title: PC3, RNA-seq, ctr2

city: Shanghai

first: Zixian

last: Wang

address: 130 Dong'an Road, Xuhui District, Shanghai

institute: Fudan University

description:
Library name: PC3-ctr2
PC3-RNAseq.count.tsv
PC3-RNAseq.tpm.tsv

data_processing:
ChIP-seq: Raw reads were assessed with FastQC (v0.11.9) and adapter/low-quality sequences removed using TrimGalore (v0.6.7). Clean reads were aligned to the GRCh38 reference genome with Bowtie2 (v2.2.5) under default parameters. Alignments with mapping quality < 30 or flagged as unmapped, secondary, or PCR/optical duplicates (−F 3844) were discarded using SAMtools (v1.9). Duplicate reads were further marked and removed with Picard (v2.25.1). Library complexity and enrichment were evaluated with ENCODE's Phantompeakqualtools (run_spp.R), providing fragment-length estimates for downstream analyses. Peak calling was performed with MACS3 (v3.0.0a6) using the options “--nomodel --shift 0 --extsize <fragment_length>” and a significance threshold of p < 1 × 10-3.
RNA-seq: The raw data quality was evaluated with FastQC (v0.11.9). Adapter sequences and low-quality bases were removed using AdapterRemoval (v2.3.2). The resulting clean reads were aligned to the human reference genome (GRCh38) with STAR (v2.7.9a), and the alignments were sorted with SAMtools (v1.13). Gene-level read counts were generated from the sorted BAM files using htseq-count (v0.13.5). Differentially expressed genes (DEGs) were identified with DESeq2, applying thresholds of |fold change| > 2 and FDR < 0.05. Transcript abundance in transcripts per million (TPM) was quantified using StringTie (v2.2.1).
scRNA-seq: The raw sequencing data were processed using Fastp to trim adapter sequences and low-quality bases, followed by alignment to the human GRCh38 reference genome using SeekOne®Tools for gene expression matrix generation. Subsequent analysis was performed using 10x Genomics Cell Ranger and the Seurat package (v5.2.1) in R (v4.4.2), beginning with stringent quality control to select high-quality cells based on the following criteria: 200 < nFeature_RNA < 7000, nCount_RNA > 1000 (excluding the top 3% of cells by nCount_RNA), mitochondrial gene content < 10%, and ribosomal gene content between 0-60%.
Assembly: hg38
Supplementary files format and content: bigwig and narrowPeak for ChIP-seq data (except for input sample); gene expression matrix for RNA-seq and scRNA-seq data.

extract_protocol:
The immunoprecipitated and input DNA was purified using the MinElute PCR Purification Kit (#28006, Qiagen). Total RNA was isolated using the EZ-10 DNAaway RNA Mini-Preps Kit (#B618133, Sangon Biotech). Single-cell RNA-Seq RNA extracted were prepared using SeekOne® Digital Droplet Single Cell 3’ library preparation kit (No.K00202, SeekGene Catalog).
ChIP-seq libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (#E7103L, NEB) according to the manufacturer’s instructions. RNA-seq libraries were prepared using the Stranded mRNA-seq Lib Prep Module (RK20349, Abclonal). Single-cell RNA-Seq libraries were prepared using SeekOne® Digital Droplet Single Cell 3’ library preparation kit (No.K00202, SeekGene Catalog).

value: Prostate cancer cell line


title: Spatial transcriptomics of mus musculus: middle-aged male brain4 mouse 8

status: Public on Aug 13 2026

type: SRA

city: Saarbrücken

first: Fabian

middle: Michael

last: Kern

state: Saarland

address: Campus E2 1

department: Center for Bioinformatics

country: Germany

institute: Saarland University

description: Library name: AGE_8_73_Brain4_GEX27_D1

library_source: transcriptomic

data_processing:
We stitched the image series from the Axio Imager KMAT brightfield microscope using the Zeiss ZEN software (v3.6) and cropped and rotated the resulting high-resolution TIFF files using Fiji. Individual sample images were color corrected where needed to match the other images.
In the next step, we ran Spaceranger (v2.1) with the demultiplexed FASTQ files and the corresponding TIFF images. In brief, Spaceranger aligns the reads to a reference genome (refdata-gex-mm10-2020-A), detects tissue under spots and the fiducial frame in the image, to create a spot x UMI count matrix. For the infection cohort, we additionally built a custom Spaceranger reference genome combining mouse (GRCm39) and Plasmodium berghei -ANKA genome (PBANKA01). For two samples the alignment of the fiducial frame was manually corrected after the automatic detection had failed initially. For the manual image QC of Visium samples we used the 10x Loupe browser tool (v6.5.0).
Based on the Spaceranger reports, we either excluded a sample completely from further analysis, or using the 10x Cloupe tool manually selected small spot regions to exclude from downstream analysis where necessary (e.g. removing corners with uneven permeabilization), and further applied criteria-based spot filtering on each sample, to eventually obtain a high-quality data set. To this end, we inspected the variable distributions for the number of UMIs, the number of read counts, mitochondrial content, as well as spot complexity (=log10(Number of features) / log10(Number of counts)) for each tissue separately, and applied the following filtering criteria on the filtered spot count matrix from Spaceranger after loading into Seurat objects: (Maximum percentage of mitochondrial read content; Minimum number of UMIs; Maximum number of UMIs; Minimum number of detected features; Maximum number of detected features; Minimum spot complexity) Brain: (35; 500; 70,000; 300; 10,000; 0.75), Bulb: (35; 500; 70,000; 300; 10,000; 0.75), Heart: (60; 1000; 25,000; 300; 5000; 0.75), Kidney: (30; 1000; 50,000; 300; 8000; 0.75) Liver: (15; 1000; 40,000; 500; 8000; 0.75), Lung: (10; 1000; 30,000; 300; 8000; 0.75) Spleen: (10; 500; 35,000; 300; 8000; 0.75).
We then rotated the images of the remaining samples to best match the desired orientation for each tissue. We then followed the default Seurat (v4.4) workflow to preprocess each sample by applying NormalizeData, FindVariableFeatures with variance stabilizing transformation for 2000 features, followed by SCTransform (v1), regressing out the percentage of mitochondrial reads and subsetting to 3000 variable features. Subsequently, we performed RunPCA on the SCT assay, FindNeighbors on up to 30 PCs, FindClusters with leiden clustering followed by RunUMAP with up to 30 dimensions. Using the cleaned list of Visium samples, we created one Seurat object for each tissue using the SCT-based integration workflow as follows: set the default assay to SCT, run SelectIntegrationFeatures with 3000 features, then PrepSCTIntegration, and FindIntegrationAnchors followed by IntegrateData, each with “SCT” set as normalization method and up to 30 dimensions. Next, we performed the standard dimension reduction workflow consisting of RunPCA, RunUMAP, FindNeighbors, and FindClusters again with up to 30 dimensions and fixed random seeds.
In case of the STOmics Stereo-seq samples, images were obtained and stitched with the Zeiss Moticam Pro 500i. Combined QC and primary analysis of sequencing and image data was performed using the STOmics analysis workflow (SAW) v1.0 to obtain quantification matrices for the mouse genome (GRCm38). The SAW pipeline was also used to accumulate DNB spot gene expression counts into the specific area and cell binnings.
For the STOmics Stereo-seq samples, we first converted the outputs of the Stereo-Seq Analysis Workflow (SAW) pipeline90 into Seurat objects for both bin200 (200x200 Stereo-seq spots) and cell bins. This is done by using the io.stereo_to_anndata function in the Stereopy package91 (v1.1.0), followed by applying the h5ad2rds.R script provided by BGI. Cell bins were obtained using image-based cell segmentation of the corresponding ssDNA-stained images. Filtering was performed using the following criteria: (Maximum percentage of mitochondrial read content; Minimum number of UMIs; Maximum number of UMIs; Minimum number of detected features; Maximum number of detected features; Minimum spot complexity) Brain (bin200): (35; 500; 100,000; 300; 10,000; 0.75), Brain (cell bin): (35; 100; 10,000; 100; 3,000; 0.75).
In line with the Visium 10x samples we applied a default Seurat preprocessing workflow for each sample. Using the cleaned list of STOmics Stereo-Seq samples, we performed the Harmony-based integration workflow as follows: set the default assay to SCT, run SelectIntegrationFeatures with 5000 features, merge the sample list into one Seurat sample, run RunPCA with 50 components, run RunHarmony on SCT assay and the PCA reduction using Sample_ID and Age as latent variables, run JoinLayers on the RNA assay. Then, we performed the sample dimension reduction workflow as in the Visium samples with up to 50 dimensions.
Assembly: GRCm39
Supplementary files format and content: tif image of H&E stained tissue slice on capture area in high resolution
Supplementary files format and content: jpg image of spaceranger aligned fiducials
Supplementary files format and content: jpg image of spaceranger detected tissue
Supplementary files format and content: h5 of filtered spot barcode-to-feature count matrix from spaceranger
Supplementary files format and content: h5 of raw spot barcode-to-feature count matrix from spaceranger
Supplementary files format and content: json of spaceranger image scale factors
Supplementary files format and content: csv of spatial enrichment results for gene features
Supplementary files format and content: png image of tissue slice on visium capture area in high resolution
Supplementary files format and content: png image of tissue slice on visium capture area in low resolution
Supplementary files format and content: csv of spaceranger spot tissue positions
Supplementary files format and content: tif image of adjacent (before or after) H&E stained tissue slice in high resolution
Supplementary files format and content: cellbin.gef cellbin feature expression matrix records the information of cells individually, including the centroid coordinate, boundary coordinates, expression of genes, and cell area.
Supplementary files format and content: gef feature expression matrix includes the whole information over a complete chip region. It only has bin1 expression counts.
Supplementary files format and content: gem gene expression matrix (GEM) stores gene spatial expression data. SAW generates multiple gene expression matrix files in the workflow, the basic format requires six columns with a header row that shows the column names. The six columns are gene ID, gene name, x coordinate, y coordinate, MID count and exon count. When it comes to cellbin GEM, the seventh column is for cell ID. The header of the expression matrix for the maximum area enclosing rectangle region has several annotation rows starting with "#" before the column rows.
Supplementary files format and content: tissue.gef Feature expression matrix under the tissue coverage region. It is also a visualization GEF that includes expression counts for bin1, 5, 10, 20, 50, 100, 150, 200.
Supplementary files format and content: tissue.gem gene expression matrix (GEM) stores gene spatial expression data for spots under tissue. SAW generates multiple gene expression matrix files in the workflow, the basic format requires six columns with a header row that shows the column names. The six columns are gene ID, gene name, x coordinate, y coordinate, MID count and exon count. When it comes to cellbin GEM, the seventh column is for cell ID. The header of the expression matrix for the maximum area enclosing rectangle region has several annotation rows starting with "#" before the column rows.
Library strategy: Spatial Transcriptomics

molecule: polyA RNA

organism: Mus musculus

source_name: Brain

growth_protocol:
For the aging cohort, male C57BL/6JN mice were shipped from the National Institute on Aging colony (Charles River) to the Stanford ChEM-H animal facility, where they were housed at 12/12h light/dark cycle in cages of 2-3 mice. Water and food were provided ad libitum.
For the infection cohort, C57BL/6 mice were bred at the animal facility of the Bernhard Nocht Institute. P. berghei ANKA parasites were obtained from the blood of sporozoite-infected C57BL/6 mice provided by the Parasitology Section of the Bernhard Nocht Institute. Male C57BL/6 mice (6–8 weeks old) were infected i.p. with 1×10^5 parasitized red blood cells. Parasitemia was determined in Giemsa-stained blood smears from tail blood. Body weight was determined at different time points.

