title: 027V02D0


title: BT183 PDX #2


title: C1_PI-_33_702

value: Pietrain


title: BT183 PDX #1


title: C2_PI-_38_706


title: A549 cells, pCMV6-Entry, empty vector control, 24 hr, replicate 2

value: Lung carcinoma epithelial cell

value: pCMV6-Entry transfected

value: 24 hr

value: control


title: A549 cells, pCMV6-Entry, empty vector control, 24 hr, replicate 1


title: siHDAC9_3

city: Jinan, Shandong

first: Shuai

last: Leng

address: 105 Jiefang Road

institute: Central Hospital Affiliated to Shandong First Medical University

data_processing:
Raw reads were processed using Trimmomatic. The reads containing ploy-N and the low quality reads were removed to obtain the clean reads.
The clean reads were mapped to reference genome using hisat 2.
FPKM value of each gene was calculated using cufflinks, and the read counts of each gene were obtained by htseq-count.
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample
Supplementary files format and content: hg38

source_name: aorta

extract_protocol:
Total RNA was extracted using using Trizol reagent following the manufacturer's protocol.
The libraries were constructed using TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions.

value: aorta

value: HASMCs

value: Vascular smooth muscle cells


title: 026V02D0

data_processing:
Base-calling, alignment and filtering were performed by the Ion Proton Server
Assembly: GRCh37
Supplementary files format and content: Counts_Leish2b_20230615.csv contains read counts for all the samples
Supplementary files format and content: vstNormCount.csv containing normalized counts (log2-like)

value: youth


title: 026V02D1


title: siHDAC9_2


title: S2_PI+_47_797

source_name: Liver SINS high, breed Pietrain resistant (PI+)

value: mixed


title: SK3CO

source_name: patient tumor tissue

value: patient tumor tissue

value: Embryonal tumor with multilayered rosettes (ETMR) cells

value: male

value: treatment naïve


title: siHDAC9_1


title: NCH3602 PDX #1

value: PDX tissue

value: female


title: 025V02D1

source_name: Whole blood

value: Whole blood

value: 7.5x10^10 viral particles


title: BCH1446


title: Control_3

value: Control


title: 025V02D0


title: MUV9N


title: Control_2


title: 014V02D1

value: adults


title: S1_PI+_48_818

value: SINS high

value: liver

value: Pietrain


title: 014V02D0


title: S3_PI+_46_745


title: Control_1

value: aorta

value: HASMCs


title: C3_PI+_40_706

source_name: Liver SINS low, breed Pietrain resistant (PI+)

value: SINS low


title: MUV3N


title: 013V02D1


title: C2_PI+_37_706

value: mixed


title: MUV45

value: patient tumor tissue


title: S3_DU_36_705

source_name: Liver SINS high breed: Duroc

value: Duroc


title: MUV30

value: Embryonal tumor with multilayered rosettes (ETMR) cells


title: KO_3

city: Shinjuku

first: Sumihiro

last: Maeda

state: Tokyo

address: 35 Shinanomachi, Tokyo

country: Japan

institute: Keio University School of Medicine

data_processing:
The read counts, fragments per kilobase of exon per million mapped fragments (FPKM), and transcripts per million (TPM) were calculated using featureCounts version 1.6.3.
The samples were clustered with the Wald method based on Euclidean distances of the normalized counts using stats (Version 3.6.1) and gplots (Version 3.0.1.1) R packages.
Assembly: hg38

source_name: neurons

growth_protocol: hiPSCs were differentiated by driving the expression of Ngn2 and microRNA9/9*/124

extract_protocol:
Total RNA was extracted with an RNeasy Kit (Qiagen, Hilden, Germany), and RNA was quantified using a NanoDrop-2000c spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
The mRNA libraries were prepared in a strand-specific manner using NEBNext Poly(A) mRNA Magnetic Isolation Module (Cat No. E7490) and NEBNext UltraTMII Directional RNA Library Prep Kit (Cat No. E7760).

