title: 12


title: 119

status: Public on Sep 23 2023

type: SRA

city: Seoul

first: Woo Young

last: Kwon

address: Yonsei-ro 50

department: Department of Surgery

country: South Korea

institute: Severance hospital

library_source: transcriptomic

data_processing:
QuantSeq 3’ mRNA-Seq reads were aligned using Bowtie2
The read count data were processed based on quantile normalization method using EdgeR within R
Gene classification was based on searches done by DAVID and Medline databases
Assembly: hg19
Supplementary files format and content: tab-delimited text files include read count values for each Sample

molecule: total RNA

organism: Homo sapiens

source_name: Human pancreas

extract_protocol:
Total RNA was isolated using Trizol reagent.
The construction of library was performed using QuantSeq 3’ mRNA-Seq Library Prep Kit

tag: cell line origin

value: Human pancreas


title: 118


title: R217

city: Los Angeles

first: Suhas

middle: G

last: Kallapur

address: 10833 Le Conte Avenue

country: USA

institute: UCLA

description:
bulk RNAseq
AM217 Ad+LPS

data_processing:
Bulk RNA-seq  The reads were mapped with STAR 2.5.3a to the Macaca mulatta genome (Mmul 8.0.1) or to the human genome (GRCh38). The counts for each gene were obtained by using the options --quantMode GeneCounts. Differential expression analyses were carried out using DESeq2. The normalized counts were obtained from the DESeq2 analysis. Principal Component Analysis (PCA) was performed with the plotPCA function in DESeq2 after regularized log transformation. Heatmaps were plotted on the log2 value of the normalized counts. Inference of GO terms, Wiki pathways, and KEGG pathways were generated using Enrichr 63.
Single-cell (sc)RNA-seq: Data processing including quality control, read alignment (to the reference genome Mmul 8.0.1), and gene quantification was conducted using the 10X Cell Ranger software (version 2.1.1). The samples were then merged into a single expression matrix using the cellranger aggr pipeline. The R package Seurat (v3.1.2) was used to cluster the cells in the merged matrix. Cells with less than 500 transcripts or 100 genes, or more than 5% of mitochondrial expression were first filtered out as low-quality cells. The Seurat function NormalizeData was used to normalize the raw counts. Variable genes were identified using the FindVariableGenes function. The ScaleData function was used to scale and center expression values in the dataset, the number of unique molecular identifiers (UMI) was regressed against each gene. Principal component analysis (PCA), and uniform manifold approximation and projection (UMAP) were used to reduce the dimensions of the data, and the first two dimensions were used in the plots. The FindClusters function was used to cluster the cells. Marker genes were found using the FindAllMarkers function for each cluster, which employs the Wilcoxon Rank Sum Test to determine the significance and the Benjamini-Hochberg Procedure to correct for multiple comparisons. Cell types were annotated based on the marker genes and their match to canonical markers. The module scores were calculated using the AddModuleScore function. Sub-clustering on the amnion cells was performed. The same functions described above were used to obtain the sub-clusters. We obtained eight high-quality scRNA-seq profiles from CAD samples. Analysis of cellular trajectory by RNA velocity was performed using the Python package scVelo 64. In one Ctrl sample we did not detect any amnion cells and therefore we excluded this sample from the analysis. Thus, final analyses were conducted on 7 samples (ctrl n=1; LPS n=3; and Adalimumab+LPS n=3). In order to illustrate the variations of cell types in amnion after intrauterine infection we used hdWGCNA package 28 to perform co-expression network analysis on genes expressed in at least 5% of the cells. MetacellsByGroups function was used to construct metacells in each treatment group.  The resulting metacells expression matrix was normalized by calling NormalizeMetacells function and then used for network analysis. In order to ensure scale-free topology of the co-expression network TestSoftPowers function was used to perform parameter sweep. The sweep identified 10 as the optimum soft power threshold to use when constructing the co-expression network by calling ConstructNetwork function with otherwise default parameters. WGCNA dendrogram was visualized with PlotDendrogram function whereas ModuleFeaturePlot was used to map harmonized module eigengenes, computed with ModuleEigengene function, onto the dimensionality reduction plot. The modules identified by hdWGCNA analysis were functionally characterized by performing enrichment tests with enrichR package. For each of the modules, a set of 100 most connected genes (ModuleConnectivity function) was tested for enrichment. Top 10 GO Biological Process terms for each module with adjusted p-value <0.05 were shown along with the enrichment score.
Assembly: Macaca mulatta genome (Mmul 8.0.1)
Assembly: Homo sapiens GRCh38
Supplementary files format and content: Bulk RNA-seq_Raw counts_Rhesus_Amnion_Presicce.txt
Supplementary files format and content: Bulk RNA-seq_Raw counts_Human_Amnion_Presicce.txt

organism: Macaca mulatta

source_name: amnion

extract_protocol:
For scRNA seq experiements of chorioamniondecidua samples were collected right after delivery, digested, and cell suspension was sorted for live cells. For both human and rhesus bulk RNA seq experiments, amnion was physically separated from chorion and decidua, snap-frozen, and RNA was harvested using Trizol reagent.
Bulk RNAseq:  Fifteen amnion samples were used (ctrl n=3; LPS n=6; and Adal+LPS n=6). The integrity of purified total RNA from amnion was assessed using the RNA High-Sensitivity Assay on the TapeStation 2200 (Agilent Technologies). 200-300 ng of starting material were used as input material for the NEBNext rRNA Depletion kit (cat# E6350). RNA libraries were then prepared using the NEBNext Ultra II RNA kit (cat# E7765). Two hundred ng of each final library was subject to capture hybridization using Illumina TruSeq Exome, (cat #20020490) using Illumina Exome Panel (cat #20020183) according to the manufacturer’s instructions. Quality control for the final libraries was performed using the DNA D1000 Assay (TapeStation 2200 - Agilent Technologies) and quantified using a Qubit dsDNA BR Assay (Life Technologies). Diluted libraries were pooled and sequenced 50 single-end on a HiSeq3000 (Illumina).
Single-cell (sc)RNA-seq: For scRNA-seq analysis, eight unseparated chorioamnion-decidua (CAD) samples were used (Ctrl n=2; LPS n=3; and Adal+LPS n=3. Note that 6/8 tissues were also used for bulk RNA-seq analysis (Supplementary Table I). Live-sorted CAD cells were microfluidically partitioned with single cell capture, barcoding, and library construction (10X genomics chromium platform, Pleasanton, California).  Single-cell RNA-seq libraries were prepared using the 10X Single Cell 3′ v2 Reagent Kits. Specifically, single cell suspensions were loaded on a Chromium Controller instrument to generate single-cell Gel Bead-In- EMulsions (GEMs). GEM-RT were performed in a Veriti 96-well thermal cycler (Thermo Fisher Scientific, Waltham, MA), following which, GEMs were harvested and the cDNAs were amplified and cleaned up with SPRIselect Reagent Kit. Indexed sequencing libraries were constructed using Chromium Single-Cell 3′ Library Kit for enzymatic fragmentation, end-repair, A-tailing, adapter ligation, ligation cleanup, sample index PCR, and PCR cleanup. The barcoded sequencing libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, MA). Sequencing libraries were loaded on a NovaSeq2 (Illumina, San Diego, CA) with a custom sequencing setting (26bp for Read 1 and 91bp for Read 2).

treatment_protocol: intra-amniontic injection of LPS or saline. Intra-amniotic + subcutaneous injection Adalimumab

tag: tissue

value: amnion

tag: cell type

value: amnion cells

tag: treatment

value: IA+SC Adal + IA LPS


title: 117


title: 116


title: R441

description:
bulk RNAseq
AM441 Ad+LPS

value: IA+subcutaneous (SC) Adalimumab (Adal) + IA LPS


title: AM442L

description:
bulk RNAseq
AM442 LPS

value: IA LPS


title: Rnase R untreated-Chronic infection with CNS pathology (CP+)-BH-1

city: Baltimore

first: Kenneth

middle: W.

last: Witwer

state: MD

address: 733 N. Broadway

department: Department of Molecular and Comparative Pathobiology

institute: Johns Hopkins University School of Medicine

description: mRNA_Yiyao9_TMM_normalized_cpm_cdna_ncrna_121

data_processing:
The raw reads were first trimmed from contaminating adapter sequences using cutadapt software. The trimmed and size-selected (>15 nt) reads were then aligned using Bowtie2 (default settings) to the manually curated M. mulatta mRNA reference containing a single (main) transcript per each gene (designated with a gene symbol, e.g., GAPDH). All reads which did not align to the above mRNA were mapped to the combined M. mulatta cDNA and ncRNA references from Ensembl (designated with transcript ID, e.g., ENSMMUT00000047080.3). The numbers of reads mapped to each transcript were extracted using eXpress software based on a previous publication (Roberts and Pachter, 2013).
circRNA identification by Sequential Alignment (CiRISeqA):  All reads aligned to the M. mulatta combined cDNA and ncRNA references from Ensembl were discarded. The remaining reads were additionally filtered from non-circular RNA sequences by removing all reads mapped to "non-modified" M. mulatta CircAtlas v.2 references (downloaded from: http://circatlas.biols.ac.cn/) using the same Bowtie settings. Next, the remaining reads were aligned to "duplicated" CircAtlas v.2 reference transcripts that correspond to sequences mapped to the junction regions of circRNAs. The numbers of reads mapped to each duplicated RNA reference were extracted using eXpress software based on a previous publication (Roberts and Pachter, 2013). See also Supplementary Figure 2.
circRNA identification by circExplorer2:  The genome and gene annotations for M. mulatta were obtained from UCSC (BCM Mmul_8.0.1/rheMac8). First, raw files were aligned to the genome using STAR (chimSegmentMin 10), and the chimeric junction files were used to count circRNAs using circExplorer2. circRNAs identified by both our custom pipeline (above) and circExplorer2 were included in the following analysis.
Assembly: M. mulatta
Supplementary files format and content: Excel files contains all mapped reads