extract_protocol:
We followed the standard protocol for fresh frozen tissues (compare document CG000239 - Rev F) using the Visium Spatial Gene Expression (GEX) Slide & Reagent Kits with recommended reagents (10x Genomics, PN-1000193). In brief, after H&E staining and imaging (10x magnification, Axio Imager KMAT - ZEISS), tissues were permeabilized with the previously defined permeabilization time. After reverse transcription on the slide and denaturation, second strand synthesis is performed. For further processing, cDNA is released via denaturation. To determine cycle number for amplification the KAPA SYBER FAST qPCR Master Mix Kit (KAPA Biosystems) is used in a qPCR. All samples in this study were amplified using 14 – 19 PCR cycles. After bead purification using SPRIselect beads (Beckman Coulter, Product No. B233318), cDNA is quantified via 2100 Bioanalyzer using the Bioanalyzer High Sensitivity DNA kit (Agilent, cat.no.:5067-4626).
For each brain, five 10µm sections (Bregma: –1.7, -2.06, -2.7, -3.52 and -4.72) identical to the Visium cohort were collected and loaded onto the StereoSeq Chip T-Slide according to manufacturer´s protocol. In short, after fixation of the tissue using ice-cold methanol, cell nuclei were stained using the ssDNA Qubit dye (Thermo Fisher) and imaged in 10x resolution in FITC channel. Subsequently, brain tissue was permeabilized for 14 min at 37°C, releasing mRNAs from the cells onto the Chip T-Slide. Reverse transcription of mRNAs was conducted for 3 hours at 42°C generating cDNA with sequence barcodes indicating their unique location on the T-Slide. Afterwards, the tissue was removed using tissue removal buffer for 10 min at 55°C and cDNA was collected from the chip o/n at 55°C. CDNA was then purified, amplified and size distribution was checked using High Sensitivity DNA Bioanalyser Kit (Agilent).
The library construction was either performed manually or using the MGISP-100 liquid handling system according to the 10x Visium user guide. In short, the following steps were performed: fragmentation, end repair, A-tailing, double sided size selection, adaptor ligation, post ligation cleanup, sample index PCR, double sided size selection; followed by post library construction QC performed using the Bioanalyzer High Sensitivity DNA kit (Agilent, cat.no.:5067-4626) and KAPA Library Quantification Kit (Roche, KK4824). Libraries were pooled in an equimolar fashion with up to 15 samples in one library, pooling only samples originating from the same organ whenever possible. All libraries were sequenced using NovaSeq 6000 (Illumina).
Library Preparation was performed using Stereo-seq Library Preparation Kit (MGI) using 30ng of cDNA and 13 cycles of amplification following the manufacturer´s protocol. Final quality check of the size distribution of the library was performed using High Sensitivity DNA Bioanalyzer Kit.

treatment_protocol:
For the aging cohort, tissues from brain, heart, kidney, liver, lung and spleen were collected after anaesthetization with 2.5% v/v Avertin and transcardial perfusion with 20 mL cold PBS and flash frozen in isopentane cooled with liquid nitrogen, all between 10:15 AM- 12:00 PM on a single day. For brain samples, the right and left hemisphere were separated before freezing and stored separately. Samples were stored at -80°C until embedding in OCT. Samples were embedded for sectioning in precooled OCT on dry ice.
For the infection cohort, tissues samples were collected on consecutive days, from 9:30 AM (Ctrl.) and 10:30 AM (day 6 post-infection) until 12 PM. To prevent lung deflation, the tracheae were sutured, and the lungs were inflated with a 1:1 mixture of PBS and OCT. The small lung lobe was separated and frozen separately from the residual lung tissue. Brains were frozen without separating the hemispheres. All tissues were simultaneously frozen while embedded in OCT, in an isopentane bath cooled with liquid nitrogen and stored at -80°C.
Cryosectioning and section mounting were performed according to the manufacturer’s protocol, in a Leica cryostat (LEICA CM3050 S) set to -15°C with a slice thickness of 16 µm (except for the spleen of the infection cohort; 10 µm). For the aging cohort, five brain sections were mounted using visual landmarks and the Allen Mouse Brain Atlas, resulting in sections from bregma –1.7, -2.06, -2.7, -3.52 and -4.72. For the infection cohort, two brain sections were mounted, namely from bregma –2.06 and bregma 3.89 (Olfactory bulb). Sections were mounted on designated capture areas and stored at -80°C until further processing. After tissue mounting on the slide, an additional 10-15 sections were collected for RNA Quality assessment. All samples used in this study had a RIN value between 3.0 and 9.0.

tag: tissue

value: Brain

tag: strain

tag: mouse id

tag: Sex

value: male

tag: age

value: 73 weeks

tag: slide

tag: area

tag: sequencing primer

tag: permeabilization time_(minutes)

tag: slice thickness_(um)

tag: cohort

value: Aging

tag: technology

value: 10x Visium

tag: genotype

value: Wildtype

tag: treatment

tag: disease

tag: disease stage


title: LUHMES pSG5-miR-873 1

data_processing:
Sample primary processing was performed with miRMaster (Fehlmann et al., Nucleic Acids Res, 2021) using standard parameters. Mapping to the genome was performed allowing up to 5 mulit-mapping hits with Bowtie. One mismatch was allowed for the quantification of isomiRs.  As output, miRMaster generated a list with the expression of 80,668 RNAs from 10 RNA classes. The data were normalized to expression in one million reads and further processed with R (R 4.0.4 GUI 1.74 Catalina build (7936)). To limit the effect of low-abundance miRNAs, we considered miRNA isoforms as expressed if at least five in 10 million mapped miRNA reads were found for at least three independent samples.
Supplementary files format and content: Reads per million mapped miRNA normalzed counts for each sample and human miRNA from miRBase v22.

molecule: total RNA

organism: Homo sapiens

source_name: LUHMES

growth_protocol: The cell lines HEK293T (ACC 635), SH-SY5Y (ACC 209) and HeLa (ACC 57) were purchased from the German collection of microorganisms and cell cultures (DSMZ). The authenticity of the cell lines was confirmed by STR fingerprinting by the supplier. Cell lines were cultivated in DMEM (Life Technologies, Darmstadt, Germany) supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml) and either 10% [v/v] FCS for 293T and HeLa cells or 20% [v/v] FCS for SH-SY5Y cells. Lund human mesencephalic (LUHMES) cells were purchased from the American Type Culture Collection (ATCC; CRL-2927). The cells were cultured as previously described by Scholz et al. We performed one passage for all cell lines after purchasing them and cryo-conserved them. All cell lines are from female. All cell lines were subcultured two times a week for no more than three months after the start of the culture.

extract_protocol:
0, 4, 8, 24, 48 h after transfections for miR-7 and 48 h for miRNAs miR-34a, -129, -133b, 873, cells were lysed by QIAzol Lysis Reagent (Qiagen, Hilden, Germany), and total RNA was isolated using the miRNeasy Mini Kit (Qiagen, Hilden, Germany).
Library preparation for small RNA sequencing was performed using the MGIEasy Small RNA Library Prep Kit according to the manufacturer’s recommendations. In detail, after 3’ and 5’ adapter ligation, RNAs were reverse transcribed and PCR amplified (21 cycles). PCR products were purified using 6% TBE gels, excising only the 110 bp band corresponding to the PCR product of miRNAs. Subsequently, twelve samples were pooled equimolar into one library, and the library was further circularized and purified. Circularized libraries were sent to BGI Hong Kong for sequencing using the standard MPS protocol as described previously (Li et al., Nucleic Acids Res, 2021).

treatment_protocol: Sequence inserts representing miRNA precursor sequences with additional nucleotides up- and downstream of the precursor sequence for mir-129 and mir-873 were either synthesized by Eurofins Genomics (Ebersberg, Germany) or PCR amplified using sequence-specific primers with human genomic DNA as a template and subsequently cloned into the expression plasmid pSG5 (Agilent Technologies, Santa Clara, California) using EcoR I and BamH I restriction sites. The expression plasmid pSG5-miR-129 harbors nucleotides 128207756-128208053[+] of human chromosome 7 (GRCh38/hg38) and pSG5-miR-873 nucleotides 28888786-28889058[-] of human chromosome 9 (GRCh38/hg38). Cloned sequences of miRNA expression plasmids were verified by Sanger sequencing. Cloning of the pSG5-miR-133b, pSG5-miR-34a and pSG5-miR-7 expression vectors was described previously (Alles et al., Nucleic Acids Res, 2019; Hart et al., Oncotarget, 2016; Kern et al., Nucleic Acids Res, 2021).  For ectopic expression of the respective miRNAs for NGS of miR-transfected HEK293T and HeLa cells, 4x105 cells were seeded out in 6-well plates. Twenty-four hours later, the cells were transfected with the respective DNA amounts (0.5 µg, 1 µg, 2 µg for miR-7, 2 µg for mir-34a, -129, -133b, 873) using PolyFect transfection reagent (Qiagen, Hilden, Germany). For the transfection of the different amounts of mir-7 (0.5 µg, 1 µg) in HEK293T cells, the pSG5-miR-7 expression plasmids were supplemented with the respective amount of pSG5 empty vector to achieve equal amounts of transfected DNA in all samples. For ectopic expression of the respective miRNAs of miR-transfected SH-SY5Y (4,5x105 cells/well) and 4x105 LUHMES (4x105 cells/well) cells for NGS, cells were seeded out in 6-well plates. Twenty-four hours later, the cells were transfected with 2 µg of the respective expression plasmids for mir-7, mir-34a, mir-129, mir-133b, mir-873 or the pSG5-empty vector using Lipofectamine™ 2000 transfection reagent (Invitrogen, Karlsbad, USA). All experiments were conducted 3 times.