value: neurons

value: hiPSC-derived neurons

value: KO/KO


title: KO_4


title: C1_PI+_39_706


title: 013V02D0

value: Whole blood


title: BCH1162

value: female

value: treatment naïve


title: 012V02D1

value: 7.5x10^10 viral particles

value: adults


title: BCH1640

value: male


title: KO_2


title: BCH1548


title: C5_DU_52_744

source_name: Liver SINS low, breed Duroc

value: liver


title: S1_DU_34_705

value: SINS high


title: 011V02D1


title: C4_DU_50_744


title: 012V02D0


title: BCH1078


title: A152T_4


title: 011V02D0


title: S2_DU_27_641


title: C2_DU_43_744


title: C3_DU_51_744


title: C1_DU_30_672


title: BCH736


title: WT_4


title: batch-3_0120-B

city: Cape Town

first: Valentin

last: Voillet

state: Western Cape

address: First Floor Wembley Square, 3 McKenzie St, Gardens

country: South Africa

institute: cape town hvtn immunology laboratory

data_processing:
The `RNASeqPipelineR` was used to perform alignment, quantification and annotation of the RNA sequencing data (RNA-seq) from the whole and mini population RNA-Seq libraries. The RNA-seq data were aligned against the human genome (hg38) using STAR (v2.4.2a), and gene expression quantification was performed using RSEM (v1.2.22).
Reads from a same sample but different batches were aggregated.
Assembly: hg38
Supplementary files format and content: wraw_counts.csv

source_name: BAL

extract_protocol:
RNA from blood samples was extracted using Paxgene RNA extraction kit and from the BAL samples we used Qiagen RNAEasy micro kit method
Libraries were prepared from RNA samples of both sample types using NEBNext kits

value: BAL

tag: patient id

tag: disease status

value: LTBI and HIV-positive


title: CTRL Infected Male 7 DPI mRNA Rep 3 [TX3_D7_M_neg]

city: University Park

first: Cory

last: Henderson

address: Room W127, Millennium Science Complex

department: molecular cellular and integrative biosciences

institute: Pennsylvania State University

data_processing:
Adapter trimming and low quality bases removed with Trimmomatic using base settings
Align reads to genome using STAR RNA sequencing aligner
Filter reads less than 50 bp and mapping quality less than 20 from further analysis
Read counts calculated using rSubread package in R
Differential expression was conducted using a negative binomial GLM with the EdgeR package in R
Genome_build: GCA_000005575
Supplementary_files_format_and_content: Contains either read counts mapping to each gene ID in the AgamP4.12 (GCA_000005575) genome build (.csv) or differential expression results (.xlsx).

organism: Anopheles gambiae

source_name: CTRL Infected Male 7 DPI_whole mosquito

growth_protocol: Mosquito colonies were reared and maintained at at 27˚C ± 1˚C, 12 hour light 12 hour dark diurnal cycle at 80% relative humidity in 30×30×30-cm cages. Ground fish flakes were used to feed larvae, and upon emergence adult mosquitoes were maintained with a 10% sucrose solution.

extract_protocol:
Mosquitoes were anesthesized with triethylamine and RNA was extracted from each individual mosquito using mirVana RNA extraction kit (Life Technologies) applying the protocol for extraction of total RNA.
The NEBNext Ultra II RNA library kit (New England Biolabs) was used for RNA-Seq library construction for total RNA.

treatment_protocol: Mosquitoes were injected either with 69 nL or O'nyong'nyong virus at 2*10^7 FFU/mL or  uninfected supernatant with a Nanoject II programmable injector (Drummond Scienific, Broomall, PA, USA).

value: Uninfected control

value: Male

tag: days post infection

value: Whole mosquito


title: batch-3_0113-B


title: WBC6-DH-Monosomes RNA-seq

city: changchun

first: lu

last: li

institute: Jilin university

data_processing:
CLC Genomics Workbench v 11.0.1
Supplementary files format and content: tab-delimited text files include RPKM values for each sample

organism: Giardia duodenalis

source_name: Giardia duodenalis

extract_protocol:
RNA was harvested using Rneasy mini plus kit (Qiagen). 1,3ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols.

value: Giardia duodenalis

tag: cell type

value: parasite

value: Giardiavirus-containing

value: Giardiavirus infection


title: A152T_3

value: neurons

value: hiPSC-derived neurons


title: batch-3_0093-B

value: LTBI and HIV-negative


title: CTRL Infected Male 7 DPI mRNA Rep 2 [TX2_D7_M_neg]


title: WBC6-polysomes RNA-seq

value: Giardiavirus-free

value: no


title: CTRL Infected Male 2 DPI mRNA Rep 2 [TX2_D2_M_neg]

source_name: CTRL Infected Male 2 DPI_whole mosquito


title: batch-3_0113-P

source_name: PBMC

value: PBMC


title: WBC6-DH-polysomes RNA-seq


title: A152T_2


title: A152T_1


title: batch-3_0073-B


title: DH-Monosomes RNA-seq

value: DH


title: WT_2


title: CTRL Infected Male 2 DPI mRNA Rep 3 [TX3_D2_M_neg]


title: CTRL Infected Male 2 DPI mRNA Rep 1 [TX1_D2_M_neg]