organism: Macaca nemestrina

source_name: Brain

extract_protocol:
BH RNA was extracted by adding Trizol (Thermo Fisher15596018) and homogenizing tissues with Lysing Matrix D (MP Biomedicals116913100) in a benchtop homogenizer (FastPrep-24, MP Biomedicals) at 4.0 m/s for 20 seconds. After homogenization, the supernatant was collected, and RNA was isolated by miRNeasy Mini Kit solutions (Qiagen217004) and Zymo-Spin IIICG Columns (Zymo Research C1006-50-G) following the manufacturer's instructions.
1 µg of BH RNAs was incubated with 1 U/µg RNase R (Lucigen RNR07250) at 37°C for 30 min. RNase R-treated RNAs were then re-isolated by RNA Clean & Concentrator™-5 (Zymo Research R1014) according to the manufacturer’s protocol. 100 ng of total BH RNA with and without RNase R treatment was then used to construct cDNA libraries by Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus (Illumina 20072063). A Ribo-Zero Plus kit was used to deplete ribosomal RNA in these samples, and library construction was then done per manufacturer’s protocol. Indexes were attached using IDT for Illumina RNA UD Indexes according to the manufacturer’s protocol. The yield and size distribution of the total RNA libraries were assessed using the Fragment Bioanalyzer™ system with DNA 1000 chip (Agilent 5067-1505). Multiplexed libraries were equally pooled to 1nM and sequenced with the NovaSeq 6000 system (Illumina) and NovaSeq 6000 S2 Reagent Kit v1.5 (300 cycles) (Illumina 20028314).

value: Brain


title: 114


title: 115


title: Rnase R untreated-Chronic infection without CNS pathology (CP-)-BH-7


title: 112


title: 113


title: AM440L

description:
bulk RNAseq
AM440 LPS


title: Rnase R untreated-Chronic infection without CNS pathology (CP-)-BH-6


title: 111


title: AM437L

description:
bulk RNAseq
AM437 LPS


title: 15


title: 110


title: Rnase R untreated-Chronic infection without CNS pathology (CP-)-BH-5


title: AM436L

description:
bulk RNAseq
AM436 LPS


title: 108


title: Rnase R untreated-Chronic infection without CNS pathology (CP-)-BH-4


title: 107


title: Rnase R untreated-Chronic infection without CNS pathology (CP-)-BH-3


title: R116

description:
bulk RNAseq
AM116 LPS


title: Rnase R untreated-Chronic infection with CNS pathology (CP+)-BH-2


title: 104


title: 106


title: 103


title: R29

description:
bulk RNAseq
AM29 LPS


title: Rnase R untreated-Chronic infection without CNS pathology (CP-)-BH-1


title: 101


title: RnaseR untreated-Acue-BH-5

value: RnaseR untreated-Acue-BH-5


title: 100


title: 10


title: AM427C

description:
bulk RNAseq
AM427 Ctrl

value: IA saline


title: RnaseR untreated-Acue-BH-4

value: RnaseR untreated-Acue-BH-4


title: 40-09-13014


title: 7-14-22733


title: 109


title: 39-09-49612


title: 38-09-50883


title: 37-09-60615


title: R209

description:
bulk RNAseq
AM209 Ctrl

value: intraamniotic (IA) saline


title: RnaseR untreated-Acue-BH-2

value: RnaseR untreated-Acue-BH-2


title: 105


title: Lrrc8af/f, Cd4-Cre mice2

city: wuhan

first: zaiqiao

last: sun

country: China

institute: wuhan university

data_processing:
Low-quality reads and reads without primers were discarded with trimmomatic (v0.39).
PCR and sequencing errors were corrected by unique molecular identifiers (UMIs).
TCR information that includes clone count, clone fraction, target sequence, CDR3 amino acid and so on, was obtained by trust4
Further data analysis of the TCR repertoire is performed by the RStudio software
Assembly: GRCm38
Library strategy: TCR-seq

organism: Mus musculus

source_name: thymus

growth_protocol: Mice were maintained in specific pathogen free (SPF) animal facility. All animal experimentation was approved by the Animal Care Committee

extract_protocol:
total RNA was extracted using Takara MiniBEST Universal RNA Extraction Kit
TCR library construction and sequencing according to SMARTer® Mouse TCR a/b Profiling Kit

treatment_protocol: Fresh thymocytes were isolated from (Lrrc8af/f, Cd4-Cre) mice and their littermate control mice (Lrrc8af/f)

value: thymus

value: thymocytes

tag: genotype

value: Lrrc8af/+ mice  with Cd4-Cre

value: no


title: AM430C

description:
bulk RNAseq
AM430 Ctrl


title: 35-10-36517


title: 34-10-38862


title: RnaseR untreated-Acue-BH-1

value: RnaseR untreated-Acue-BH-1


title: Lrrc8af/f control mice7

value: Lrrc8af/+ mice  WT


title: Lrrc8af/f, Cd4-Cre mice1


title: Rnase R untreated-Uninfected-BH-5

value: Rnase R untreated-Uninfected-BH-5


title: 31-10-47825

value: Human pancreas


title: 30-10-50615


title: Rnase R untreated-Uninfected-BH-4

value: Rnase R untreated-Uninfected-BH-4


title: 29-10-54071


title: 28-11-10161


title: 27-11-24922


title: Rnase R untreated-Uninfected-BH-3

value: Rnase R untreated-Uninfected-BH-3


title: Lrrc8af/f control mice4


title: 26-11-34806


title: RnaseR untreated-Acue-BH-3

value: RnaseR untreated-Acue-BH-3


title: 25-11-52929


title: Rnase R untreated-Uninfected-BH-2

value: Brain

value: Rnase R untreated-Uninfected-BH-2


title: Lrrc8af/f control mice3


title: Rnase R untreated-Uninfected-BH-1

value: Rnase R untreated-Uninfected-BH-1


title: 36-10-24489


title: Chronic infection with CNS pathology (CP+)-EVs-4

description:
Yiyao8_EV_combined
EVs22

data_processing:
Raw reads were first trimmed from polyA-tails using cutadapt software, and the PCR duplicates were removed by collapsing identical sequences with seqkit. De-duplicated reads were trimmed from the 5'-UMI sequences and size-selected using cutadapt software.
Trimmed and size-selected (>15 nt) readswere aligned to custom-curated hg38 reference transcriptomes using Bowtie,allowing 1 mismatch tolerance (-v 1 option) as follows.
First, reads were mapped to RNA species with low sequence complexity and/or high repeat number:rRNA,tRNA,RN7S,snRNA,snoRNA, scaRNA,vault RNA, RNY, and mitochondrial chromosome (mtRNA).Unmapped reads were aligned sequentially tomature miRNA,pre-miRNA, protein-coding mRNA transcripts (mRNA), and long non-coding RNAs (lncRNAs).Unmapped reads were aligned to the remaining transcriptome (otherncRNAs: mostly pseudogenes and non-protein-coding parts of mRNAs). Finally, all remaining unmapped reads were aligned to the human genome reference (rest hg38) corresponding to introns and intergenic regions.
Assembly: Hg38
Supplementary files format and content: Excel file contains all mapped reads

extract_protocol:
bdEVs were separated from frozen occipital cortex tissues using our published protocol with minor modifications (Huang et al., 2022a, 2022b, 2020). Before extraction, a small (∼50 mg) piece of tissue was stored at –80°C for later RNA extraction from brain homogenate (BH).  bdEV RNAs were extracted by adding Trizol LS (Thermo Fisher 10296028) to 100 μL bdEV resuspension. After phase separation, RNAs were purified by miRNeasy Mini Kit solutions (Qiagen217004) and Zymo-Spin I Columns (Zymo ResearchC1003–50) according to the manufacturer's instructions.  BH RNA was extracted by adding Trizol (Thermo Fisher15596018) and homogenizing tissues with Lysing Matrix D (MP Biomedicals116913100) in a benchtop homogenizer (FastPrep-24, MP Biomedicals) at 4.0 m/s for 20 seconds. After homogenization, the supernatant was collected, and RNA was isolated by miRNeasy Mini Kit solutions (Qiagen217004) and Zymo-Spin IIICG Columns (Zymo Research C1006-50-G) following the manufacturer's instructions.
RNA was resuspended in 40 μL Rnase-free water, and 8 µl was used for small RNA libraries construction by the D-Plex Small RNA-seq Kit (Diagenode C05030001). Indexes were attached using the D-Plex Single Indexes for Illumina - Set A (Diagenode C05030010) according to the manufacturer’s protocol.

tag: group


title: Lrrc8af/f control mice2


title: MDA-MB-231 cells-NFIC1_rep3

city: Chang Chun

first: Xin

last: Hu

address: 2699 Qianjin road,china

institute: Jilin university

data_processing:
The library preparations were sequenced on Novaseq6000-1.7.5, RTA3 software was used for base-calling, and paired-end reads were generated.
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data(clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30, GC-content and sequence duplication level of the clean data were calculated. All the downstream analyses were based on clean data with high quality
Gene expression levels were calculated by fragments per kilobase of transcript per million fragments mapped (FPKM)
Differential expression analysis of two groups was performed using the DESeq R package (1.10.1). The resulting P values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate (FDR). Genes withfold change ≥2 or ≤0.5, false discovery rate (FDR) <0.05 were selected as differentially expressed gene.
Assembly: ensembl CRCh38
Supplementary files format and content: FPKM

source_name: MDA-MB-231 cells

growth_protocol: MDA-MB-231 cell line was cultured in DMEM (Gibco) supplemented with 10% FBS and streptomycin/penicillin (100 U/mL) at 37˚C in an atmosphere of 5% CO2.