tag: cell line

value: LUHMES

value: Female

tag: plasmid

tag: concentration

tag: time

value: 48 hr


title: WSUDLCL2 - DMSO - D2 - rep3

city: Summit

first: Junfei

last: Zhao

state: NJ

address: 556 Morris Ave

country: USA

institute: Bristol Myers Squibb

description: Library name: 7357-CH-0168

data_processing:
We used featureCounts 2.0.6 to count the number of reads on gene level.
Assembly: GRCh38
Supplementary files format and content: readcount on gene level

source_name: cell line

extract_protocol:
Cells for RNAseq were prepared according to instructions from Discovery Life Sciences.
Five million cells per sample were collected, washed, fixed, and stored at -80 C prior to shipping to Discovery Life Sciences where downstream RNAseq was carried out.

tag: tissue

value: cell line

value: DMSO

tag: batch


title: Niche, CH05

city: Toronto

first: Soheil

last: Jahangiri

state: Ontario

address: 610 University Ave

department: Princess Margaret Cancer Centre

country: Canada

institute: University Health Network

description:
Library name: HS_5
10x Genomics Single Cell 3’ V3

library_source: transcriptomic single cell

data_processing:
Raw FASTQ files were processed using CellRanger version 8.0.1 (10X Genomics). RNA counts were generated by aligning to the GRCh38 (hg38) reference using SC3Pv3 or SC3Pv4 for 10Xv3 or 10Xv4 3’ pair end sequencing respectively. HTO counts were generated using the same method, by aligning to their respective barcodes.
Assembly: GENCODE human reference genome v38
Supplementary files format and content: Gene expression – Raw counts

source_name: Bone Marrow

extract_protocol:
Niche fraction - Digested cell suspension were depleted for hematopoietic cells using automated magnetic cell separation system (AUTOMACS). Cells were stained with biotin anti-human CD45 and biotin anti-human glycophorin A (Biolegend).  Depleted fraction was stained for 1 hour at 4 degrees with the following antibodies: CD45-BUV395 (BD), GlyA-FITC (Biolegend), CD38-PECy7 (Biolegend), CD34-APCCy7 (Biolegend), NGFR-AF647 (Biolegend) and CD31-PE (Biolegend) and FVS575V was used for dead cell exclusion. Cells were sorted on a BD Aria Fusion. Two populations were sorted: CD45-GlyA-CD38-  niche fraction and CD45lowCD34+ HSPC fraction. Niche fraction was resuspended in PBS + 0.04% BSA and loaded into the 10x Genomics Chip G or X.  Hematopoietic fraction - Frozen mononuclear cell fractions were thawed using X-VIVO medium (Lonza) supplemented with 50% FBS and 110 mg/mL DNase. The suspension was centrifuged at 1200 rpm for 8 mins, cells were resuspended in RPMI with 10% FBS and counted. 4-5x106 MNC cells were resuspended in staining buffer (2% BSA,0.01% Tween in PBS) in conical bottom polypropylene FACS tubes. Cells were incubated with Fc Blocking reagent at a dilution of 1:10 (FcX, BioLegend) for 10 minutes on ice. After FcX block incubation, 0.1ug (per 1x106 cells) of unique TotalSeq™-B Hashing antibody (Biolegend) was added and incubated for 30 minutes on ice. Cells were washed 5 times with 1 ml Staining buffer (2%BSA/0.01%Tween, PBS), spined at 4˚C for 5 minutes at 350g. After the final wash, cells were resuspended in FACS buffer (PBS 2% FBS) and stained with the following antibodies: Lineage cocktail-FITC, CD45-PECy7, CD34-APCCy7 for 1 hour at 4 degrees. DAPI was used for dead cell exclusion. Cells were filtered and sorted on a BD Aria Fusion. Three hematopoietic populations were sorted: Mature cells (Lineage+), committed progenitors (Lineage-CD45+) and hematopoieitc progenitors (Lineage-CD45lowCD34+).  Samples were pooled evenly for two library preparations: Mature library (Mature cells and committed progenitors) and HSPC library. Pooled samples were delivered to the Princess Margaret Genomics Center (PMGC) for single cell sequencing preparation using the 10X platform.
We used Chromium Next GEM Single Cell 3’ GEM, Library & Gel Bead Kit v3.1 and Chromium GEM-X Single Cell 3', Library & Gel Bead Kit v4 and followed the protocol as provided by 10x Genomics. Sorted HSPC fraction was frozen until further use.

value: Bone Marrow

tag: disease state

value: CHIP

tag: Sex

value: Male

tag: compartment

value: BM Niche


title: WSUDLCL2 - DMSO - D2 - rep2

description: Library name: 7357-CH-0167


title: Bulk RNA-seq of brain endothelial cells CTRL-1

city: Stanford

first: Julia

last: Belk

state: California

address: 450 Serra Mall

institute: Stanford University

description:
Library name: CTRL_1
RNA-seq

data_processing:
Adapter trimming was performed using fastp (v0.20.1) and reads were aligned to the GRCm39 reference genome using hisat2 (v2.1.0). To count reads overlapping genes, featureCounts v2.0.6 was used, and the output was loaded into R (v4.2.2) for downstream analysis.
Assembly: GRCm39
Supplementary files format and content: counts table

growth_protocol: 6-week-old C57BL/6 mice mice were anesthetized with a ketamine/xylazine injection and perfused with 20ml of ice-cold PBS. Cortices were homogenized with GentleMACS tissue dissociator (Mylteni Biotech) in prewarmed DMEM supplemented with 2% Bovine Calf Serum (BCS, HyClone), 25µg/ml Liberase DL, 100µg/ml Dispase, 0.5mg/ml Papain, 10µg/ml DNase-I (all enzymes from Sigma) for 30 min at 37°C. After homogenization the cell suspension was filtered through a 70 μm strainer and myelin depletion was performed in 30% isotonic percoll (800g for 20 min at 4°C). Brain cell pellets were stained with anti-CD45-AlexaFluor 647 (1:200), anti-CD31-PE (1:100), and anti-Ly6C-APCCy7 (1:200) for 30 minutes on ice (all antibodies from Biolegend). DAPI was used for live/dead cells discrimination. The DAPI−CD45−CD31+Ly6C+ population was sorted on FACS Aria-III (BD Bioscience) using a 100 µm nozzle. Approximately 600,000 brain endothelial cells (BEC) per sample were sorted (two brains were pooled for each sample). BECs were collected into Endothelial Cell Medium (Cell Biologics, M1168), plated onto a Matrigel-coated 24-well plate and expanded for 12 days in Endothelial Cell Medium at 37°C with 5% CO2. Matrigel was removed using Cell Recovery Solution (Corning) prior to RNA extraction.

extract_protocol:
Cells were lysed in 450µl of lysis buffer for RNA extraction. The lysate was mixed by pipetting and briefly shaken for 1 minute at room temperature. Lysates were collected, and RNA was purified using the RNeasy Micro Kit (Qiagen 74004), following the manufacturer’s protocol. Purified RNA was stored at -80°C until analysis.
Library preparation was performed with 5-10ng of total RNA per sample. ds-cDNA was made using the low-input SMARTseq mRNA kit (Takarabio) according to manufacturer’s protocol. cDNA was fragmented using a Covaris E220 sonicator using peak incident power 18, duty factor 20%, cycles per burst 50 for 120 seconds. 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 15 cycles using primers incorporating unique dual index tags. Libraries were pooled and sequenced on an Illumina NovaSeqX Plus (2x150 sequencing recipe), targeting 50M reads per sample. Basecalls and demultiplexing were performed with Illumina’s bcl2fastq software with a maximum of one mismatch in the indexing read.

treatment_protocol: On the day of the experiment, cells were incubated in serum-free medium for 2 hours prior to stimulation with recombinant mouse GAS6 (250 ng/mL; R&D Systems) and recombinant mouse IGF-I (200 ng/mL; PeproTech), or PBS as a control. After 6 hours stimulation, the medium was removed, and wells were washed with PBS.

tag: cell type

value: Endothelial cells

value: Wild-type

value: PBS


title: LUHMES pSG5-miR-129 1


title: WSUDLCL2 - DMSO - D2 - rep1

description: Library name: 7357-CH-0166


title: Bulk RNA-seq of brain endothelial cells STIM-3

description:
Library name: STIM_3
RNA-seq

value: Brain

value: IGF-1 + GAS6


title: Niche, CH02

description:
Library name: HS_3
10x Genomics Single Cell 3’ V3


title: LUHMES pSG5-miR-34a 1


title: SUDHL4 - BMS158_2.5_CC282_50 - D2 - rep3

description: Library name: 7357-CH-0165


title: Niche, NC05

description:
Library name: HS_2
10x Genomics Single Cell 3’ V3

value: Non-Carrier


title: LUHMES pSG5-miR-7 1


title: PC3, ChIP-seq, Input

country: China

description: Library name: PC3-Input

library_source: genomic

molecule: genomic DNA

value: Prostate cancer cell line


title: SUDHL4 - BMS158_2.5_CC282_50 - D2 - rep2

description: Library name: 7357-CH-0164


title: SM11_NFIX_HCKO_2x

city: HOUSTON

first: Sanjana

last: Murali

state: TX

address: 7600 Kirby Drive

institute: Baylor College of Medicine

description: Library name: SAMPLE 11

data_processing:
Quality control was performed using fastQC (v0.10.1) and MultiQC (v0.9) . Reads were mapped to the mouse genome mm10 assembly using STAR (v2.5.0a). In R(v3.5.2) Bioconductor packages GenomicAlignments (v1.16.0) and GenomicFeatures (v1.32.2) were used to build count matrices (Lawrence et al., 2013). UCSC transcripts were downloaded from Illumina iGenomes in GTF file format. Normalization and differential gene expression analysis were performed using DESeq2 (v1.20.0). Gene Ontologies were determined using Enrichr and visualized using GOplot (v1.0.2).
Assembly: Reads were mapped to the mouse genome mm10 assembly using STAR (v2.5.0a).
Supplementary files format and content: csv file with raw counts for each sample

source_name: Hippocampus

extract_protocol:
RNA was extracted from sorted cells using RNeasyMicro Kit (Cat.No. 4004, QIAGEN). RNA integrity (RINR8.0) was assessed using the High Sensitivity RNA Analysis Kit (DNF-472-0500, Agilent) on a 12-Capillary Fragment Analyzer.
cDNA synthesis and Illumina sequencing libraries with 8-bp single indices were constructed from 10 ng total RNA using the Revelo RNA-seq High Sensitivity kit (Cat.No. 30184149, TECAN).