title: P301S_3


title: batch-3_0052-B


title: WT_1


title: WBC6-Monosomes RNA-seq


title: KO_1

value: KO/KO


title: 11TPn

city: Balice

first: Ewelina

last: Semik-Gurgul

address: Krakowska 1

department: Department of Animal Molecular Biology

country: Poland

institute: National Research Institute of Animal Production

library_source: genomic

data_processing:
Raw sequencing reads were first subjected to quality control using FastQC software (Babraham Bioinformatics, UK)
Low-quality reads (phred-quality of 20 and a minimum read length of 36 were required to pass the filter) and fragments containing adapter sequences were filtered with the use of FlexBar software v. 3.5.0 (Dodt et al., 2012).
The alignment to the reference genome (ARS-UCD1.2) was performed using the bisulfite mapping software known as BSMAP v. 2.9.0 (Yuanxin and Wei, 2009), with default options recommended for RRBS data.
The percentage of methylation of individual CpG sites with coverage higher than 5 reads was determined using Methylation Caller software provided in the BSMAP package.
Assembly: ARS-UCD1.2
Supplementary files format and content: Files containing the percentage of methylation of individual CpG sites determined using Methylation Caller software followed by the conversion in R to be viable as input files for Methylkit software

organism: Bos taurus

source_name: parenchyma tissue

extract_protocol:
DNA was isolated from tissue using using Sherlock AX kit (A&A Biotechnology, Poland).
The RRBS libraries construction was performed using Ovation® RRBS Methyl-Seq System 1–16 (NuGEN Technologies, San Carlos, CA, United States), following the manufacturer’s protocol. Sequencing was performed using an Illumina NextSeq550 system with a High Output kit v2.5 and 1 × 75 sequencing cycles. To control the quality of the reads obtained and increase RRBS library complexity at early sequencing cycles, 30% phiX genomic DNA (Illumina, San Diego, California) was added to the libraries.

treatment_protocol: Three experimental groups: Control group (n= standard nutrition , estimated by Nutrient Requirement of Dairy Cattle (NRC, 2001); Restricted group (n=6) restriction of the daily ration of milk replacer (MR); and Unlimited group (n=6) administration of MR ad libitum 3x a day for 30 min. The collected material was transferred into tubes with RNAlater and stored at -80°C.

value: fat pad tissue

tag: treatment/diet

value: Unlimited nutrition


title: ONNV Infected Male 7 DPI mRNA Rep 3 [TX3_D7_M_plus]

source_name: ONNV Infected Male 7 DPI_whole mosquito

value: ONNV infected


title: DH-polysomes RNA-seq


title: P301S_2


title: CTRL Infected Male 7 DPI mRNA Rep 1 [TX1_D7_M_neg]

value: Uninfected control

value: Male


title: batch-3_0050-B

value: BAL

value: LTBI and HIV-positive


title: batch-3_0036-B


title: P301S_1


title: ONNV Infected Male 7 DPI mRNA Rep 2 [TX2_D7_M_plus]

value: Whole mosquito


title: 15TPn


title: 9TPn


title: ONNV Infected Male 7 DPI mRNA Rep 1 [TX1_D7_M_plus]


title: batch-3_0021-Bbis


title: ONNV Infected Male 2 DPI mRNA Rep 3 [TX3_D2_M_plus]

source_name: ONNV Infected Male 2 DPI_whole mosquito


title: A4_different_Input

city: Tempe

first: Vel

last: Murugan

state: Arizona

address: 727 E. Tyler St.

department: Biodesign Institute

institute: Arizona State University

data_processing:
Cutadapt (v 1.18) was used to trim raw sequencing reads.
BWA (v 0.7.17 50) was used to align reads to the human reference genome (GRCh38.p12, hg38).
MethylQA (v 0.2.1) was used to generate genome density for MeDIP-Seq data and reads mapping and CpG coverage statistics.
MACS2 (v 2.2.6) were used to call peaks with input as a control.
Assembly: hg38
Supplementary files format and content: narrowPeak (except for Input sample) (http://genome.ucsc.edu/FAQ/FAQformat.html#format12)