extract_protocol:
The total RNA was harvested using Trizol reagent. Illumina NEBNext Ultra Directional RNA Library Prep Kit(NEB) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: MDA-MB-231 cells were transiently transfected with pcDNA3.1 (Control) or pcDNA3.1-NFIC1 (NFIC1) for 48 h using Lipofectamine 2000.

tag: cell line

value: MDA-MB-231 cells

value: Breast cancer cells

value: NFIC1 overexpression

value: Transient transfection with pcDNA3.1-NFIC1


title: 19-12-26174


title: Chronic infection with CNS pathology (CP+)-EVs-3

description:
Yiyao8_EV_combined
EVs21


title: MDA-MB-231 cells-NFIC1_rep2


title: 20-12-23419


title: MDA-MB-231 cells-NFIC1_rep1


title: 9-14-15462


title: Lrrc8af/f control mice5

value: thymus

value: thymocytes


title: Lrrc8af/f control mice6

value: no


title: Lrrc8af/f, Cd4-Cre mice6

value: Lrrc8af/+ mice  with Cd4-Cre


title: Lrrc8af/f control mice1

value: Lrrc8af/+ mice  WT


title: Lrrc8af/f, Cd4-Cre mice7


title: Chronic infection with CNS pathology (CP+)-EVs-2

description:
Yiyao8_EV_combined
EVs20


title: 18-12-28361


title: MDA-MB-231 cells-Control_rep3

value: Control

value: Transient transfection with pcDNA3.1


title: Chronic infection with CNS pathology (CP+)-EVs-1

description:
Yiyao8_EV_combined
EVs19


title: 8-14-20281

value: Human pancreas


title: MDA-MB-231 cells-Control_rep2


title: 6-14-24091


title: Chronic infection without CNS pathology (CP-)-EVs-7

description:
Yiyao8_EV_combined
EVs18


title: Lrrc8af/f, Cd4-Cre mice4


title: 24-11-54370


title: MDA-MB-231 cells-Control_rep1


title: 5-14-32957


title: Lrrc8af/f, Cd4-Cre mice3


title: 4-14-33419


title: KO Colon snRNAseq

city: Augusta

first: Jiliang

last: Zhou

state: GA

address: 1120 15th street

institute: Augusta University

description: nuclear RNA

data_processing:
Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 500ng to 1ug of total RNA. Ribosomal RNA was removed by an RNase-H method using RiboErase kits (Kapa Biosystems). mRNA was then fragmented in reverse transcriptase buffer and heating to 94 degrees for 8 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.
RNA-seq reads were then mapped to Ensembl release 76 top-level assembly using STAR version 2.0.4b. The raw count for genes annotated in Ensembl GRCm38.76 were calculated with Subread:featureCount version 1.4.5 and reads per kilobase per million mapped reads (RPKM) was obtained to quantify relative expression of transcripts. Differential expression analysis was performed with the R package edgeR.
The Cell Ranger Single Cell software suite 3.0 (10X Genomics) using STAR aligner was employed to align sequencing reads to the mouse pre-mRNA reference genome (mm10) and generated BAM files as well as filtered gene-barcode matrices on the USC High Performance Cluster. The cellranger count function was used to create filtered gene-cell expression matrices removing cell barcodes not represented in nuclei.
Assembly: mm10
Supplementary files format and content: tsv files include RPKM values for each Sample ...
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample

source_name: Colonic muscularis

extract_protocol:
The colonic muscularis were isolated by manually, and flash frozen on liquid nitrogen. Total RNA was harvested using TRIZOL reagent. Single nuclei were extracted following the protocol provided by 10X Genomics (CG000375).
For bulk RNA-seq, RNA libraries were prepared for sequencing using standard Illumina protocols. For snRNA-seq, nuclei RNA libraries were processed according to the 10X Genomic single cell 3’ protocol. Completed 10X libraries were sequenced using standard Illumina protocols.

tag: strain

value: Colon

value: Carmn iKO

tag: seq method

value: snRNA


title: Chronic infection without CNS pathology (CP-)-EVs-5

description:
Yiyao8_EV_combined
EVs16

value: Brain


title: WT Colon snRNAseq

description: nuclear RNA

value: WT mTmG


title: SCD1KO_naive CD4 T

city: Shanghai

first: Lin

last: Liangyu

state: Shanghai

address: 320 Yueyang Road

institute: Shanghai Institutes for Biological Sciences, Chinese Academy of Science

library_source: genomic

data_processing:
Raw Reads was filtered by Trimmomatic software with default settings to remove adaptor and low quality reads.
Paired‐end reads were aligned to mouse genome mm10 using the BWA program (Cambridge, MA, USA)
To create ATACseq coverage plots, the locations of the mapped ATACseq reads were extended to 150 bp to represent sequenced fragments, renormalized (to reads per million, rpm) and reformatted in the bigWig file format.
For annotation, MACS2 software was used for peak calling with a cutoff q value < 0.05.
bigWig files were generated using the genomicRanges R package. Score represents the normalized coverage of DNA fragments at a given genomic coordinate. narrowPeak files were generated using MACS v2 with default settings.
Assembly: mm 10
Supplementary files format and content: bigWig files
Supplementary files format and content: narrowPeak

molecule: genomic DNA

source_name: spleen

growth_protocol: Naive CD4 positive T cells were sorted by naive T cell  magnetic beads isolation kit from mice spleen; DN3 cells were sorted from mice thymus  in two steps. Firstly, depleting lineage cells and CD4 positive cells by magnetic beads. Secondly, flow sorting to select Lin-CD4-CD8-CD44-Cd25+ cells

extract_protocol:
Cells were washed with PBS and incubated in lysis buffer. Nuclear extraction was conducted based on method developed by Jason et al. (PMID: 25559105).
Libraries were prepared using 1X NEBNext High-Fidelity PCR Master Mix (New England Biolabs, MA). The PCR products was purified by MinElute PCR Purification Kit (Qiagen).

treatment_protocol: Purified cells were directly subjected to the ATAC-seq without any treatment.

value: spleen

value: primary cells

value: CD4 naive T cells

value: SCD1 deficient

value: no additional treatement


title: 33-10-42513


title: SCD1KO_DN3

value: thymus

value: DN3 thymocytes


title: 32-10-45876


title: Lrrc8af/f, Cd4-Cre mice5

value: thymocytes

value: Lrrc8af/+ mice  with Cd4-Cre

value: no


title: SCD1HZ_DN3

value: Littermate control


title: SCD1HZ_naive CD4 T


title: 3-14-38658

value: Human pancreas


title: H1075(2)

description:
bulk RNAseq
H1075(2)

value: amnion

value: amnion cells

value: inflammation


title: H1077

description:
bulk RNAseq
H1077


title: H1081

description:
bulk RNAseq
H1081


title: Chronic infection without CNS pathology (CP-)-EVs-6

description:
Yiyao8_EV_combined
EVs17

value: Brain


title: 22-12-07920


title: wt-j-t6

source_name: Jejunum muscularis

value: Jejunum

value: WT mTmG

value: bulkRNA


title: H1069

description:
bulk RNAseq
H1069


title: wt-j-t7


title: H1062

description:
bulk RNAseq
H1062


title: H1068A

description:
bulk RNAseq
H1068A


title: 21-13-11184


title: wt-j-t1


title: 17-12-57966


title: iko-j-t4

value: Carmn iKO


title: 16-12-59503


title: H1053

description:
bulk RNAseq
H1053


title: 23-12-00006


title: H1076

description:
bulk RNAseq
H1076

value: no inflammation


title: 15-13-15400


title: iko-j-t1


title: H1064A

description:
bulk RNAseq
H1064A


title: H1071

description:
bulk RNAseq
H1071


title: metastasis-5

type: RNA

city: Chengdu

first: Jianling

last: Xia

address: No.32, Section 2, West 1st ring road, Qingyang District, Chengdu

institute: Sichuan Provincial People Hospital

description: Gene expression data from hepatocellular carcinoma with extrahepatic metastasis

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Drosophila Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.

data_processing: The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100.

label: biotin

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions.