value: Hippocampus

value: KO


title: Niche, NC03

description:
Library name: HS_1
10x Genomics Single Cell 3’ V3


title: LUHMES pSG5 1

value: LUHMES

value: Female


title: SUDHL4 - BMS158_2.5_CC282_50 - D2 - rep1

description: Library name: 7357-CH-0163

value: cell line


title: APOE 4 knock-in replicate 3

description:
Library name: E4_3
10x Genomics

data_processing:
Fastq files from scRNA-seq data were trimmed and aligned to the GRCm39 reference genome (refdata-gex-GRCm39-2024-A available from 10X Genomics) using the “cellranger count” pipeline (v8.0.1).
Assembly: GRCm39
Supplementary files format and content: Count matrix from cellranger count (h5 format)

extract_protocol:
Brain immune cells were isolated as described in the respective method section. The entire sample preparation protocol was carried out either on ice, or at 4°C. Briefly, after perfusion in ice-cold PBS, brain cortices were dissected and mechanically homogenized. Cell suspension was filtered through a 70 μm strainer and pellets were resuspended in 30% isotonic percoll for myelin depletion (centrifugation at 800g for 20 min). To sort brain macrophages, cells were stained with CD45-PE (1:200), CD11b-APC (1:100), and lineage exclusion mix (Ly6C-FITC 1:200, Ly6G-FITC 1:200, CD43-FITC 1:100, CD44-FITC 1:100, NK1.1-FITC 1:100) for 30 min (all antibodies from Biolegend). For live/dead discrimination, a 1 mg/ml of DAPI (Sigma) at 1:5000 final dilution was added to the sample immediately prior to sorting. Sorting was performed on FACS Aria-III (BD Bioscience) using a default 85µm nozzle. Brain macrophages were sorted in one collection tube containing 300µl of PBS + 0.4% UltraPure BSA (Thermo Fisher) + 1U/µl of Recombinant RNasin Ribonuclease Inhibitor (Promega). Sorted cells were washed in PBS and resuspended into PBS + 0.04% UltraPure BSA buffer at a concentration of approximately 1,000 cells/µl.
Single-cell libraries were prepared using the Chromium GEM-X Single Cell 3' v4 protocol, targeting 20,000 cells/sample. Briefly, cDNA was prepared after the GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps. Purified cDNA was amplified for 11-13 cycles before being cleaned up using SPRIselect beads, and cDNA concentration was determined on the Bioanalyzer. GEX libraries were prepared as recommended by the Chromium GEM-X - Single Cell 3' Reagent Kits v4 (CG000731) with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform, the Chromium GEM-X Single Cell 3' Kit v4 - 16 rxns (PN-1000691), Chromium GEM-X Single Cell 3' Chip Kit v4 - 4 chips (PN-1000690) and Dual Index Kit TT Set A, 96 rxns (PN-1000215) were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer’s protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeq X Plus instrument. Normalized libraries were sequenced on a NovaSeq X Plus Flow Cell using the 151x10x10x151 sequencing recipe according to manufacturer protocol. Read 1 was trimmed to the 10x Genomics recommendation of 28bp. A median sequencing depth of 50,000 reads/cell was targeted for each gene expression library.

value: primary cells

value: Macrophages


title: SH-SY5Y pSG5 3

value: 48 hr


title: APOE 4 knock-in replicate 2

description:
Library name: E4_2
10x Genomics


title: Hematopoietic, (NC01,CH01,NC04)

description:
Library name: HM_3
10x Genomics Single Cell 3’ V4

value: Bone Marrow

value: Mixed

value: Hematopoietic


title: SUDHL4 - BMS158_2.5 - D2 - rep2

description: Library name: 7357-CH-0161


title: APOE 4 knock-in replicate 1

description:
Library name: E4_1
10x Genomics


title: SH-SY5Y pSG5-miR-7 3


title: Subject 1.30037, Visit 6

city: Palo Alto

first: Dmitri

middle: A

last: Kazmin

state: CA

address: 240 Pasteur Dr

department: Institute for Immunity, Transplantation and Infection

description: Library name: 1.30037.Visit.6

data_processing:
Sequencing data were aligned to a composite reference of GRCh38 with Ensemble annotation version 100 and SARS-CoV-2 reference assembly MN985325.1 using STAR v2.7.3a. Transcript abundance estimates were calculated by using the
STAR “quantMode GeneCounts” option using the algorithm of htseq-count.
Assembly: GRCh38-100, MN985325.1
Supplementary files format and content: Tab delimited text with counts for each sample in columns and transcript in rows.

source_name: Whole blood

growth_protocol: Whole blood collected in Paxgene tubes. No growth

extract_protocol:
Blood was collected into PAXgene Blood RNA tubes (BD Biosciences) and the RNA was extracted using the MagMAX for Stabilized Blood Tubes RNA Isolation Kit, compatible with PAXgene Blood RNA Tubes (ThermoFisher Scientific). RNA quality was assessed using a TapeStation 4200 and one microgram of total RNA was subjected to globin transcript depletion using the human GLOBINclear Kit (ThermoFisher Scientific).
Ten nanograms of the globin-depleted RNA was used as input for cDNA synthesis using the Clontech SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio) according to the manufacturer’s instructions. Amplified cDNA was fragmented and appended with dual-indexed bar codes using the Nextera XT DNA Library Preparation kit (Illumina). Libraries were validated by capillary electrophoresis on a TapeStation 4200 (Agilent)

treatment_protocol: Whole blood collected 1, 2, 3 or 7 days after primary, secondary or tertiary vaccinations

value: Whole blood

tag: subject

tag: visit

tag: day post_primary

tag: day post_prior_vaccition

tag: prior vaccition

value: Secondary

tag: infection status

value: Infected

tag: infection category

value: Report says after

tag: study

value: AZ


title: Hematopoietic, (NC03,NC05,CH02)

description:
Library name: HM_1
10x Genomics Single Cell 3’ V3


title: SH-SY5Y pSG5-miR-34a 3

tag: cell line

tag: plasmid


title: APOE 3 knock-in replicate 3

description:
Library name: E3_3
10x Genomics

value: Brain

tag: genotype


title: Subject ZA0010822, Visit 7

description: Library name: ZA0010822.Visit.7

tag: tissue

value: Uninfected

value: Healthy

value: NX


title: SH-SY5Y pSG5-miR-129 3

value: Female

tag: concentration

tag: time


title: SUDHL4 - BMS158_1.25_CC282_50 - D2 - rep3

description: Library name: 7357-CH-0159

value: cell line

tag: treatment

tag: batch


title: APOE 3 knock-in replicate 2

description:
Library name: E3_2
10x Genomics

value: primary cells

tag: cell type


title: HSPC, (NC02,CH06, NC07, CH04)

description:
Library name: HH_4
10x Genomics Single Cell 3’ V4

tag: disease state

tag: Sex

tag: compartment

value: HSPC


title: SH-SY5Y pSG5-miR-133b 3

value: 48 hr


title: APOE 3 knock-in replicate 1

description:
Library name: E3_1
10x Genomics

value: Macrophages


title: HSPC, (NC01,CH01,NC04)

description:
Library name: HH_3
10x Genomics Single Cell 3’ V4

value: Bone Marrow

value: Mixed


title: SH-SY5Y pSG5-miR-873 3


title: SUDHL4 - BMS158_1.25_CC282_50 - D2 - rep1

description: Library name: 7357-CH-0157


title: Subject 1.20076, Visit 3

description: Library name: 1.20076.Visit.3

value: Whole blood

tag: subject

tag: visit

tag: day post_primary

tag: day post_prior_vaccition

value: Primary

value: After


title: E19Diaphragm_AGO2_CDN2

city: New York

first: Benjamin

last: Kleaveland

state: NY

address: 1300 York Ave

department: Pathology and Lab Medicine

institute: Weill Cornell Medicine

description:
Library name: E19Diaphragm_AGO2_CDN2
small RNA

data_processing:
Reads were trimmed of adaptor sequence using cutadapt and filtered using fastq_quality_filter (FastX Toolkit; http://hannonlab.cshl.edu/fastx_toolkit/) with the parameters “-q 30 –p 100” to ensure that all bases had an accuracy of 99.9%. The first 19 nt of each read were matched to a dictionary of miRNA sequences downloaded from TargetScanMouse Release 8.0, requiring no mismatches between the read and the miRNA dictionary, using custom python scripts. Differential expression of the small-RNA-seq libraries was performed using the nf-core 24.10.1 pipeline and DESeq2 without the lfcShrink function and independent filtering = false.
Assembly: TargetScanMouse Release 7
Supplementary files format and content: comma-separated text file of DESeq2 output includes miRNA name (mirna), baseMean, log2 fold change, lfcSE, stat, p-value, adjusted p-value, raw counts, and normalized counts

source_name: Diaphragm

extract_protocol:
Total RNA was extracted from mouse tissues using TRI Reagent (ThermoFisher) according to the manufacturer’s protocol with the following modifications. Mouse tissues were rapidly dissected after CO2-based euthanasia and flash frozen in Eppendorf tubes in liquid N2. Tissue was transferred to a 15 mL conical tube, 1–2 mL of TRI Reagent was added, and the tissue was homogenized with a TissueRuptor (Qiagen) and disposable probes. Samples were transferred to Eppendorf tubes (1 mL per tube) and phase separated with 200 μL chloroform (J.T. Baker Analytical). After isopropanol precipitation and two 70% ethanol washes, total RNA was resuspended in water.
Small-RNA sequencing libraries were prepared with 2 μg of total RNA from each diaphragm (n = 2–3 biological replicates per genotype). RNAs that co-migrated within the range of 17 and 32 nt radiolabeled internal standards run in adjacent lanes were isolated on a 15% polyacrylamide urea gel. Gel slices were macerated and RNA was eluted overnight at 4C in 400 μL of 0.3 M NaCl. After Spin-X column (Corning CLS8170) cleanup, RNA was precipitated for >2 hours at –20C with 3 μL of GlycoBlue (ThermoFisher AM9515) and 1 mL of 100% ethanol and then resuspended in 8 μL RNase-free H2O. All 8 μL of size selected RNA was used as input for the NEBNext Low-bias Small-RNA Library Prep Kit (NEB E3420). Libraries were prepared, according to manufacturer’s instructions, with 1:3 dilutions of both 5′ and 3′ adapters. After amplifying libraries with 11 cycles of PCR, bead-based size selection was performed to further enrich libraries with miRNA-sized inserts. Libraries were sequenced on a NovaSeqX (Illumina) with single-end 50 bp reads and ~26 million reads per sample.