extract_protocol:
Frozen PBMCs were centrifuged twice at 600x g for 10 minutes at 20°C and washed with RPMI+ 20% FBS. The cell pellet was resuspended in PBS to achieve a final volume of 7,500 cells per 1µL of suspension. An equal volume of the MNase digestion buffer (0.02 U/µL MNase (Thermo Scientific, Cat. No: 88216) in 2x lysis buffer was added to the cell suspension and gently vortexed. The cell suspension was then incubated on ice for 20 minutes, followed by incubation at 55°C water bath for 10 minutes. The digestion reaction was terminated using 30 mM EGTA. Then the digested chromatin was protease k (Ambion™) treated at 55°C and then pre-cleared with 1:2 KAPA Pure beads (Roche Cat. No: 07983298001). The quality and quantity of fragmented DNA were verified using agarose gel (1.5%) electrophoresis and Qubit® 4.0 Fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, U.S), respectively.
Illumina KAPA LTP Library Preparation Kit was employed for library construction (Cat. No: KK8233). Purified, fragmented DNA was end-repaired to produce 5′-phosphorylated and blunt-ended dsDNA fragments. Unique Dual Indexes (UDIs) were ligated to 3'-A-tailed library fragments holding appropriate adapter sequences on both ends. DNA fragments were subjected to methylated DNA immunoprecipitation, followed by library amplification.

value: Input


title: batch-3_0021-B


title: 7TPn


title: ONNV Infected Male 2 DPI mRNA Rep 1 [TX1_D2_M_plus]


title: A12_different_Input


title: A5_different_Input


title: batch-3_0013-B


title: 16TPn

value: Restricted nutrition


title: 6TPn


title: 3TPn


title: CTRL Infected Female 7 DPI mRNA Rep 3 [TX3_D7_F_neg]

source_name: CTRL Infected Female 7 DPI_whole mosquito

value: Female


title: A7_equal_Input


title: batch-3_0003-B

value: LTBI and HIV-negative


title: CTRL Infected Female 7 DPI mRNA Rep 2 [TX2_D7_F_neg]


title: batch-2_0099-P


title: 13TPn


title: A5_equal_Input


title: 10TPn


title: A7_different_Input


title: batch-3_0024-B


title: CTRL Infected Female 7 DPI mRNA Rep 1 [TX1_D7_F_neg]


title: batch-2_0099-B


title: ONNV Infected Male 2 DPI mRNA Rep 2 [TX2_D2_M_plus]


title: 8TPn


title: A12_equal_Input


title: batch-2_0093-P


title: CTRL Infected Female 2 DPI mRNA Rep 3 [TX3_D2_F_neg]

source_name: CTRL Infected Female 2 DPI_whole mosquito


title: CTRL Infected Female 2 DPI mRNA Rep 2 [TX2_D2_F_neg]


title: 5TPn


title: batch-2_0088-B


title: batch-2_0088-P


title: ONNV Infected Female 7 DPI mRNA Rep 3 [TX3_D7_F_plus]

source_name: ONNV Infected Female 7 DPI_whole mosquito


title: 19TPn

value: Control


title: A5_different_IP

value: 5 methylcytosine


title: CTRL Infected Female 2 DPI mRNA Rep 1 [TX1_D2_F_neg]


title: A7_different_IP

value: PBMC


title: batch-2_0076-P


title: A4_equal_Input


title: 21TPn


title: A12_different_IP


title: batch-2_0076-B


title: 18TPn


title: A7_equal_IP


title: ONNV Infected Female 7 DPI mRNA Rep 1 [TX1_D7_F_plus]


title: A4_different_IP


title: batch-2_0073-P


title: ONNV Infected Female 2 DPI mRNA Rep 3 [TX3_D2_F_plus]

source_name: ONNV Infected Female 2 DPI_whole mosquito


title: 11PPPn

source_name: fat pad tissue

value: parenchyma tissue


title: A5_equal_IP


title: batch-2_0069-P


title: 15PPPn


title: ONNV Infected Female 2 DPI mRNA Rep 2 [TX2_D2_F_plus]


title: batch-2_0069-B


title: ONNV Infected Female 2 DPI mRNA Rep 1 [TX1_D2_F_plus]


title: A4_equal_IP


title: 9PPPn


title: A12_equal_IP


title: ONNV Infected Female 7 DPI mRNA Rep 2 [TX2_D7_F_plus]


title: 7PPPn


title: batch-2_0057-P


title: batch-2_0050-B


title: A19_individual_Input


title: 3PPn


title: 6PPTN


title: A18_individual_Input


title: A15_individual_Input


title: A16_individual_Input


title: 2PADn


title: batch-2_0038-B


title: 16PPPn


title: human adipose derived stem cells maintained in adipocyte maintenance Medium rep3

city: Tsukuba

first: Rui

last: Wang

address: Tennodai 1-1-1

institute: University of Tsukuba

description: Gene expression data from human adipose derived stem cells maintained in adipocyte maintenance Medium for 7 days

hyb_protocol: The cartridge array hybridization was performed using the Clariom cartridge array (Clariom S array, Human; ThermoFisher Scientific).

scan_protocol: Scanning was performed using GeneChip Scanner (ThermoFisher Scientific).