tag: subject id

value: patient #12


title: metastasis-3

source_name: hepatocellular carcinoma with extrahepatic metastasis

value: patient #10


title: metastasis-6

value: patient #13

value: hepatocellular carcinoma with extrahepatic metastasis


title: metastasis-4

value: patient #11


title: H1044A

description:
bulk RNAseq
H1044A


title: metastasis-7

value: patient #14


title: 12-13-34274


title: iko-j-m1


title: metastasis-2

value: patient #9


title: metastasis-free-5

description: Gene expression data from metastasis-free hepatocellular carcinoma

value: patient #5


title: wt-c-t7

value: Colon


title: 14-13-21498

value: Human pancreas


title: metastasis-free-7

value: patient #7


title: 13-13-33514


title: iko-c-t6


title: H1030B

description:
bulk RNAseq
H1030B

value: amnion

value: amnion cells


title: metastasis-1

value: patient #8


title: metastasis-free-6

value: patient #6


title: H1023

description:
bulk RNAseq
H1023


title: wt-c-t6

value: WT mTmG

value: bulkRNA


title: H1031

description:
bulk RNAseq
H1031

value: no inflammation


title: 11-13-66636


title: iko-c-t4

value: Carmn iKO


title: metastasis-free-3

value: patient #3


title: wt-c-t1


title: metastasis-free-1

value: patient #1


title: 10-14-10780


title: 392Neg

status: Public on Sep 22 2023

city: Oklahoma City

first: Kyla

last: Tooley

state: OK

address: 825 NE 13th St

department: Genes and Human Disease

institute: Oklahoma Medical Research Foundation

data_processing:
Following sequencing, reads were trimmed, aligned, differential expression statistics and correlation analyses were performed in Strand NGS software package (Agilent)
Reads were aligned against the Mm10 build of the mouse genome (2014.11.26). Alignment and filtering criteria included: adapter trimming, fixed 2bp trim from 5’ and 6bp from 3’ ends, a maximum number of one novel splice allowed per read, a minimum of 90% identity with the reference sequence, a maximum of 5% gap, trimming of 3’ end with Q<30.
Alignment was performed directionally with Read 1 aligned in reverse and Read 2 in forward orientation. Reads were filtered based on the mapping status and only those reads that aligned normally (in the appropriate direction) were retained.
Normalization was performed with the DESeq algorithm
Transcripts with an average read count value >20 in at least 100% of the samples in at least one group were considered expressed at a level sufficient for quantitation per tissue and those transcripts below this level were considered not detected/not expressed and excluded, as these low levels of reads are close to background and are highly variable.
For statistical analysis of differential expression, a one-way ANOVA with the factor of TRAP fraction and a Benjamini-Hochberg Multiple Testing Correction followed by Student-Newman Keuls post hoc test were used. For those transcripts meeting this statistical criterion, a fold change >|1.25| cutoff was used to eliminate those genes which were statistically significant but unlikely to be biologically significant and orthogonally confirmable due to their very small magnitude of change.
Assembly: mm10
Supplementary files format and content: Tab-delimited text file contains RPKM values for each sample.

source_name: Hippocampus

extract_protocol:
RNA from the hippocampus of one hemisphere of Tam-induced Camk2a-cre/ERT2+;NuTRAP+ mice was isolated via the TRAP method (Input, Negative, and Positive fractions). Tissue was minced into small pieces and homogenized in 100 ul ice-cold homogenization buffer (50 mM Tris, pH 7.4; 12 mM MgCl2; 100 mM KCl; 1% NP-40; 1 mg/mL sodium heparin; 1 mM DTT) supplemented with 100 ug/mL cycloheximide (#C4859-1ML, Millipore Sigma), 200 units/mL RNaseOUT™ Recombinant Ribonuclease Inhibitor (#10777019; Thermofisher), and 1X cOmplete™, EDTA-free Protease Inhibitor Cocktail (#11836170001; Millipore Sigma) with a Kimble pellet pestle cordless motor with one pulse of 10 sec/each on ice. An additional 300 uL of homogenization buffer was added and homogenized with pellet pestle cordless motor with one pulse of 10 sec/each (on ice). Volume was taken up to 1.5 mL with homogenization buffer and centrifuged at 12,000 x g for 10 minutes at 4°C. After centrifugation, 100 uL of the supernatant was saved as input. The remaining supernatant was transferred to a 2 mL round-bottom tube and incubated with 5 ug/uL of anti-GFP antibody (ab290; Abcam) at 4°C with end-over-end rotation for one hour. Dynabeads™ Protein G for Immunoprecipitation (#10003D; Thermofisher) were washed three times in 1 mL ice-cold low-salt wash buffer (50mM Tris, pH 7.5; 12mM MgCl2; 100mM KCl; 1% NP-40; 100μg/ml cycloheximide; 1mM DTT). After removal of the last wash, the homogenate/antibody mixture was transferred to the 2 mL round-bottom tube containing the washed Protein-G Dynabeads and incubated at 4°C with end-over-end rotation for an additional two hours. Magnetic beads were collected using a DynaMag-2 magnet and the unbound- ribosomes and associated RNA saved as the “negative” fraction. Beads were then washed three times with 1 mL of high-salt wash buffer (50mM Tris, pH 7.5; 12mM MgCl2; 300mM KCl; 1% NP-40; 100μg/ml cycloheximide; 2mM DTT). Following the last wash, 350 uL of Buffer RLT (Qiagen) supplemented with 3.5 uL 2-β mercaptoethanol was added directly to the beads and incubated with mixing on a ThermoMixer (Eppendorf) for 10 minutes at room temperature. The beads were magnetically separated and the supernatant containing the target bead-bound ribosomes and associated RNA was transferred to a new tube. 350 uL of 100% ethanol was added to the tube (“positive” fraction) and then loaded onto an RNeasy MinElute column. RNA was isolated using RNeasy Mini Kit (#74104, Qiagen), according to manufacturer’s instructions. RNA was quantified with a Nanodrop 2000c spectrophotometer (Thermofisher Scientific) and its quality assessed by HSRNA screentape with a 2200 Tapestation analyzer (Agilent Technologies)
The NEBNext Ultra II Directional Library Prep Kit for Illumina (#NEBE7760L; New England Biolabs Inc., Ipswich, MA) was used on 100 ng of total RNA for the preparation of strand-specific sequencing libraries from each TRAP-isolated RNA sample (input, negative fraction, and positive fraction), according to manufacturer's instructions. Briefly, polyA containing mRNA was purified using oligo-dT attached magnetic beads. mRNA was chemically fragmented and cDNA synthesized. For strand-specificity, the incorporation of dUTP instead of dTTP in the second strand cDNA synthesis does not allow amplification past this dUTP with the polymerase. Following cDNA synthesis, each product underwent end repair process, the addition of a single ‘A’ base, and finally ligation of adapters. The cDNA products were further purified and enriched using PCR to make the final library for sequencing. Library sizing was performed with HS DNA ScreenTape (#5067-5582; Agilent Technologies) and libraries were quantified by Qubit dsDNA HS Assay Kit (ThermoFisher Scientific). The libraries for each sample were pooled at 4 nM concentration and sequenced using an Illumina NextSeq 6000 system (PE75bp) at the Oklahoma Medical Research Foundation Genomics Facility.

value: Hippocampus

value: Neuron

tag: fraction

value: Negative

tag: library prep_kit

value: NEBNext Ultra II Directional RNA Library Prep Kit for Illumina


title: 396Input

value: Input


title: 392Pos

value: Positive


title: 396Pos


title: metastasis-free-4

value: patient #4


title: metastasis-free-2

value: patient #2


title: 391Input


title: 391Neg


title: 389Pos


title: iko-c-m1

value: Colon


title: 392Input


title: H1024

description:
bulk RNAseq
H1024

value: amnion

value: amnion cells


title: 396Neg


title: HFD PCN 27 S8

city: Oulu

first: Jukka

last: Hakkola

address: Aapistie 5

department: Research Unit of Biomedicine

country: Finland

institute: University of Oulu

description: HFD PCN 27

data_processing:
Base calling was done with Real-Time Analysis (RTA) software version 2.
75 base pair paired-end reads were aligned to mouse Ensembl reference genome GRCm38.95 with HISAT2
Reads per gene were counted with HTSeq
Differentially expressed genes were determined with DESeq2 with a fold change cut off of <-0.6 or >0.6
Genome_build: mm10
Supplementary_files_format_and_content: .txt files which include differentially expressed genes, reads per gene in all samples and log2 fold changes.

source_name: duodenum

growth_protocol: 6-week old mice were kept on regular chow or high-fat diet for 18-weeks to induce obesity and obesity-related metabolic impairments.

extract_protocol:
Total RNA was extracted with Rneasy Lipid Tissue Mini Kit (Qiagen). Samples were DNAse treated with Rnase-free Dnase set (Qiagen)
cDNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Obese mice were treated with PCN (50 mg/kg intraperitoneally) or vehicle for four days once a day.

tag: diet

value: High-fat diet (HFD)

value: intraperitoneal 50 mg/kg PCN for four days once a day


title: HFD 30 S9

description: HFD 30


title: HFD Vehicle 50 S11

description: HFD Vehicle 50

value: intraperitoneal vehicle (30% DMSO in corn oil) for four days once a day


title: HFD Vehicle 55 S12

description: HFD Vehicle 55


title: 389Input


title: Chow 19 S6

description: Chow 19

value: Regular chow


title: HFD 38 S10

description: HFD 38


title: Chow 17 S5

description: Chow 17


title: 389Neg


title: HFD Vehicle 23 S7

description: HFD Vehicle 23


title: HFD PCN 5 S2

description: HFD PCN 5


title: 11 HFD PCN S12

description: 11 HFD PCN

data_processing:
Base calling was done with Real-Time Analysis (RTA) software version 2.
75 base pair paired-end reads were aligned to mouse Ensembl reference genome GRCm38.95 with HISAT2
Reads per gene were counted with HTSeq
Differentially expressed genes were determined with DESeq2 using Benjamini-Hochberg corrected adjusted p-value cutoff of <0.1
Genome_build: mm10
Supplementary_files_format_and_content: .txt files which include differentially expressed genes, reads per gene in all samples and log2 fold changes.