value: Diaphragm

tag: Stage

tag: background

value: C57BL/6J and C57BL/6N


title: HSPC, (NC06,CH05,CH03)

description:
Library name: HH_2
10x Genomics Single Cell 3’ V3


title: Subject 1.30011, Visit 8

description: Library name: 1.30011.Visit.8

tag: prior vaccition

value: Secondary

tag: infection status

tag: infection category

value: Report says infected

tag: study

value: AZ


title: SH-SY5Y pSG5 2

tag: cell line

tag: plasmid

tag: concentration

tag: time


title: E19Diaphragm_AGO2_CDN1

description:
Library name: E19Diaphragm_AGO2_CDN1
small RNA

tag: tissue

tag: genotype


title: SH-SY5Y pSG5-miR-7 2

tag: Sex


title: HSPC, (NC03,NC05,CH02)

description:
Library name: HH_1
10x Genomics Single Cell 3’ V3

tag: disease state

tag: compartment

tag: batch


title: SUDHL4 - CC282_50 - D2 - rep2

description: Library name: 7357-CH-0155

tag: treatment


title: Neutrophil, control, rep10

city: Guangzhou

first: Sen

last: Li

address: 9 Jinsui Road, Tianhe District

institute: Guangzhou Women and Children's Medical Center

description: Library name: Sample 22

data_processing:
Sequence reads were trimmed for adaptors and low-quality sequence using fastp (version 0.18.0), then mapped to to the human reference GRCh38 with HISAT2 (version 2.1.0) with default parameters. The mapped reads were assembled using StringTie (version 1.3.1) in a referencebased approach. The expected counts and TPM values were determined using RSEM software.
Assembly: GRCh38
Supplementary files format and content: csv file includes expected counts for each sample
Supplementary files format and content: csv file includes TPM values for each sample

source_name: Peripheral blood neutrophils isolated from infants without BPD at postnatal day 14, RNA-seq

extract_protocol:
Neutrophils were isolated from peripheral blood using the EasySep Direct Human Neutrophil Isolation Kit (STEMCELL Technologies). Then, total RNA was extracted from neutrophils using TRIzol reagent (Vazyme Biotech).
RNA libraries for RNA-seq were constructed using Hieff NGS® Ultima Dual-mode mRNA Library Prep Kit (Yeasen) according to the manufacturer’s instructions.

value: Peripheral blood

tag: cell type

value: Neutrophil

value: WT


title: SUDHL4 - CC282_50 - D2 - rep1

description: Library name: 7357-CH-0154

value: cell line


title: SH-SY5Y pSG5-miR-34a 2

value: Female


title: E19Diaphragm_AGO2_PN2

description:
Library name: E19Diaphragm_AGO2_PN2
small RNA

tag: Stage

tag: background


title: Subject 1.20081, Visit 8

description: Library name: 1.20081.Visit.8

tag: subject

tag: visit

tag: day post_primary

tag: day post_prior_vaccition

tag: prior vaccition

tag: infection status

value: Uninfected

value: Healthy


title: Subject 1.20027, Visit 3

description: Library name: 1.20027.Visit.3

tag: infection category

tag: study


title: Subject 1.20028, Visit 3

description: Library name: 1.20028.Visit.3

tag: tissue


title: Mtb, phoT::Tn, lactate, pH 5.7, Rep B

city: East Lansing

first: Robert

last: Abramovitch

state: MI

address: 2215 Biomedical And Physical Sciences

institute: Michigan State University

description: Library name: Lac_Tn_5B_S7_L001_R1_001.fastq.gz

data_processing:
After the sequencing runs, reads were demultiplexed and converted fastq format using Illumina bcl2fastq (v1.8.4) script.  The reads in the raw files were then subjected to trimming of low quality bases and removal of adapter sequences using Trimmomatic (v0.30) with a 4bp sliding window, cutting when the read quality dropped below 15, or read length was less than 36 bp.  Trimmed reads were aligned to the WT erdman genome using bowtie with the -S option to produce SAM files as output.
SAM files produced by bowtie were converted to BAM files and coverage depth was calculated using SAM tools resulting in 98% coverage.  Aligned reads were then counted gene feature in WT erdman genome using the HTSeq software suite.  Data were normalized by estimating effective library sizes using robust regression within the DEseq package.  Statistical analysis and differential gene expression was performed in R studio by fitting a negative binomial model to each set of conditions and testing for differences utilizing the DESeq package.
Assembly: M. tuberculosis Erdman (ATCC 35801), GCA\_000350205.1
Supplementary files format and content: Processed files are presented as an .xlsx spreadsheet with calculated CPMs of each Rep presented.
Supplementary files format and content: Dataset_GEO.xlsx

organism: Mycobacterium tuberculosis

source_name: Bacterial Cell

growth_protocol: Mtb cultures were cultured for 3 days  in T-75 standing flasks in a 37C incubator (with 5% CO2).

extract_protocol:
RNA was extracted using the method Rohde, Abramovitch and Russell, Cell Host and Microbe, 2007. Briefly,  RNA was stabilized and isolated using a guanidine thiocyanate-based lysis buffer as previously described. Pelleted mycobacteria were lysed using 5 μg/ml lysozyme, hot Trizol, and physical disruption with silica beads in a BeadBeater. Total RNA was isolated by chloroform extraction followed by direct application to QIAGEN RNeasy column purification. Residual DNA contamination was removed using Turbo DNAfree DNase (Ambion).  RNA quality and integrity were examined using an Agilent Bioanalyzer prior to subjecting samples to rRNA depletion using the Epicentre Ribo-Zero depletion kit.
DNA libraries were constructed using the illumina true seq RNA library preparation kit omiting the polyA selection step.  After library quality control, samples were pooled and sequenced in one lane Illumina HiSeq 2500 Rapid Run flow (v1) in 50 bp, single end read format (SE50).

treatment_protocol: 8 conditions conditions, and three strains,WTERDMAN, phoR::Tn and phoT::Tn were examined with two biological replicates. The conditions examined include: 1)WT, 2mM propionate acidic, pH 7.0; 2) WT, 2mM propionate acidic, pH 5.7, 3) phoR::TN, 2mM propionate, pH 7.0, 4) phoR::TN, 2mM propionate, pH 5.7; 5) WT, 4 mM lacate,  pH7.0, 6) WT, 4 mM lactate, pH 5.7; 7) phoT::Tn 4 mM lactate, pH 7.0, 8) phoT::Tn, 4 mM lacate, pH 5.7.   Following, a 3 day incubation, total bacterial RNA was extracted.

value: Bacterial Cell


title: Neutrophil, control, rep9

description: Library name: Sample 21

tag: genotype


title: SH-SY5Y pSG5-miR-129 2

tag: cell line

tag: Sex

tag: plasmid

tag: concentration

tag: time

value: 48 hr


title: E19Diaphragm_AGO2_PN1

description:
Library name: E19Diaphragm_AGO2_PN1
small RNA

tag: Stage


title: 293T 24h pSG5-miR-7 1

value: 24 hr


title: SUDHL4 - BMS158_1.25_CC282_50 - D2 - rep2

description: Library name: 7357-CH-0158

tag: treatment

tag: batch


title: Neutrophil, control, rep8

description: Library name: Sample 20

tag: cell type


title: SUDHL4 - BMS158_1.25 - D2 - rep3

description: Library name: 7357-CH-0153


title: SH-SY5Y pSG5-miR-133b 2


title: WT65034_1

city: Ribeirão Preto

first: Matheus

middle: Mertz

last: Ribeiro

state: São Paulo

address: Av. Prof. Dr. Zeferino Vaz - Vila Monte Alegre, Ribeirão Preto - SP, 14040-900

department: Faculty of Pharmaceutical Sciences of Ribeirão Preto

country: Brazil

institute: University of São Paulo

description:
Library name: WT65034_1
Column name in RNAseq_Macrophage.csv : WT65034

data_processing:
Data processing  Raw sequencing data generated from multiple sequencing lanes were concatenated for each biological sample prior to downstream analyses. Sequence quality was assessed using FastQC, and adapter sequences and low-quality bases were removed using fastp. Filtered paired-end reads were aligned to the appropriate reference genome using HISAT2. Fungal reads were aligned against the Aspergillus nidulans FGSC A4 (ASM1142v1) or Aspergillus fumigatus A1163 reference genomes, while host reads were aligned against the *Mus musculu* reference genome (GRCm39). Alignment files were sorted and indexed using SAMtools. Gene-level read counts were generated using featureCounts based on the corresponding genome annotations (GTF/GFF3). Differential gene expression analyses were performed using the DESeq2 package in R. Raw counts were normalized using the median-of-ratios method, and differential expression was assessed using the Wald test with Benjamini–Hochberg correction for multiple testing. Genes with an adjusted *P* value < 0.05 were considered significantly differentially expressed. Genome build:*Mus musculus GRCm39;Aspergillus nidulans FGSC A4 (ASM1142v1); Aspergillus fumigatus A1163.  Supplementary files format and content: Tab-delimited text files containing raw gene counts.
Assembly: GCA_000002655.1 (A. fumigatus Af293) - GCA_000001635.9 (Mus musculus GRCm39) -  GCA_000011425.1 (A. nidulans A4)
Supplementary files format and content: RNAseq_Macrophage.csv
Supplementary files format and content: RNAseq_fumigatus.csv
Supplementary files format and content: RNAseq_nidulans.csv