data_processing:
The raw data were normalized following the signal space transformation robust multi-chip analysis (SST-RMA) algorithm. Further, gene-level analysis was performed using the Limma Bioconductor package. For differential expression analysis, a One-Way ANOVA followed by an empirical Bayes correction was performed. The detected above background (DABG) cutoff was set to 0.05. The positive vs negative area under the curve (AUC) value was set at greater than or equal to 0.7.
Signal intensity of all the samples derived from Transcriptome analysis console software (sheet 2 ). For calculating average value, standard deviation, fold change, and Anova p-value of signal intensity of replicates, Transcriptome Analysis Console software (CEL files). filter criteria was set as p value < 0.05 (one-way between-subjects ANOVA) and fold change > 2 (in linear space)
signal space transformation robust multi-chip analysis (SST-RMA) algorithm (Transcriptome Analysis Console, version 4)

label: biotin

label_protocol: Biotinylated cRNA were prepared according to the standard WT PLUS Reagent Kit protocol (ThermoFisher Scientific) from 100 ng/ uL total RNA

growth_protocol: hASCs were obtained from ATCC® (SCRC-4000TM, Manassas, VA, USA). The culture process and differentiation process was using the same procedure as detailed described in our previous research (Wang et al., 2023). In brief, the cells were cultured in DMEM/F12 medium (11320033, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% GlutaMax (35050061, Thermo Fisher Scientific, USA), and 1% 2-mercaptoethanol (21985023, Thermo Fisher Scientific, USA). The culture was maintained at 37 degrees Celsius in a 5% CO2-containing atmosphere. Seeding was initiated at a density of 5000 viable cells per cm2, and passaging occurred when the cells reached 80% confluence.  Adipogenic differentiation was initiated on the second day after reaching cell confluence, involving a 7-day induction phase followed by a 14-day maintenance period. The induction mixture included 100 nM insulin (093-06351, Wako, Japan), 1 μM dexamethasone (10008980, Cayman Chemical, Ann Arbor, MI, USA), 0.5 mM IBMX (10008978, Cayman Chemical, USA), and 1 μM rosiglitazone (Herbers et al., 2022). For maintenance, a DMEM-F12-based medium was supplemented with 100 nM insulin and 1 μM dexamethasone, with or without the addition of chemicals during the maintenance process. Both the induction and maintenance media were refreshed every 4 days over a 21-day period.

extract_protocol: Total RNA was extracted using ISOGEN (Nippon Gene Co., Ltd, Tokyo, Japan) following the manufacture’s instruction.

treatment_protocol: Treatment was added after adipocyte inducing process for 14 days in maintenance status. Maintenance consisted of a DMEM-F12 mix supplemented with 100 nM insulin and 1 μM dexamethasone, with or without other chemicals, renewed every 4 days for a 21-day period.

value: human adipose-derived stem cells


title: human adipose derived stem cells maintained in adipocyte maintenance Medium rep2

source_name: human adipose derived stem cells maintained in adipocyte maintenance Medium for 7 days


title: 13PPPn


title: batch-2_0036-P


title: A13_individual_Input


title: 10PPPn


title: batch-2_0024-B


title: batch-2_0052-B


title: 4PTPn

value: fat pad tissue


title: human adipose derived stem cells treated with oleuropein rep3

description: Gene expression data from human adipose derived stem cells were treated with 20 uM oleuropein for 14 days


title: human adipose derived stem cells maintained in adipocyte maintenance Medium rep1


title: A7_individual_Input


title: human adipose derived stem cells treated with oleuropein rep2

source_name: human adipose derived stem cells were treated with 20 uM oleuropein for 14 days


title: batch-2_0013-B


title: 4PPPn


title: batch-2_0038-Bbis


title: A5_individual_Input


title: 21PPPn


title: batch-2_0003-B


title: A4_individual_Input


title: batch-1_0120-P


title: human adipose derived stem cells treated with oleuropein rep1


title: A2_individual_Input


title: human adipose derived stem cells maintained in adipocyte maintenance Medium_rep3


title: batch-1_0120-B


title: 18PPPn


title: human adipose derived stem cells maintained in adipocyte maintenance Medium_rep2


title: A12_individual_Input


title: batch-1_0113-P


title: A19_individual_IP


title: 2PARn


title: human adipose derived stem cells treated with oelocanthal_rep3

description: Gene expression data from human adipose derived stem cells were treated with 20 uM oleocanthal for 14 days