source_name: skeletal muscle

extract_protocol:
Muscle pieces were lysed to RNAzol RT (Sigma-Aldrich) and total RNA was extracted per manufacturer's protocol. Samples were DNAse treated with Rnase-free Dnase set (Qiagen)
cDNA libraries were prepared for sequencing using standard Illumina protocols

tag: characteristics

value: diet:High-fat diet (HFD)

value: treatment:intraperitoneal 50 mg/kg PCN for four days once a day


title: 1-14-63846

value: Human pancreas


title: 391Pos

value: Hippocampus

value: Neuron


title: HFD PCN 11 S4

description: HFD PCN 11


title: Chow 8 S3

description: Chow 8


title: 38 HFD S5

description: 38 HFD


title: HFD 4 S1

description: HFD 4


title: 5 HFD PCN S2

description: 5 HFD PCN


title: 15 HFD Vehicle S11

description: 15 HFD Vehicle

value: treatment:intraperitoneal vehicle (30% DMSO in corn oil) for four days once a day


title: 19 Chow S9

description: 19 Chow

value: diet:Regular chow


title: 17 Chow S10

description: 17 Chow


title: 27 HFD PCN S7

description: 27 HFD PCN


title: 23 Vehicle S8

description: 23 Vehicle


title: S6: Chow 19

data_processing:
Base calling was done with Real-Time Analysis (RTA) software version 2.
75 base pair paired-end reads were aligned to mouse Ensembl reference genome GRCm38.95 with HISAT2
Reads per gene were counted with HTSeq
Differentially expressed genes were determined with DESeq2 using Benjamini-Hochberg corrected adjusted p-value cutoff of <0.05
Genome_build: mm10
Supplementary_files_format_and_content: .txt files which include differentially expressed genes, reads per gene in all samples and log2 fold changes.

extract_protocol:
Liver pieces were lysed to RNAzol RT (Sigma-Aldrich) and total RNA was extracted per manufacturer's protocol. Samples were DNAse treated with Rnase-free Dnase set (Qiagen)
cDNA libraries were prepared for sequencing using standard Illumina protocols

value: duodenum


title: S7: HFD Vehicle 23


title: S5: Chow 17


title: S8: HFD PCN 27


title: 30 HFD S6

description: 30 HFD


title: 4 HFD S4

description: 4 HFD


title: S11: HFD Vehicle 50


title: Liver HFD PCN 698

source_name: liver

value: PXR deficient

value: Liver


title: Liver HFD Vehicle 697

value: intraperitoneal 30% DMSO in corn oil for four days once a day


title: S10: HFD 38


title: 8 Chow S1

description: 8 Chow


title: S2: HFD PCN 5


title: S9: HFD 30


title: 55 HFD Vehicle S3

description: 55 HFD Vehicle


title: S1: HFD 4


title: Liver HFD Vehicle 705


title: Liver HFD 669


title: Liver HFD PCN 706


title: Liver HFD 659


title: Liver Chow 707


title: S4: HFD PCN 11


title: S3: Chow 8


title: Liver HFD PCN 688


title: Liver HFD Vehicle 682


title: Wild_Type_Replicate2

city: New Haven

first: Vikas

last: Gupta

address: 1 Gilbert Street

institute: Yale School of Medicine

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v7.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Common Bile Duct

extract_protocol:
Whole bile ducts were isolated and micro dissected in petri dishes to remove all adjoining tissue. Ducts were placed in 1 ml of HBSS with 1.8 mM Ca, 0.8 mM Mg, 1 mg dispase (Roche), 2 mg protease from Bacillus licheniformis (Sigma) and cut into small pieces with a microdissecting scissors. These were incubated for 15 minutes at 37 C. This solution was removed and the remaining bile duct chunks were placed in a solution with 1x PBS, 5mM EDTA, and 0.5% BSA and incubated at 37 C for another 10 minutes. This was filtered through a 100 μm filter.
This single cell suspension was washed twice with a 1x PBS, 0.04% BSA solution prior to GEM and library generation according to manufacturer’s protocol (10X Genomics, Chromium Single Cell 3' Reagent Kits User Guide, v3 Chemistry)

value: Common Bile Duct

value: All

value: wild type


title: Liver HFD 696

value: High-fat diet (HFD)


title: CD8, HTO-derived cDNA

city: Würzburg

first: hao

last: wu

address: Versbacher stress 7-9

country: Germany

institute: institute for systemsimmunology, wurzburg university

description:
Hashtag-derived oligonucleotide (HTO)
HTO

library_source: other

data_processing:
Pre-processing of sequencing results to generate count matrices (gene expression, ADT and barcode counts) was performed using the 10x genomics Cell Ranger pipeline where ADT and HTO sequences were concatenated and treated as Custom library with default settings (v3.0.2) and aligning to the mm10 genome build and Ensembl 93 annotations.
Further processing was done with Seurat v3.0.0 (cell and gene filtering, hashtag identification, clustering, differential gene expression analysis).
Assembly: mm10
Supplementary files format and content: 10x Genomics output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz
Library strategy: CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by sequencing)

molecule: protein

source_name: Lymph nodes

extract_protocol:
C57BL/6 host mice were infected with 4x10^6 IU LCMV clone13 and and CD4 T cell were depleted with 200µg GK1.5 anti-CD4 antibody (d-1 and d1). At day 23 post infection, lymph nodes were used to isolate CD8+ tdTomato+ (HIF-1a-deficient) and tdTomato+GFP+ (WT) P14 donor cells by FACS sorting. Each sample was labelled with one specific hashtag (1:400 dilution) and various CITE-seq antibodies (1:800 dilution).
Library was prepared using Chromium Single Cell 3ʹ v2 protocol (10x Genomics). Cellular mRNA and antibody-derived oligos were reverse-transcribed and indexed with a shared cellular barcode. Indexed cDNAs were then pooled and amplified by PCR according to the Chromium Single Cell 3’ v2 protocol (10x Genomics) and with specific primers amplifying the antibody-derived tags. SPRI bead size selection was performed in order to separate both the mRNA-derived cDNA (>300bp) and the antibody-derived tagged cDNAs (180bp). For the mRNA derived cDNA library preparation, we further proceeded according to the manufacturer’s instructions for Single Cell 3’ v2 protocol (10x Genomics). For antibody-derived tagged library, we used the KAPA HiFI HotStart Library Amplification Kit (Roche) with the following primers and amplification program:
-CITE-seq library: amplification was performed at 95°C for 3’ followed by ten cycles at 95°C f¬¬or 20’’; 60°C for 30’’; 72°C for 20’’ and final elongation 72°C for 5’ with 5’-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3’ Small RNA RPI1 primer as i7 primer (Illumina).
-HASH-seq library: amplification was performed at 95°C for 3’ followed by ten cycles 95°C for 20’’; 64°C for 30’’; 72°C for 20’’ and final elongation 72°C for 5’ with 5’-CAAGCAGAAGACGGCATACGAGATCGAGTAATGTGACTGGAGTTCAGACGTGTGC-3’ TruSeq D701_s primer as i7 primer (Illumina).
The following 5’- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC-3’ SI-PCR primer as i5 primer (Illumina) was used for both CITE- and HASH- seq library amplification.
Following the final bead purification, all three libraries were pooled as 80% mRNA library, 10% ADT library and 10% HTO library before sequencing.

value: Lymph nodes

value: CD8 T cells

tag: library type

value: HTO

tag: antibodies/tags

value: Anti-mouse Hashtag1/TotalSeq-A0301; Anti-mouse Hashtag2/TotalSeq-A302;


title: WT

description: 3' mRNA library: read1 file contains cell barcode and UMI; read2 file contains transcript

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v2.1.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: 10x Genomics output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz

molecule: polyA RNA

extract_protocol:
C57BL/6 mice were infected with 2x10^6 IU LCMV clone13 after 2 times CD4 T cell depletion (d-2 and d0) with 300µg GK1.5 anti-CD4 antibody. At the chronic infection phase (> d21), splenocytes were isolated and CD8+, CD44+, PD-1high cells were sorted using a FACS AriaIII cell sorter and directly subjected to scRNA-sequencing.
Library was performed according to the manufacter’s instructions (single cell 3’ v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.

value: CD8+, CD44+,PD-1high T cell

value: spleen

value: untreated


title: S12: HFD Vehicle 55

value: intraperitoneal vehicle (30% DMSO in corn oil) for four days once a day


title: Liver Chow 670

value: Regular chow


title: Liver Chow 695


title: Wild_Type_Replicate3

description: 10x Genomics


title: CD8, mRNA-derived cDNA

description:
3' mRNA library: read1 file contains cell barcode and UMI; read2 file contains transcript
poly A RNA

value: mRNA


title: subcutaneous adipose tissue,DJB,4 months 3

city: Jinan

first: Zitian

last: Liu

address: 107 Wenhua west road

department: Department of General Surgery

institute: Qilu Hospital of Shandong University

data_processing:
A cDNA library constructed by technology from the pooled RNA from < sample description > samples of < research species > was sequenced run with Illumina Novaseq TM 6000 sequence platform.
Using the Illumina paired-end RNA-seq approach, we sequenced the transcriptome, generating a total of millon 2 x 150 bp paired-end reads. Reads obtained from the sequencing machines includes raw reads containing adapters or low quality bases which will affect the following assembly and analysis
Thus, to get high quality clean reads, reads were further filtered by Cutadapt (https://cutadapt.readthedocs.io/en/stable/, version:cutadapt-1.9). The parameters were as follows:1) removing reads containing adapters;2) removing reads containing polyA and polyG;3) removing reads containing more than 5% of unknown nucleotides (N);4) removing low quality reads containing more than 20% of low quality (Q-value≤20) bases. Then sequence quality was verified using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/, 0.11.9). including the Q20, Q30 and GC-content of the clean data.
Assembly: mRatBN7.2
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

organism: Rattus norvegicus

source_name: subcutaneous adipose tissue

growth_protocol: With the condition that constant temperature is 24 to 26 °C, humidity is 50% to 60%, and light/dark alternates each 12 h,kept in the Laboratory Animal Center of Qilu Hospital, were separately housed in independently ventilated cages.