source_name: murine macrophages

extract_protocol:
Total RNAs of cultures of early dual biofilm interactions of 24 and 48 h were extracted by the TRIzol method, treated with RQ1 RNase-free DNase I (Promega), and purified using the RNAeasy kit (Qiagen) according to the manufacturer’s instructions. The total RNA was quantified using a NanoDrop, and its integrity was analyzed using an Agilent 2100 Bioanalyzer. All RNA had a minimum RNA integrity number value of 8.0. For RNA sequencing, the cDNA libraries were constructed using the TruSeq Total RNA with Ribo Zero (Illumina, San Diego, CA, USA).
mRNA library  preparation (poly A enrichment)

tag: sample group

value: murine macrophages

value: Macrophage WT + A. nidulans 65034

value: Coculture


title: Control rep1 - primary human pulmonary microvascular endothelial cells

city: jinan

first: yanan

last: li

state: 山东省

address: lixia

department: medicine

institute: Shandong University

description: Library name: A1

data_processing:
1) Raw sequencing data in FASTQ format were generated by CASAVA base calling. Quality scores are encoded in Qphred+33 format (Illumina 1.8+). 2) Quality control was performed using FastQC (v0.11.9) to assess base quality score distribution, GC content, adapter contamination, and duplicate levels. 3) Adapter trimming and low-quality read filtering (Q < 20) were performed using Trimmomatic (v0.39). 4) Clean reads were aligned to the human reference genome GRCh38 (hg38) using HISAT2 (v2.2.1). 5) Gene expression quantification was performed using featureCounts (v2.0.1) to generate raw read counts.
Assembly: GRCh38 (hg38)
Supplementary files format and content: Processed data files are provided as tab-delimited text files. Each file contains gene-level expression counts: first column is gene symbol/Ensembl ID, subsequent columns represent raw read counts per sample.

source_name: lung

growth_protocol: Primary human pulmonary microvascular endothelial cells (HPMVECs) were obtained from iCell Bioscience Inc, Shanghai. Cells were cultured in endothelial cell medium (ECM, Sciencell) supplemented with 5% fetal bovine serum (FBS, Sciencell), 1% penicillin/streptomycin, and 1% endothelial cell growth factor. Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂ and 95% air. Only cells at passages 3 to 5 and at approximately 80% confluence were used for subsequent experiments.

extract_protocol:
Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. RNA integrity and concentration were assessed using Agilent 2100 Bioanalyzer. Only samples with RNA integrity number (RIN) ≥ 7.0 were used for library construction.
Library construction was performed using total RNA. Polyadenylated mRNA was enriched using Oligo(dT) magnetic beads. The enriched mRNA was then fragmented using divalent cations in Fragmentation Buffer. First-strand cDNA was synthesized using random oligonucleotides as primers and M-MuLV reverse transcriptase. Second-strand cDNA synthesis was performed using RNaseH and DNA polymerase I. The double-stranded cDNA was purified, end-repaired, A-tailed, and ligated with sequencing adapters. AMPure XP beads were used to select cDNA fragments of 370-420 bp. The final library was amplified by PCR and purified again with AMPure XP beads. Library quality was assessed using Qubit 2.0 Fluorometer and Agilent 2100 Bioanalyzer, and library concentration was quantified by qRT-PCR (effective concentration > 1.5 nM).

treatment_protocol: Cells were divided into three groups: (A) Control group (no treatment), (B) Overexpression group (transduced with adenovirus carrying FKBP5 provided by Vigene Biosciences), and (C) Knockdown group (transfected with siFKBP5 targeting FKBP5 provided by Boshang Biotechnology). Transfection/transduction was performed according to the manufacturers' protocols. Cells were harvested 48 hours post-treatment for RNA extraction.

value: lung

value: primary cell

value: control


title: SUDHL4 - BMS158_1.25 - D2 - rep2

description: Library name: 7357-CH-0152


title: Secondary disease 4

city: Omaha

first: Kate

last: Hyde

state: NE

address: 985802 Nebraska Medical Center

institute: University of Nebraska Medical Center

description: Library name: SPrelapse4

data_processing:
The original fastq format reads were trimmed by Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) to remove adapters, terminal unknown bases (Ns) and low quality 3’ regions (Phred score < 20).
The trimmed fastq files were processed by FastQC (Andrews S. (2010). FastQC: a quality control tool for high throughput sequence data; available online at:http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and MultiQC for quality control (QC).
The QC passed trimmed fastq files were mapped to GRCm38 reference genome using STAR aligner and RSEM to quantifiy the mapped reads
Assembly: GRCm38
Supplementary files format and content: Tab-delimited text files include raw counts for each sample
Supplementary files format and content: Tab-delimited text file with raw gene counts for all genes and all samples

source_name: Spleen

extract_protocol:
For RNA extractions, after harvesting samples were homognised into single cells to break down the nucleoprotein complex using TRIZOL. They were treated briefly with chloroform and RNA percipitation was done to separate the aqueous phase using isopropanol. The RNA was washed couple of times with 75% ethanol. Once RNA pellet was seen, supernatant removed and pellet was air dried. The pellets were resuspended in RNAse free water. This was then store in -80C with 500ul Trizol reagent.
RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturer’s instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads.  Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).

value: Spleen

value: Leukemia cells

tag: transduction

value: CM cells transduced with shRNA

value: Doxycycline (Dox)


title: Neutrophil, control, rep7

description: Library name: Sample 19

value: Peripheral blood

value: Neutrophil

value: WT


title: 293T 48h pSG5-miR-7 2µg 3

value: Female


title: SH-SY5Y pSG5-miR-873 2


title: SUDHL4 - BMS158_1.25 - D2 - rep1

description: Library name: 7357-CH-0151

value: cell line


title: Secondary disease 3

description: Library name: SPrelapse3

treatment_protocol: For RNA extractions, after harvesting samples were homognised into single cells to break down the nucleoprotein complex using TRIZOL. They were treated briefly with chloroform and RNA percipitation was done to separate the aqueous phase using isopropanol. The RNA was washed couple of times with 75% ethanol. Once RNA pellet was seen, supernatant removed and pellet was air dried. The pellets were resuspended in RNAse free water. This was then store in -80C with 500ul Trizol reagent.


title: Neutrophil, control, rep6

description: Library name: Sample 18


title: SH-SY5Y pSG5-miR-873 1

value: 48 hr


title: SUDHL4 - DMSO - D2 - rep3

description: Library name: 7357-CH-0150

value: DMSO


title: SH-SY5Y pSG5-miR-133b 1


title: SUDHL4 - DMSO - D2 - rep2

description: Library name: 7357-CH-0149


title: Secondary disease 1

description: Library name: SPrelapse1


title: SH-SY5Y pSG5-miR-129 1


title: Neutrophil, control, rep4

description: Library name: Sample 16


title: WSUDLCL2 - BMS158_2.5_CC282_5 - D1 - rep3

description: Library name: 7357-CH-0129


title: Spleen dox sample 3

description: Library name: SPdox3


title: Neutrophil, control, rep2

description: Library name: Sample 14


title: SH-SY5Y pSG5-miR-7 1


title: Spleen dox sample 1

description: Library name: SPdox1


title: Neutrophil, control, rep1

description: Library name: Sample 13


title: WSUDLCL2 - BMS158_2.5_CC282_5 - D1 - rep2

description: Library name: 7357-CH-0128


title: SH-SY5Y pSG5 1


title: WSUDLCL2 - BMS158_2.5_CC282_5 - D1 - rep1

description: Library name: 7357-CH-0127


title: Spleen control sample 3

description: Library name: SPC3

value: CM expressing primary cells

value: Control (sucrose)


title: Neutrophil, BPD, rep12

description: Library name: Sample 12

source_name: Peripheral blood neutrophils isolated from infants with BPD at postnatal day 14, RNA-seq