title: A18_individual_IP


title: batch-1_0113-B


title: 1PARn


title: A16_individual_IP


title: batch-1_0099-P


title: 15Wn

source_name: liver tissue

value: liver tissue

value: Unlimited nutrition


title: A15_individual_IP


title: human adipose derived stem cells treated with oelocanthal_rep1

source_name: human adipose derived stem cells were treated with 20 uM oleocanthal for 14 days


title: batch-1_0099-B

source_name: BAL


title: 9Wn


title: A13_individual_IP


title: human adipose derived stem cells treated with oelocanthal_rep2


title: KM12SM_2

city: Madrid

first: Javier

last: Robles

state: Madrid

address: Calle Ramiro de Maetzu 9

department: Biomedicine

country: Spain

institute: Biological Research Centre Margarita Salas

hyb_protocol: “Clariom™ S Array, human” (Applied Biosystems).

scan_protocol: “GeneChip® Scanner 3000 7G” (Applied Biosystems).

data_processing: Data analysis was performed with Transcriptome Analysis Console (TAC) Software.

label_protocol: Samples were terminal labeled with “GeneChip® WT PLUS Reagent Kit” (Applied Biosystems).

growth_protocol: Cells were grown in DMEM containing 10% FBS (Invitrogen) and antibiotics at 37 °C in a 5% CO2 humidified atmosphere.

extract_protocol: Total RNA was isolated using NucleoSpin RNA kit (Macherey-Nagel). Quality assessment of the RNA was assessed with an Agilent 2100 bio-analyzer.

treatment_protocol: Cell lines were silenced by using TRCN00000555660 shRNA from Open Biosystems.

tag: condition

value: Colorectal cancer


title: KM12SM_1


title: human adipose derived stem cells maintained in adipocyte maintenance Medium_rep1


title: A12_individual_IP

tag: treatment


title: batch-1_0093-B


title: 3Wn


title: 6Wn


title: A7_individual_IP


title: batch-1_0088-P


title: 11Wn


title: 16Wn


title: A5_individual_IP


title: batch-1_0088-B


title: KM12SM-sh60_1


title: KM12SM_3


title: 13Wn


title: A4_individual_IP


title: batch-1_0076-P


title: batch-1_0093-P


title: A2_individual_IP


title: batch-1_0076-B


title: ATC patient, scRNA

city: Chengdu

first: Heng

last: Xu

state: Sichuan

address: 17#, section 3, South Renmin Road

institute: Sichuan University

description: 10x Genomics

data_processing:
Raw bcl files were demultiplexed using mkfastq (Cell Ranger v3.1.0) to generate FASTQ files. FASTQ were mapped to the human reference genome (GRCh38 v3.0.0) using count application (Cell Ranger v6.0.2) with default settings. Read10× function from the Seurat package (version 4.1.1) was used to merge all sample data into an aggregate Seurat object. Cells were filtered by several criteria: less than 15% of mitochondrial gene expression, minimal expression of 200 genes per cell, and nFeature RNA less than 4000. After filtering, we obtained 13,564 cells. We ran Seurat ScaleData and NormalizeData with default parameters. The variable genes were identified by the FindVariableFeatures function of Seurat package. We corrected batch effects between samples with RunHarmony function in harmony (version 0.1.0) R packages (44). Last, we clustered cells using the FindNeighbors and FindClusters functions and performed nonlinear dimensional reduction with the RunUMAP (resolution = 0.4) function with default settings. Differentially expressed genes (DEGs) between every pair of clusters were identified by using FindMarkers with default settings.
Assembly: genome GRCh38
Supplementary files format and content: The count matrix of all cells

source_name: Thyroid

extract_protocol: The Chromium Single Cell 3′ Library & Gel Bead Kit v2 (PN120237), Chromium Single Cell 3’ Chip Kit v2 (PN-120236), and Chromium i7 Multiplex Kit (PN-120262) were used according to the manufacturer’s instructions in the Chromium Single Cell 3′ Reagents Kits v2 User Guide. The single-cell suspension was washed twice with 1× phosphate-buffered saline (PBS) + 0.04% BSA. Cell number and concentration were confirmed with TC20 Automated Cell Counter. Cells were subjected immediately onto a 10× Genomics Chromium Controller machine for gel beads-in-emulsion (GEM) generation. Barcoded complementary DNAs (cDNAs) were prepared using a 10× Genomics Chromium Single Cell 3′ reagent kit (V2 chemistry), which was subsequently recovered, purified, and amplified to generate sufficient quantities for library preparation. Library quality and concentration were assessed using Agilent Bioanalyzer 2100. Libraries were run on the NovaSeq platform of Illumina for PE150 sequencing.