extract_protocol:
Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer's procedure. Then 10 ug of total RNA was extracted according to the RNA-Seq sample preparation kit for constructing sequencing library.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol:
The dosage of STZ was determined based on therats'weights (0.3%,30 mg/kg)
duodenal-jejunal bypass operation

value: subcutaneous adipose tissue

value: subcutaneous adipocyte

value: WT

value: HFD+DJB


title: subcutaneous adipose tissue,DJB,4 months 5


title: subcutaneous adipose tissue,DJB,4 months 2


title: visceral adipose tisse,DJB,4 months 6

source_name: visceral adipose tisse

value: visceral adipose tisse

value: visceral adipocyte


title: subcutaneous adipose tissue,DJB,4 months 1


title: visceral adipose tisse,DJB,4 months 5


title: subcutaneous adipose tissue,DJB,4 months 4


title: subcutaneous adipose tissue,T2DM,4 months 4

value: HFD


title: CD8, ADT-derived cDNA

description:
Antibody-derived oligonucleotide library: read1 file contains cell barcode and UMI; read2 file contains Antibody Derived Tag reads
Antibody-derived oligonucleotide (ADT)

value: Lymph nodes

value: ADT

value: Anti-mouse IL-33Ra/TotalSeq-A0837 ; Anti-mouse/human KLRG1/TotalSeq-A0250 ; Anti-mouse CD185/TotalSeq-A0846 ; Anti-mouse CD278/TotalSeq-A0847; Anti-mouse CD103/TotalSeq-A0201; Anti-mouse CD49b/TotalSeq- A0421; Anti-mouse CD5/TotalSeq-A0111; Anti-mouse/rat/human CD27/TotalSeq-A0191; Anti-mouse CD127/TotalSeq-A0198; Anti-mouse CD200R/TotalSeq-A0807


title: visceral adipose tisse,DJB,4 months 4


title: subcutaneous adipose tissue,T2DM,4 months 5


title: visceral adipose tisse,DJB,4 months 1


title: visceral adipose tisse,DJB,4 months 2


title: subcutaneous adipose tissue,T2DM,4 months 1


title: visceral adipose tisse,T2DM,4 months 4


title: subcutaneous adipose tissue,T2DM,4 months 3


title: G4_CM_d50

city: Tokyo

first: Shugo

last: Tohyama

address: 35 Shinanomachi, Shinjuku

country: Japan

institute: Keio University

description: Gene expression in  hiPSC(253G4)-derived cardiomyocytes on day 50

hyb_protocol: 0.6 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a Agilent fragmentation buffer and was hybridized at 65°C for 17 hr using Gene Expression Hybridization Kit.

scan_protocol: Microarray slides were scanned at 3 μm resolution using an Agilent SureScan (G4900DA).

data_processing: The scanned images were analyzed with Feature Extraction Software 11.5.1.1(Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.

source_name: hiPSC(253G4)-derived cardiomyocytes on day 20

label_protocol: Agilent one-color Microarray-Based Gene Expression Analysis Low Input Quick Amp Labeling protocol

growth_protocol: hiPSC(253G4 line)- derived cardiomyocytes were cultured in low-glucose medium (MEMα) plus 5% fetal bovine serum (FBS) and samples were collected at three time points: 20 days, 35 days, and 50 days after the initiation of differentiation induction. Subsequently we performed microarray analysis on each samples.

extract_protocol: Total RNA was isolated from cell culture supernatant using a ReliaPrep™ RNA Cell Miniprep System (Promega) according to the manufacturer’s instructions.

value: hiPSC(253G4)-derived cardiomyocytes

tag: time point


title: visceral adipose tisse,T2DM,4 months 5


title: subcutaneous adipose tissue,T2DM,4 months 2


title: visceral adipose tisse,T2DM,4 months 6


title: Wild_Type_Replicate1

description: 10x Genomics

value: Common Bile Duct

value: All

value: wild type


title: G4_CM_d35

description: Gene expression in  hiPSC(253G4)-derived cardiomyocytes on day 35


title: visceral adipose tisse,T2DM,4 months 2


title: G4_CM_d20

description: Gene expression in  hiPSC(253G4)-derived cardiomyocytes on day 20


title: visceral adipose tisse,T2DM,4 months 3


title: visceral adipose tisse,T2DM,4 months 1

value: visceral


title: ChIPseq_rtf1MO_rep1_input

first: Adam

middle: David

last: Langenbacher

state: CA

address: 615 Charles E Young Dr. S., BSRB 454

department: Molecular, Cell, and Developmental Biology

institute: University of California Los Angeles

data_processing:
Reads were mapped to the danRer11 genome using bowtie2 (options: --local -X 1000). The makeUCSCfile (options: -norm 1e7 -fsize 500e6) and bedGraphToBigWig functions in the HOMER application were used to generate bigwig files for viewing.
Assembly: danRer11
Supplementary files format and content: bigwig files for individual samples

organism: Danio rerio

source_name: whole embryo

extract_protocol:
Fixation and preparation of chromatin was carried out using the Covaris truChIP Chromatin Shearing Kit and 7 minutes of sonication in a Covaris E220 ultrasonicator using the recommended settings.  Immunoprecipitations were carried out on approximately 5 ug of chromatin (minimum 2.5 ug, maximum 8.8 ug) using 5 ul of total RNA Pol II antibody (Cell Signaling D8L4Y).
Libraries were prepared using the NEBNext Ultra II DNA Library Kit for Illumina (NEB) and sequenced with an Illumina Novaseq system to produce 50 bp paired-end reads.

treatment_protocol: Rtf1MO embryos were injected with 0.5 ng rtf1 morpholino and 1 ng p53 morpholino to knockdown Rtf1 function.  Embryos were treated with 3 ug/ml flavopiridol from 60-70% epiboly until the 10-12 somite stage.

value: whole embryo

value: rtf1 mutant

tag: chip antibody


title: ChIPseq_rtf1MO_flav_rep1_input

tag: tissue

value: flavopiridol


title: ChIPseq_control_rep1_input

value: control


title: ChIPseq_rtf1MO_rep3

value: D8L4Y (Cell Signaling)


title: ChIPseq_rtf1MO_flav_rep2


title: ChIPseq_rtf1MO_rep2


title: ChIPseq_control_rep2


title: ChIPseq_control_rep3


title: visceral adipose tisse,DJB,4 months 3

tag: cell type

value: WT

value: HFD+DJB


title: ChIPseq_rtf1MO_flav_rep3

tag: treatment


title: ChIPseq_control_rep1

tag: genotype


title: KK_RNA_rep1

city: Beijing

first: Meng

last: Wang

address: No. 5 Yiheyuan Road

department: School of Life Sciences

institute: Peking University

description:
KK.log2FC.bigwig
KK.enhancer.bed

data_processing:
STARR-seq data analysis: The raw sequencing data for reporter RNAs and plasmid DNAs were trimmed with Trimmomatic (version v0.39) to remove adaptors. The cleaned reads were mapped to human reference genome GRCh38 using bwa mem (version 0.7.17). The mapped reads were filtered with minimum mapping quality of 30 and minimum fragment length of 250. Potential PCR duplicates were kept. The signal tracks were generated with deeptools bamCoverage (version 3.5.2).
Active enhancer analysis:  To calculate enhancer activities, we split the human GRCh38 reference genome into 350bp nonoverlap bins and counted the number of RNA fragments and DNA fragments in each bin for each replicate with bedtools (version 2.31.0). We filtered the bins with DNA fragments count per million less than 1 in either replicate. Then we calculated the p-value and fold change of RNA over DNA using DESeq2 (version 1.38.3) by leveraging the two replicates of RNA and DNA and using the DNA count as control. For DESeq2, local fit type was used. Bins with raw p-value < 0.05 and log2 fold change > 0 were considered as active enhancers. Bins with p-value < 0.05 and log2 fold change < 0 were considered as negative sequences. Bins with p-value > 0.95 and the absolute value of log2 fold change < log2(1.1) were considered as neutral sequences.
Assembly: GRCh38
Supplementary files format and content: bigwig: signals across the genome
Supplementary files format and content: bed: peak regions

source_name: cell line

growth_protocol: Both K562 and A549 cell lines were cultured with 1640 RPMI medium with 10% FBS, Pen/Strep (Gibco 15140-163, 500μl per 50ml) and L-Glutamine (Gibco 25030-164, 500μl per 50ml). The cells were incubated at 37℃ with 5% CO2. The K562 cells were split into 0.4*106 cells/ml when the density reached 0.8*10^6 cells/ml. The A549 cells were split 1:4 when growing into 90% confluent. Each cell type had two independent cell cultures.

extract_protocol:
The RNAs were extracted using QIAGEN RNeasy Midi Kit (QIAGEN 75144) and mRNAs were selected with Invitrogen Dynabeads mRNA Purification Kit (Invitrogen 61006) following the manufactory’s protocol. The mRNAs were treated with Turbo DNase I (Ambion AM2238) and cleaned up using QIAGEN RNeasy MinElute clean up kit (QIAGEN 74204) and eluted in 60μl RNase-free water. The transfected plasmid DNA was extracted from the cells using QIAGEN Plasmid Plus Midi kit (QIAGEN 12943).
Reverse transcription of the mRNAs was performed with 59μl eluted mRNA, 5μl dNTP and 1μl 10μM reporter gene specific primer: CTCATCAATGTATCTTATCATGTCTG using the Invitrogen SuperScript III First-Strand Synthesis System (Invitrogen 18080051) following the manufactory’s protocol. The cDNA was treated with RNase A and cleaned up with AMPure XP beads (Beckman A63882). Junction PCR was performed to enrich the reporter cDNA with forward primer TCGTGAGGCACTGGGCAG*G*T*G*T*C, reverse primer CTTATCATGTCTGCTCGA*A*G*C and 2X Kapa HiFi HotStart ReadyMix (Kapa KK2611). The sequencing ready library was generated by PCR with the junction PCR product, Universal PCR Primer for Illumina and index primer for Illumina (NEB E7335S), and 2X Kapa HiFi HotStart ReadyMix (Kapa KK2611). The PCR product was purified using SPRIselect beads (Beckman B23318). The sequencing ready plasmid library was generated by PCR with the extracted plasmid, Universal PCR Primer for Illumina and index primer for Illumina (NEB E7335S), and 2X Kapa HiFi HotStart ReadyMix (Kapa KK2611). The PCR product was purified using SPRIselect beads (Beckman B23318).