title: WSUDLCL2 - BMS158_2.5 - D1 - rep3

description: Library name: 7357-CH-0126


title: HeLa pSG5 3

source_name: HeLa

value: HeLa


title: Spleen control sample 2

description: Library name: SPC2


title: Neutrophil, BPD, rep11

description: Library name: Sample 11


title: HeLa pSG5-miR-7 3


title: Spleen control sample 1

description: Library name: SPC1


title: HeLa pSG5-miR-34a 3


title: WSUDLCL2 - BMS158_2.5 - D1 - rep1

description: Library name: 7357-CH-0124


title: Neutrophil, BPD, rep10

description: Library name: Sample 10


title: Neutrophil, BPD, rep9

description: Library name: Sample 9


title: Bone marrow dox sample 3

description: Library name: BMdox3

source_name: Bone marrow

value: Bone marrow


title: HeLa pSG5-miR-129 3


title: WSUDLCL2 - BMS158_1.25_CC282_5 - D1 - rep3

description: Library name: 7357-CH-0123


title: Bone marrow dox sample 2

description: Library name: BMdox2


title: Neutrophil, BPD, rep8

description: Library name: Sample 8


title: HeLa pSG5-miR-133b 3


title: WSUDLCL2 - BMS158_1.25_CC282_5 - D1 - rep2

description: Library name: 7357-CH-0122


title: Bone marrow dox sample 1

description: Library name: BMdox1


title: Neutrophil, BPD, rep7

description: Library name: Sample 7


title: WSUDLCL2 - BMS158_1.25_CC282_5 - D1 - rep1

description: Library name: 7357-CH-0121


title: HeLa pSG5-miR-873 3


title: WSUDLCL2 - CC282_5 - D1 - rep3

description: Library name: 7357-CH-0120


title: Bone marrow control sample 2

description: Library name: BMC2

value: CBFB::MYH11 (CM) expressing primary cells


title: Neutrophil, BPD, rep5

description: Library name: Sample 5


title: HeLa pSG5-miR-7 2


title: WSUDLCL2 - CC282_5 - D1 - rep2

description: Library name: 7357-CH-0119


title: Bone marrow control sample 1

description: Library name: BMC1


title: Neutrophil, BPD, rep4

description: Library name: Sample 4


title: WSUDLCL2 - CC282_5 - D1 - rep1

description: Library name: 7357-CH-0118


title: Mouse, scRNA-seq, TKT knockout 2

description: Library name: Mus-sc-TKO2

source_name: Mouse prostate cancer tissue

value: Mouse prostate cancer tissue

value: PtenPC-/- TktPC-/-


title: Neutrophil, BPD, rep3

description: Library name: Sample 3


title: Bone marrow control sample 3

description: Library name: BMC3

value: Leukemia cells


title: HeLa pSG5-miR-129 2


title: WSUDLCL2 - BMS158_1.25 - D1 - rep3

description: Library name: 7357-CH-0117


title: Neutrophil, BPD, rep6

description: Library name: Sample 6


title: E19Diaphragm_AGO2_PN3

description:
Library name: E19Diaphragm_AGO2_PN3
small RNA

value: Diaphragm

tag: background

value: C57BL/6J and C57BL/6N


title: HeLa pSG5 2


title: Mouse, scRNA-seq, TKT knockout 1

description: Library name: Mus-sc-TKO1


title: Neutrophil, BPD, rep2

description: Library name: Sample 2

value: Peripheral blood


title: HeLa pSG5-miR-133b 2


title: WSUDLCL2 - BMS158_1.25 - D1 - rep2

description: Library name: 7357-CH-0116


title: Mouse, scRNA-seq, wild type 2

description: Library name: Mus-sc-WT2

value: PtenPC-/-


title: Neutrophil, BPD, rep1

description: Library name: Sample 1

value: Neutrophil

value: WT


title: HeLa pSG5-miR-873 2

value: Female


title: WSUDLCL2 - BMS158_1.25 - D1 - rep1

description: Library name: 7357-CH-0115

value: cell line


title: Mouse, scRNA-seq, wild type 1

description: Library name: Mus-sc-WT1


title: Mock infected, PMNs, no acyclovir, rep 2

city: Ithaca

first: Joel

middle: D

last: Baines

address: 235 Hungerfordhill Rd

institute: Cornell University

description:
Library name: Mock_2
the samples were run paired end, but only the L1_1 was processed to match the quiescent samples which were all run as single-end

data_processing:
All PRO-seq data was probed and quantified using the SeqMonk Software (70, 71) and the .bam files from the PRO-seq 2.0 alignment. The Hep2 data sets were normalized with both cellular RPM values and Drosophila spike-in values as we and others previously demonstrated that cellular reads fluctuate as HSV-1 infection progresses in epithelial and fibroblast cells (67, 72). Viral read data from the PMN infections were normalized to the cellular RPM amounts, as these values were consistent over all time points, acute and quiescent.  They also showed more consistency than the Drosophila spike-in, likely because we reduced the amount of spike-in used for the PMN infections to increase the sequencing depth for viral reads.  We were initially thinking that viral reads would be extremely low which, in retrospect, was not necessary and along with human error could have been source of the unnecessary variation in the spike-in.  For the cellular RPM normalization, the total human PRO-seq reads for each sample were divided by 1,000,000 and then used to determine the scaling factor for the data.  The data from each sample was scaled down to match the sample with the lowest cellular reads before dividing by the probe length for the density calculations.
Assembly: hg38 concatenated to the HSV-1 17 syn+ and drosophila dm3 genomes available here: https://github.com/Baines-Lab/Public/tree/main/HSV-1
Supplementary files format and content: .bam and .bw files from the Pro_seq 2.0 alignment https://github.com/Danko-Lab/proseq2.0
Library strategy: PRO-Seq

molecule: nuclear RNA

source_name: LUHMEs

growth_protocol: LUHMES cells were purchased from ATCC (catalog no. CRL-2927) and cultured as previously described. Briefly, 10cm cell culture dishes and T75 flasks were coated with poly-l-ornithine hydrobromide (Sigma, catalog no. P3655) followed by fibronectin (Sigma, catalog no. F2006), rinsed with water, and allowed to fully air dry before cell plating. LUHMES cells were maintained in proliferation media (DMEM : F12, ATCC, catalog no. 30-2006) containing 1% N2 supplement (ThermoFisher Sci., catalog no. A1370701), 1X Penicillin-Streptomycin-Glutamine solution (ThermoFisher Sci., catalog no. 10378016) and recombinant human FGF-basic (Fibroblast Growth Factor, Peprotech, catalog no. 100-18B; reconstituted at 100 μg/mL in 10mM Tris pH 7.6 plus 0.1% BSA) at 40ng/mL final concentration. To induce cellular differentiation, LUHMES were cultured in DMEM : F12 plus N2 supplement media containing 1 μg/mL tetracycline hydrochloride (Sigma catalog no. T7660), 1mM N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (Sigma, catalog no. D0627) and 2ng/mL recombinant human GDNF (Glial cell-derived neurotrophic factor; R&D Systems, catalog no. 212-GD-010). All experiments were conducted with LUHMES neuronal cells following 7 days post-induction of differentiation.

extract_protocol:
Nuclie were isolated as previously described (Birkenheuer, CH 2020)
Frozen nuclei were brought to 37℃, spiked with Drosophila S2 nuclei at a ratio of 1 / 1000 Drosophila / Human nuclei, and pre-warmed run-on reaction buffer (10 mM Tris-HCl [pH8], 300 mM KCl, 1% Sarkosyl, 5mM MgCl2, 1mM DTT, 100 μM biotin-11-ATP, -CTP, -GTP, and -UTP [Perkin-Elmer], and 0.4 U / μL) was added to each sample.  Reactions were incubated at 37℃ with constant shaking for 3 minutes. Reactions were stopped by adding 1.8 mL of Trizol LS (Thermo Fisher). RNA was isolated by adding 520 μL chloroform to each resuspended sample. Incubation times and centrifugation times were performed following the manufacturer’s guidelines. RNA was precipitated with a 2:1 volume of ethanol and the pellets were washed and stored in 700 μL of 75% ethanol before sequencing library preparation. PRO-seq library preparation The biotinylated RNA was resuspended in nuclease free water and hydrolyzed with 0.2N NaOH in a final volume of 30 μL. After 20 min, the NaOH was neutralized with the addition of 1M Tris-HCl (pH 6.8), and samples were run through a Bio-Rad P-30 column to remove any unincorporated biotinylated nucleotides. Biotinylated RNA was isolated using streptavidin M280 Dynabeads (Thermo Fisher) following previously published protocols.  The 3’RNA adapter (5’Phos-GAUCGUCGGACUGUAGAACUCUGAAC-3’InvdT) was ligated to the RNA using 10 U/μL of T4 RNA ligase I (New England BioLabs [NEB]) as described. After an additional round of M280-Streptavidin bead binding and washing, RNA 5’-pyrophosphohydorlase (RPPH) (NEB) was used to remove any 5’ caps on the biotinylated RNA. After cap removal, T4 polynucleotide kinase (PNK) (NEB) in its associated reaction buffer was added directly to the RPPH mix to repair the 5’-hydroxyl group. Trizol purified RNA was ligated to the 5’adapter (5’-CCUUGGCACCCGAGAAUUCCA-3’). RNA was reverse transcribed and PCR amplified using barcoded sequencing primers  for an experimentally determined number of cycles for each library preparation as previously described. Libraries were run through an 8% polyacrylamide gel, and fragments 140 nt and larger were extracted from the gel and submitted for sequencing.The barcoded libraries from the acute infections were pair-end sequenced on the NovaSeq X Plus PE150 platform.  For comparison to the quiescent infections, the paired end data from the acute infections was analyzed as the single-end data.  Sequences were aligned to a concatenated index file containing the hg38 human genome, the dm3 Drosophila melanogaster genome, and the HSV-1 strain 17 genome (NC_001806.2) with masked terminal repeats (https://github.com/Baines-Lab/Public/tree/main/HSV-1) using the Danko PRO-seq2.0 pipeline (https://github.com/Danko-Lab/proseq2.0) (68). This algorithm uses cut adapt to trim the adapter sequences and bwa to perform the alignment, it then converts the .BAM file into a bigwig (.bw) that reports the read counts only for the 5’ most nucleotide in the read, this in combination with the high concentration of biotinylated nucleotides used in the run on provides nucleotide resolution

treatment_protocol: LUHMEs were plated at 40,000 cells/cm2 onto coated dishes and flasks and cells were allowed to proliferate for two days at which time they were switched into differentiation media. At 6 days post-differentiation (8 days post plating), PMNs for the quiescence experiments were pre-treated with 50µM Acyclovir (ACV).   All infections both acute and quiescent were performed with HSV-1 17syn+ at a MOI equal to 3 on day 10 post plating (8 days post differentiation). The PMNs slated for quiescence were cultured in the presence of ACV for 48 hr after infection, at which time the media was changed without further exposure to ACV. Nuclei were prepared from acutely infected, non-acyclovir treated PMNs at 1, 3, 6 and 24 hr post infection, and the quiesently infected PMNs were collected at 10 days post-infection (8 days post acyclovir removal) There were five independent experiments for the quiescent time points, and two independent experiments for each of the acute infection time points.  Each experiment consisted of five T75 flasks with an approximate number of neurons per experiment totaling 3-3.5x108.

value: LUHMEs

value: post-mitotic neurons derived from myc inhibition by tetracyclin

value: infected with HSV-1 strain 17 syn + at an MOI of 3


title: HeLa pSG5-miR-873 1

value: HeLa

value: 48 hr


title: WSUDLCL2 - DMSO - D1 - rep3

description: Library name: 7357-CH-0114

value: DMSO


title: HeLa pSG5-miR-34a 2


title: Bulk RNA-seq of primary endothelial cells STIM-3

description:
Library name: STIM_3
RNA-seq

data_processing:
Reads were trimmed with fastp
Reads were aligned to the GRCm39 genome with hisat2
featureCounts was used to quantify read counts overlapping each gene
DESeq2 was used to analyze differential genes
Assembly: GRCm39
Supplementary files format and content: counts matrix

source_name: Aorta

growth_protocol: 3-month-old C57BL/6 mice were anesthetized with a ketamine/xylazine injection and perfused via the left ventricle with 1ml of room-temperature PBS containing heparin (1,000 U/ml), immediately followed by perfusion with 5ml of ice-cold PBS to remove residual blood cells. The thoracic aorta was dissected using micro-dissection tools and transferred to a Petri dish containing ice-cold PBS. Residual adipose tissue was carefully removed with surgical forceps, and the aorta was cut into 3–4 segments of approximately 1 cm each. Segments were then opened longitudinally using micro scissors and placed lumen-side down in a 6-well plate pre-coated with Matrigel (Corning). The aorta segments were immediately incubated at 37°C with 5% CO2 in 200µl of Endothelial Cell Medium (Cell Biologics, M1168), prepared according to the manufacturer’s instructions. After 4 hours, 2ml of additional medium was added to each well. After 4-5 days, the sections were carefully removed from the Matrigel, and adherent endothelial cells were left to expand for an additional 4-5 days. Cells were then dissociated from the Matrigel using Cell Recovery Solution (Corning), re-plated in a 24-well plate coated with 0.1% gelatin and expanded for 2 days, as described above.