value: Thyroid

tag: disease state

value: Anaplastic thyroid cancer (ATC)

value: Tyrosine kinase inhibitors (TKI) + radiotherapy + immunotherapy (pre/post-treatment)


title: 8Wn


title: batch-1_0073-P

value: LTBI and HIV-positive


title: 5Wn


title: batch-1_0073-B

value: BAL


title: 4Wn

value: Restricted nutrition


title: RPE1_Taxol_rep3

city: Grand Forks

first: Motoki

last: Takaku

state: ND

address: W221 Biomedical Sciences 1301 N. Columbia Road

institute: University of North Dakota

data_processing:
Adapter sequences were removed with Trimmomatic
Reads were mapped to the hg38 genome using STAR aligner (Version 2.7.1a)
Mapped read counts per gene were collected by Subread featureCounts (version 1.6.4).
Assembly: hg38
Supplementary files format and content: coverage tracks in bigWig format
Supplementary files format and content: DESeq2 output format

growth_protocol: DMEM  + 10% FBS

extract_protocol:
RNeasy Mini Kit
TruSeq stranded mRNA k

treatment_protocol: 24 hours with 10 μM CMPD1

tag: agent

value: Taxol


title: KM12SM-sh60_3


title: SW620_1


title: SW620_2


title: RPE1_Taxol_rep2


title: 21Wn

tag: tissue


title: RPE1_Taxol_rep1

tag: cell line


title: KM12SM-sh60_2


title: 10Wn


title: SW620_4


title: 18Wn


title: RPE1_CMPD1_rep3


title: 2Wn


title: SW620_5


title: RPE1_CMPD1_rep2


title: 1Wn


title: batch-1_0057-B

value: LTBI and HIV-negative


title: RPE1_CMPD1_rep1


title: SW620_3


title: SW620_6


title: RPE1_control_rep3

value: 0 h


title: RPE1_control_rep2


title: SW620-SCR_2


title: batch-1_0050-P


title: batch-1_0052-B


title: SW620-SCR_1


title: batch-1_0057-P


title: batch-1_0069-B


title: batch-1_0069-P


title: RPE1_control_rep1


title: batch-1_0050-B


title: SW480 sh RNA 2

city: Guangzhou

first: Yuyi

last: Huang

address: Changgang Street

institute: The Second Affiliated Hospital of Guangzhou Medical University

description:
The sequencing strategy is PE150
480 m

data_processing:
Raw fastq were quality control use fastp.
Expression of genes and transcripts were quantified using Salmon and Tximport.
DESeq2 used to perform differential expression genes analysis.
Gene set enrichment analysis were performed by clusterProfiler.
Assembly: GRCh38
Supplementary files format and content: Read counts of genes for all RNA-Seq samples in csv format.
Supplementary files format and content: TPM of genes for all RNA-Seq samples in csv format.

source_name: SW480 sh RNA 2 cells

growth_protocol: All cell lines were maintained in L15 supplemented with 1×107 U/L penicillin, 10 mg/L streptomycin, and 10 % fetal bovine serum

extract_protocol:
Whole RNA was extracted by Trizol
Construction of transposase RNA library for Illumina high-throughput sequencing platform

value: SW480 sh RNA 2 cells

value: Colorectal cancer cells

value: Knock down


title: batch-1_0046-P

value: PBMC


title: p22252-s030_RGu18-ROt18-LM98_D8_BAL_GEX

city: Atlanta

first: Gregory

middle: K

last: Tharp

state: GA

address: 954 Gatewood Dr

department: Developmental and Cognitive Neuroscience

institute: Yerkes National Primate Research Center

description: Library name: p22252-s030_RGu18-ROt18-LM98_D8_BAL_GEX

data_processing:
The cellranger v6.1.0 (10X Genomics) pipeline was used for processing the 10X sequencing data and the downstream analysis was performed using the Seurat v4.0.4 R package. A composite reference comprising of Mmul10 from Ensembl release 100 and SARS-CoV2 (strain MT246667.1 - NCBI) was used for alignment with cellranger. The percentage of SARS-CoV-2 reads was determined using the PercentageFeatureSet for SARS-CoV2 genes. For BAL samples, a total of 107,830 cells across all animals passed quality control (QC) and were used for analyses. For lung samples, a total of 101,766 cells passed upstream QC and were used for analysis. For single-cell RNA-Seq, approximately 20,000 cells were loaded onto the 10X Genomics Chromium Controller in the BSL3 facility using the Chromium NextGEM Single Cell 5’ Library & Gel Bead kit according to manufacturer instructions
Gene expression and HTO counts are combined in the cellranger output files.
Assembly: Mmul10, Ensembl annotatation version 100
Assembly: MT246667.1
Supplementary files format and content: cellranger output files: mRNA_and_HTO_barcodes.tsv.gz, mRNA_and_HTO_features.tsv.gz, mRNA_and_HTO_matrix.mtx.gz