treatment_protocol: The STARR-seq screening was performed as described previously (Muerdter et al. 2018). Each plasmid library was transfected into K562 or A549 cells by electroporation using Lonza SF Cell Line 4D-Nucleofector X Kit (Lonza V4XC-2012) with Lonza 4D-Nucleofector. For each library, 20 electroporation reactions were performed with 5*10^6 cells and 10μg plasmid library per reaction, following the manufactory’s protocol. After transfection, the cells were cultured for 24h at 37℃ in culture mediums without Pen/Strep. Then the cells were harvested and 3/4 of the cells were used for RNA extraction while the other 1/4 were used for plasmid DNA extraction.

value: cell line

value: transfected with K562 DNA


title: ChIPseq_rtf1MO_flav_rep1


title: KK_DNA_rep1

description:
KK.log2FC.bigwig
KK.enhancer.bed

data_processing:
library strategy: plasmid DNA seq
STARR-seq data analysis: The raw sequencing data for reporter RNAs and plasmid DNAs were trimmed with Trimmomatic (version v0.39) to remove adaptors. The cleaned reads were mapped to human reference genome GRCh38 using bwa mem (version 0.7.17). The mapped reads were filtered with minimum mapping quality of 30 and minimum fragment length of 250. Potential PCR duplicates were kept. The signal tracks were generated with deeptools bamCoverage (version 3.5.2).
Active enhancer analysis:  To calculate enhancer activities, we split the human GRCh38 reference genome into 350bp nonoverlap bins and counted the number of RNA fragments and DNA fragments in each bin for each replicate with bedtools (version 2.31.0). We filtered the bins with DNA fragments count per million less than 1 in either replicate. Then we calculated the p-value and fold change of RNA over DNA using DESeq2 (version 1.38.3) by leveraging the two replicates of RNA and DNA and using the DNA count as control. For DESeq2, local fit type was used. Bins with raw p-value < 0.05 and log2 fold change > 0 were considered as active enhancers. Bins with p-value < 0.05 and log2 fold change < 0 were considered as negative sequences. Bins with p-value > 0.95 and the absolute value of log2 fold change < log2(1.1) were considered as neutral sequences.
Assembly: GRCh38
Supplementary files format and content: bigwig: signals across the genome
Supplementary files format and content: bed: peak regions

molecule: other


title: KK_RNA_rep2

description:
KK.log2FC.bigwig
KK.enhancer.bed


title: KK_DNA_rep2

description:
KK.log2FC.bigwig
KK.enhancer.bed


title: KA_RNA_rep1

description:
KA.log2FC.bigwig
KA.enhancer.bed

value: transfected with A549 DNA


title: KA_DNA_rep2

description:
KA.log2FC.bigwig
KA.enhancer.bed


title: KA_DNA_rep1

description:
KA.log2FC.bigwig
KA.enhancer.bed


title: AK_DNA_rep1

description:
AK.log2FC.bigwig
AK.enhancer.bed


title: AK_DNA_rep2

description:
AK.log2FC.bigwig
AK.enhancer.bed


title: AK_RNA_rep1

description:
AK.log2FC.bigwig
AK.enhancer.bed


title: KA_RNA_rep2

description:
KA.log2FC.bigwig
KA.enhancer.bed


title: AA_RNA_rep1

description:
AA.log2FC.bigwig
AA.enhancer.bed


title: AA_RNA_rep2

description:
AA.log2FC.bigwig
AA.enhancer.bed


title: AK_RNA_rep2

description:
AK.log2FC.bigwig
AK.enhancer.bed


title: AA_DNA_rep2

description:
AA.log2FC.bigwig
AA.enhancer.bed


title: ChIP_H3K4me1_K562_rep1


title: AA_DNA_rep1

description:
AA.log2FC.bigwig
AA.enhancer.bed


title: ChIPseq_rtf1MO_rep1


title: ChIP_H3K4me1_K562_rep2


title: ChIP_H3K4me1_A549_rep2


title: ChIP_H3K4me1_A549_rep1


title: TSCEV3

city: Chicago

first: Morgan

last: Zenner

state: IL

address: 909 South Wolcott Ave

department: Pathology

institute: University of Illinois at Chicago College of Medicine

description:
TSC3_pz_exo_EtOH_ACAGTG
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_no_serum_CPM_tmm_normalizedcounts_no_piR.xlsx

data_processing:
GeneGlobe data analysis center
Reads were trimmed using cutadapt, which removed the 3' adapters and low-quality bases
MicroRNA sequences with less than 16 bp and UMI sequences less than 10 bp were removed
MicroRNAs were mapped to miRBase using bowtie, which allowed up to two mismatches
Assembly: miRBase version 22
Supplementary files format and content: Excel files (CPM_tmm_normalizedcounts)

source_name: Prostate

growth_protocol: Patient-derived prostate epithelial cells were maintained in PrEBM media in humidified atmosphere with 5% CO2 at 37C. Patient-derived prostate stromal cells were maintained in MCDB media with 5% FBS and DHT and Fgfb in humidified atmosphere with 5% CO2 at 37C. Patient-derived prostate tissue slices were maintained in PrEBM media in humidified atmosphere with 5% CO2 at 37C.

extract_protocol:
Primary epithelial cell, stromal cell, and tissue slice RNA was harvested using miRNeasy  Mini kit (Qiagen). Primary epithelial cell EV, stromal cell EV, tissue slice EV, and serum EV RNA was harvested using the Serum/Plasma miRNeasy kit (Qiagen). 100 ng of total RNA was used for the construction of cell and tissue sequencing libraries. 5 uL of RNA was used for  the construction of cell EV, tissue EV, and serum EV sequencing libraries.
microRNA libraries for smallRNA-seq were prepared using the QIAseq miRNA Library Kit following manufacturer's protocols. Libraries were diluted to 10 nM for sequencing.
smallRNA-seq by HiSeq 4000 single read 100 nucleotides

treatment_protocol: Prostate tissue slices, epithelial cells, and stromal cells were grown to 70% confluence and then cultured in EV-free media for two days. Evs were isolated from the cell media by differential ultracentifugation.

value: Prostate

value: primary tissue slice

tag: sample type

value: extracellular vesicles (from tissue slice)

value: tissue slice culture in PrEBM for two days


title: TSCEV4

description:
TSC6_pz_exo_CGTACG
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_no_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: TSCEV2

description:
TSC3_ca_exo_EtOH_GATCAG
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_no_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: TSCEV1

description:
TSC4_ca_Day_2_EtOH_exo_GTGAAA
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_no_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: PrS EV4

description:
TCspz_exo_CTATAC
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_no_serum_CPM_tmm_normalizedcounts_no_piR.xlsx

value: primary stromal cell

value: extracellular vesicles (from stromal cells)

value: primary cell culture in MCDB media


title: PrS EV2

description:
BWspz_exo_CGGAAT
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_no_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: PrS EV3

description:
RJspz_exo_ATTCCT
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_no_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: PrS EV1

description:
BWsca_exo_CCAACA
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_no_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: PrE EV2

description:
CBpz_exo_CTCAGA
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_no_serum_CPM_tmm_normalizedcounts_no_piR.xlsx

value: primary epithelial cell

value: extracellular vesicles (from epithelial cells)

value: primary cell culture in PrEGM media


title: PrE EV4

description:
WTca_exo_GACGAC
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_no_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: PrE EV3

description:
TCpz_exo_CTAGCT
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_no_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: TSC4

description:
TSC6_pz_Day_0_GTTTCG
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
non_ev_CPM_tmm_normalizedcounts_no_piR.xlsx


title: TSC2

description:
TSC3_ca_Day_0_CGATGT
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
non_ev_CPM_tmm_normalizedcounts_no_piR.xlsx


title: PrE EV1

description:
BWca_exo_CATTTT
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_no_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: TSC1

description:
TSC3_pz_Day_0_ATCACG
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
non_ev_CPM_tmm_normalizedcounts_no_piR.xlsx


title: PrS4

description:
TCspz_CACTCA
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
non_ev_CPM_tmm_normalizedcounts_no_piR.xlsx


title: PrS3

description:
RJspz_ATGAGC
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
non_ev_CPM_tmm_normalizedcounts_no_piR.xlsx


title: PrS1

description:
BWsca_CAACTA
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
non_ev_CPM_tmm_normalizedcounts_no_piR.xlsx


title: PrS2

description:
BWspz_CACCGG
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
non_ev_CPM_tmm_normalizedcounts_no_piR.xlsx


title: PrE4

description:
WTca_CATGGC
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
non_ev_CPM_tmm_normalizedcounts_no_piR.xlsx


title: PrE3

description:
TCpz_CACGAT
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
non_ev_CPM_tmm_normalizedcounts_no_piR.xlsx


title: PrE2

description:
CBpz_CAGGCG
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
non_ev_CPM_tmm_normalizedcounts_no_piR.xlsx


title: Engrafted 8

city: La Jolla

first: Raymond

middle: Ma

state: California

address: 2880 Torrey Pines Scenic Dr

department: Bioengineering

institute: University of California San Diego

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
rg3

scan_protocol: Immobilized digitial barcode reads from probes hybridized with RNA sample were read on cartridge surface with the Nanostring nCounter Digital Analyzer.