organism: Macaca mulatta

extract_protocol:
To collect non necropsy BAL, a 14Fr, sterile, single-use pediatric suction catheter (Covidien #37424) was inserted into the trachea and directed into a mainstem bronchi. The catheter was advanced until it was secured into a distal subsegmental bronchus. 35-70 mL of physiological saline was instilled into the bronchus and then manually aspirated back into a syringe to obtain a minimum of 20 mL of lavage fluid and placed into a 50mL conical. If less than 20mL was recovered, an additional 10-15mL was administered again and a second attempt was taken and placed into a second 50mL conical. BAL was then filtered through a 70uM strainer (Falcon #352350). 1mL of BAL fluid and cells were taken to perform viral load testing by adding RNazol and Acetic Acid into a 5mL microcentrifuge tube. After vigorous vortexing, samples were frozen for later RNA extraction using the RNAzol BD kit. After viral loads were taken, BAL was spun at 300 x g for 10 minutes to pellet the cells. BAL supernatant was collected for MSD and plaque assay. BAL cells were then lysed with the ACK lysis buffer for 10 minutes at 37˚C and the reaction was quenched using DPBS. Cells were then spun and counted in the same manner as PBMCs. At least 100,000 cells were set aside for 10x sequence and the remaining cells were divided between the isotype control and stained panel. At necropsy, an additional 100mL of Post Mortem BAL fluid was taken by placing a large sterile irrigation syringe with the plunger removed into the lungs. Approximately 150 mL of sterile 1 x PBS is poured into the lungs via the syringe to infuse both sides. The syringe is then removed and plunger replaced. The syringe goes back into the trachea opening and the lavage fluid is pulled back and dispensed into 50mL conicals. Post mortem BAL sample is processed the same way as non-necropsy through a 70uM filter and lysing. After samples for viral load, supernatant and 10x were taken for pre-mortem samples, the cells for pre and post mortem were combined together for staining. At necropsy, at least 5 million cells were stained for both the isotype and staining panel with any remaining frozen down using the same freezing media as PBMCs. Caudal lung is processed by following the steps indicated in Voix et al 2023 with the following changes. During the 1 hour 37˚C incubation, samples were placed on a 50mL tube rotator at 10 rpm (Thermo Scientific # 888810001). Samples were run at least 6 times on the pre-loaded M_Lung_02_01 to ensure proper blending of the tissue. After, the tissue is strained over 100uM filters (Falcon # 352360) and the digestion reaction is quenched with R10. Cells are then centrifuged at 1100 x g for 10 minutes. The supernatant is removed and cells are washed again with R10 and centrifuged at 1100 x g for 10 minutes. The cells are lysed using 10mL of ACK lysis and incubated at 37˚C for 10 minutes. Lysing was then quenched using DPBS and cells were spun down at 1100 x g for 10 minutes. Cells were then counted using the same manner as PBMCs and aliquoted for 10x sequencing, bulk RNA sequencing and freezing. Cells were used for 10x sequencing and the extra were saved in the same manner as PBMCs.  Spleen was processed in a similar manner to the lung but without the digestion step. Spleen was dissected using blunt scissors and added to C tubes with 5mL of R10. Using the same M_Lung 02_01 protocol run at least 6 times, spleen was blended then passed through a 100uM filter. Due to the nature of the sample, it is then passed through a 70uM filter and then 40uM filter (Falcon #352340) to remove the debris. Cells were then washed using R10 and then spun at 750 x g for 10 minutes to pellet. Supernatant was removed and the cells were washed with DPBS then centrifuged at 750 x g for 10 minutes. Cells were lysed with 20mL of ACK for 10 minutes. The lysing reaction was quenched with DPBS and cells were pelleted following the same speed and time. Cells were counted and frozen for later use.
Single cell suspensions were prepared and loaded onto the 10X Genomics Chromium Controller in the BSL3 facility using the Chromium NextGEM Single Cell 5’ Library & Gel Bead kit to capture individual cells and barcoded gel beads within droplets (113). The libraries were prepared according to manufacturer instructions, including the preparation of feature barcode libraries for hashtag detection. They were then sequenced on an Illumina NovaSeq 6000 with a paired-end 26x91 configuration targeting a depth of 50,000 reads per cell. Cell Ranger software was used to perform demultiplexing of cellular transcript data, as well as mapping and annotation of UMIs and transcripts for downstream data analysis.

tag: subject

value: multiplexed

tag: dpi

tag: library type

value: mRNA