data_processing: Data was exported through the manufacturer nSolverTM Analysis Software 4.0 and analyzed through custom R scripts under R versions 4.04 with normalized counts determined by the NanostringDiff package and thresholding outside of one standard deivation from the mean signal from the negative control probes.  Specific scripts uploaded to Wang, Raymond M. (2022). NanoString Differential Expression Analysis with NanoStringDiff package (Version 6). Zenodo. 10.5281/zenodo.7190262

label: Codeset barcode consisting of 4 fluorophores for identifying each specific hybridized gene probe.

source_name: Subcutaneous myocardial matrix hydrogel injection and nearby dorsal skin

label_protocol: 100 ng of total RNA per sample were labeled with Immunology Panel Reporter CodeSet solution along with relevant buffers and master mix based on manufacturer instructions.

growth_protocol: Male mast cell deficient mice (w-sh mice: B6.Cg-KitW-sh/HNihrJaeBsmJ, Jackson Laboratory, Sacramento, CA), mast cell engrafted mice and wild-type C57BL6/J were housed till 11-13 week old before performing procedures.  Hydrogels were isolated at 11 days post-injection based on latest timepoint for discernible presence hydrogel degradation profile and analyzed for gene expression analysis by Nanostring Mouse Immunology Panel.  Mast cell-engrafted mice were created by delivering eight to ten 100 µL subcutaneous injections of bone marrow differentiated mast cells in DMEM totaling 4 million cells.  Injections were at evenly spaced intervals throughout the dorsal region of 4-6 week old deficient mice.

extract_protocol: 11 days post-injection, mice were euthanized by sodium

treatment_protocol: Mice received 2x subcutaneous injections in the dorsal region of porcine-derived decellularized myocardial matrix hydrogels.

tag: Sex

value: male

value: w-sh mice:B6.Cg-KitW-sh/HNihrJaeBsmJ

tag: age

value: 12-14 weeks old


title: Engrafted 6

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
rg1

tag: strain


title: Engrafted 5

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
rm48c


title: Engrafted 7

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
rg2


title: Engrafted 3

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
rm47a


title: PrE1

description:
BWca_CAAAAG
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
non_ev_CPM_tmm_normalizedcounts_no_piR.xlsx

value: WT


title: SERUM20

description:
30_V1_TAATCG
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_serum_CPM_tmm_normalizedcounts_no_piR.xlsx

source_name: Serum

value: Serum

value: extracellular vesicles (from serum)

value: fresh collection


title: SERUM19

description:
26_V1_CGGAAT
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: Engrafted 1

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
rm44c


title: SERUM18

description:
25_V1_CATTTT
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: SERUM16

description:
23_V1_CACGAT
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: Deficient 8

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
km45a


title: Deficient 6

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
kme3c


title: SERUM15

description:
22_V1_CAACTA
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: Deficient 5

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
kg3


title: TSC3

description:
TSC4_ca_Day_0_AGTCAA
primary sequencing analysis (mapping) TISSUE_CELLS.xlsx
non_ev_CPM_tmm_normalizedcounts_no_piR.xlsx

value: Prostate


title: SERUM14

description:
20_V1_ATTCCT
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: SERUM13

description:
19_V1_GGTAGC
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: Deficient 2

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
kme4c


title: Deficient 3

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
km47c


title: Deficient 4

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
kg2


title: Deficient 1

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
km44c


title: Engrafted 4

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
rm45c


title: Engrafted 2

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
rm47c


title: SERUM12

description:
18_V1_CGTACG
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: SERUM11

description:
17_V1_GTGGCC
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: SERUM17

description:
24_V1_CAGGCG
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: SERUM10

description:
16_V1_GTCCGC
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: Wild-type 5

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
wme4c


title: SERUM7

description:
13_V1_CTTGTA
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: SERUM9

description:
15_V1_CCGTCC
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: Wild-type 8

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
wg2c


title: Wild-type 7

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
wg1c


title: Deficient 7

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
km46c


title: SERUM8

description:
14_V1_AGTTCC
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: Wild-type 3

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
wm46c


title: Wild-type 6

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
wme5c


title: Wild-type 2

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
wm45c


title: SERUM6

description:
11_V1_GATCAG
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: SERUM5

description:
10_V1_CAGATC
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: Wild-type 4

description:
Samples were processed according to manufacturer instructions.  In brief, RNA sample concentrations were measured on a Qubit 3.0 Fluorometer with a QubitTM RNA HS Assay kit.  70 µL of hybridization buffer was mixed with Immunology Panel Reporter CodeSet solution, and 8 µL of this master mix was mixed in a separate reaction vessel with 100 ng of RNA per tissue sample and RNA-free water up to 13 µL total.  2 µL of Capture ProbeSet was added to each vessel, mixed and placed on a thermocycler at 65ºC for 16-48 hours before being maintained at 4ºC for less than 24 hours.  Nanostring nCounter Prep Station performed automated fluidic sample processing to purify and immobilize hybridized sample to cartridge surface.  Digital barcode reads were analyzed by Nanostring nCounter® Digital Analyzer.
wm47c


title: SERUM2

description:
5_V1_CTATAC
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: SERUM4

description:
8_V1_ACAGTG
Qiagen GeneGlobe Primary Analysis Summary_SERUMEV.xlsx
ev_CPM_tmm_normalizedcounts_no_piR.xlsx
ev_serum_CPM_tmm_normalizedcounts_no_piR.xlsx


title: PHH in MPCC treated with  FFA and HGF followed by reversal

first: Karissa

last: Cottier

state: Maryland

address: 1450 S. Rolling Rd.

institute: BioIVT

description: Test (Group6)

data_processing:
Values for triplicate samples for treatment and 6 samples for control group were averaged for the analysis
The normalization and all the data analysis were performed according to the manufacturers instructions using their web-based software package: https://geneglobe.qiagen.com/us/analyze
For the normalization it uses the average of the arithmetic mean of five housekeeping genes: B2M, HPRT1 , RPLP0, GAPDH, ACTB.
Target gene signals normalized to housekeeping genes; 2^-deltaCt, where deltaCt = (Ct_Target − Ct_HKG)]
The web-based software package automatically performs all deltadeltaCt based fold-change calculations from the uploaded raw threshold cycle data.
Data from triplicate samples (6 samples for control group) were grouped and the web based software determined fold change for each treatment group as compared to the control group
Matrix normalized worksheet reports normalized signal (against housekeeping genes). Web based analysis software perfomed normalization automatically against housekeeping genes.
Fold Change worksheet reports test/control (such as FFA/control) ratios.

label: SYBR green

source_name: Primary Human Hepatocytes  and 3T3-J2 mouse stromal cells in MPCC

label_protocol: PCR assays were performed using the RT2 Profiler PCR Array Human Fatty Liver (Qiagen, Germantown, MD) following the Manufacturer’s instructions. Reverse transcription was performed using the RT2 First Strand Kit (Qiagen, Germantown, MD). Quantitative real-time PCR were performed (Quantstudio5, Applied Biosystems, MA) with 40 cycles at 95 oC for 15 seconds and 60 oC for 60 seconds.

growth_protocol: MPCC (HEPATOPAC) cultures were created using microfabrication processes to generate an organized co-culture containing cryopreserved primary human hepatocytes (PHH) from BioIVT (Baltimore, MD) and 3T3-J2 murine embryonic fibroblasts. Cryopreserved primary human hepatocytes from  donor lot TWJ were thawed at 37°C, counted, and plated on collagen-patterned 96 or 24 well plates. The cells were allowed to adhere until the following day, when 3T3-J2 fibroblasts were introduced to the culture plates. The resulting culture contained multiple hepatocyte islands having a 500 μm diameter spaced 1200 μm apart, center-to-center; with 3T3-J2 murine fibroblasts filling the area surrounding the hepatocyte islands. MPCC cultures were stabilized in serum containing medium for 7 days. At maturity, approximately 3,100 hepatocytes and 15,000 fibroblasts were present/well in a 96 well plate and about 21,000 hepatocytes and 90,000 fibroblasts were present/well in a 24 well plate. Treatment was initiated on day 7 after initiation of culture manufacture.

extract_protocol: RNA was extracted from treated  and control samples using the RNAeasy kit (Qiagen, Germany) as per manufacturer’s instructions. RNA was quantified spectrophotometrically, and all samples were normalized to the lowest concentration in the group. cDNA synthesis was performed using the RT2 First Strand Kit (Qiagen, Germany).

treatment_protocol: Cells were treated with control medium containing 1.0g/L glucose, 1.1 µM,  and 10% bovine serum or steatosis inducing media containing 0.5 mM free fatty acid (FFA) (Palmitate/Oleate 2:1, Sigma), 10g/L glucose and 1.0g/L fructose (HGF), or a combination of FFA and HGF with media exchanges every other day. Cells were treated with steatosis inducers continuously until day 7 of treatment or switched back to control media on day 4 for 3 days, until day 7 of treatment (reversal samples).

value: 0.5mM FFA and 10.0g/L glucose and 1.0g/L fructose

tag: treatment duration

value: 4 days + 3 days in control medium

tag: hepatocyte vendor

value: TWJ

tag: hepatocyte lot

value: BioIVT

tag: group

value: FFA + HGF + reversal