extract_protocol:
Cells were lysed in 450µl of lysis buffer for RNA extraction. The lysate was mixed by pipetting and briefly shaken for 1 minute at room temperature. Lysates were collected, and RNA was purified using the RNeasy Micro Kit (Qiagen 74004), following the manufacturer’s protocol. Purified RNA was stored at -80°C until analysis.
Library preparation was performed with 10ng of total RNA per sample. ds-cDNA was made using the low-input SMARTseq mRNA kit (Takarabio) according to manufacturer’s protocol. cDNA was fragmented using a Covaris E220 sonicator using peak incident power 18, duty factor 20%, cycles per burst 50 for 120 seconds. 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 15 cycles using primers incorporating unique dual index tags. Libraries were pooled and sequenced on an Illumina NovaSeqX Plus (2x150 sequencing recipe), targeting 50M reads per sample. Basecalls and demultiplexing were performed with Illumina’s bcl2fastq software with a maximum of one mismatch in the indexing read.

value: Aorta

value: Endothelial cells

value: Wild-type

value: IGF-1 + GAS6


title: Mock infected, PMNs, no acyclovir, rep1

description:
Library name: Mock_1
the samples were run paired end, but only the L1_1 was processed to match the quiescent samples which were all run as single-end


title: WSUDLCL2 - DMSO - D1 - rep2

description: Library name: 7357-CH-0113


title: HeLa pSG5-miR-133b 1


title: Bulk RNA-seq of primary endothelial cells STIM-2

description:
Library name: STIM_2
RNA-seq


title: 24hr acute infected, PMNs, no acyclovir, rep 2

description:
Library name: Inf_24hr_2
the samples were run paired end, but only the L1_1 was processed to match the quiescent samples which were all run as single-end


title: Neutrophil, control, rep5

description: Library name: Sample 17


title: HeLa pSG5-miR-129 1


title: WSUDLCL2 - DMSO - D1 - rep1

description: Library name: 7357-CH-0112


title: Bulk RNA-seq of primary endothelial cells STIM-1

description:
Library name: STIM_1
RNA-seq


title: 24hr acute infected, PMNs, no acyclovir, rep 1

description:
Library name: Inf_24hr_1
the samples were run paired end, but only the L1_1 was processed to match the quiescent samples which were all run as single-end


title: HeLa pSG5-miR-34a 1


title: SUDHL4 - DMSO - D2 - rep1

description: Library name: 7357-CH-0148


title: SUDHL4 - BMS158_2.5_CC282_50 - D1 - rep3

description: Library name: 7357-CH-0111


title: SH-SY5Y pSG5-miR-34a 1


title: Neutrophil, control, rep3

description: Library name: Sample 15


title: Bulk RNA-seq of primary endothelial cells CTRL-3

description:
Library name: CTRL_3
RNA-seq

value: PBS


title: 6hr actue infected, PMNs, no acyclovir, rep 2

description:
Library name: Inf_6hr_2
the samples were run paired end, but only the L1_1 was processed to match the quiescent samples which were all run as single-end


title: HeLa pSG5-miR-7 1


title: SUDHL4 - BMS158_2.5_CC282_50 - D1 - rep2

description: Library name: 7357-CH-0110


title: Bulk RNA-seq of primary endothelial cells CTRL-2

description:
Library name: CTRL_2
RNA-seq


title: 6hr actue infected, PMNs, no acyclovir, rep 1

description:
Library name: Inf_6hr_1
the samples were run paired end, but only the L1_1 was processed to match the quiescent samples which were all run as single-end


title: Spleen dox sample 2

description: Library name: SPdox2

value: Spleen

value: Leukemia cells

value: CM cells transduced with shRNA

value: Doxycycline (Dox)


title: HeLa pSG5 1

value: Female


title: SUDHL4 - BMS158_2.5_CC282_50 - D1 - rep1

description: Library name: 7357-CH-0109

value: cell line


title: HEK293T pSG5 3


title: 3hr acute infected, PMNs, no acyclovir, rep 2

description:
Library name: Inf_3hr_2
the samples were run paired end, but only the L1_1 was processed to match the quiescent samples which were all run as single-end

value: LUHMEs


title: SUDHL4 - BMS158_2.5 - D1 - rep3

description: Library name: 7357-CH-0108


title: Maf-flox : Cribb1-Cre brain macrophages (pool of two samples stained with TotalSeq-B Hashtag reagent B0301 or B0302) ADT

description:
Library name: KO_ADT
10x Genomics

library_source: other

data_processing:
FASTQ files were trimmed, filtered, and aligned using the cellranger count pipeline from 10X genomics. Antibody hashtags and GEX were quantified together using the feature barcode workflow.
Filtered counts matrices containing transcript counts (GEX) and antibody hashtag counts (ADT) for each single cell were imported into Seurat for downstream processing.
Hashtags were used to assign each cell to a unique replicate sample.
Cells were clustered and visualized using Seurat.
Assembly: GRCm39
Supplementary files format and content: Filtered cell counts matrix generated by 10X cellranger count pipeline for GEX and ADT counts.
Library strategy: CITE-seq

molecule: other

source_name: Brain

extract_protocol:
Brain immune cells were isolated as described in the respective method section. The entire sample preparation protocol was carried out either on ice, or at 4°C. Briefly, after perfusion in ice-cold PBS, brain cortices were dissected and mechanically homogenized. Cell suspension was filtered through a 70 μm strainer and pellets were resuspended in 30% isotonic percoll for myelin depletion (centrifugation at 800g for 20 min). Suspension of brain cells was stained with the TotalSeq-B anti-mouse Hashtag reagent (Biolegend, B0301 and B0302) at 1:200 dilution in Fc block prior to adding the staining mix with fluorophore conjugated antibodies. To sort brain macrophages, cells were stained with CD45.2-PE (1:200), CD11b-APC (1:100), and lineage exclusion mix (Ly6C-FITC 1:200, Ly6G-FITC 1:200, CD43-FITC 1:100, CD44-FITC 1:100, NK1.1-FITC 1:100) for 30 min (all antibodies from Biolegend). Anti-CD45.2-PE was used to avoid competition with the anti-CD45 hashtag. For live/dead discrimination, a 1 mg/ml of DAPI (Sigma) at 1:5000 final dilution was added to the sample immediately prior to sorting. Sorting was performed on FACS Aria-II (BD Bioscience) using a default 85µm nozzle. Brain macrophages from two mice per genotype (stained with either B0301 or B0302 reagents) were sorted in one collection tube containing 400µl of PBS + 0.4% UltraPure BSA (Thermo Fisher) + 1U/µl of Recombinant RNasin Ribonuclease Inhibitor (Promega). Sorted cells were washed in PBS and resuspended into PBS + 0.04% UltraPure BSA buffer at a concentration of approximately 1,000 cells/µl.
Single-cell libraries were prepared using the Chromium Next GEM Single Cell 3' HT (high throughput) v3.1 protocol in super-loading mode, targeting 40,000 cells/sample. Briefly, cDNA was prepared after the GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps. Purified cDNA was amplified for 11-13 cycles before being cleaned up using SPRIselect beads, and cDNA concentration was determined on the Bioanalyzer. GEX libraries were prepared as recommended by the Chromium Next GEM Single Cell 3' HT Reagent Kits v3.1 (Dual Index) (CG000416) with appropriate modifications to the PCR cycles based on the calculated cDNA concentration.  For sample preparation on the 10x Genomics platform, the Chromium Next GEM Single Cell 3' HT Kit v3.1 - 48 rxns (PN-1000348), Chromium Next GEM Chip M Single Cell Kit - 16 rxns (PN-1000371) and Dual Index Kit TT Set A, 96 rxns (PN-1000215) were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer’s protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeq X Plus instrument.  Normalized libraries were sequenced on a NovaSeq X Plus Flow Cell using the 151x10x10x151 sequencing recipe according to manufacturer protocol.  Read 1 was trimmed to the 10x Genomics recommendation of 28bp. A median sequencing depth of 50,000 reads/cell was targeted for each gene expression library.

value: Brain

value: Macrophages

value: Maf-flox :Cribb1-Cre


title: 3hr acute infected, PMNs, no acyclovir, rep 1

description:
Library name: Inf_3hr_1
the samples were run paired end, but only the L1_1 was processed to match the quiescent samples which were all run as single-end

value: post-mitotic neurons derived from myc inhibition by tetracyclin

value: infected with HSV-1 strain 17 syn + at an MOI of 3


title: SUDHL4 - BMS158_2.5 - D1 - rep2

description: Library name: 7357-CH-0107

source_name: cell line


title: HEK293T pSG5-miR-7 3

value: 48 hr


title: Maf-flox : Cribb1-Cre brain macrophages (pool of two samples stained with TotalSeq-B Hashtag reagent B0301 or B0302) GEX

description:
Library name: KO_GEX
10x Genomics


title: HEK293T pSG5-miR-34a 3


title: 1hr acute infected, PMNs, no acyclovir, rep2

description:
Library name: Inf_1hr_2
the samples were run paired end, but only the L1_1 was processed to match the quiescent samples which were all run as single-end


title: SUDHL4 - BMS158_2.5 - D1 - rep1

description: Library name: 7357-CH-0106


title: Maf-flox brain macrophages (pool of two samples stained with TotalSeq-B Hashtag reagent B0301 or B0302) ADT

description:
Library name: WT_ADT
10x Genomics

value: Maf-flox


title: HEK293T pSG5-miR-129 3


title: SUDHL4 - BMS158_1.25_CC282_50 - D1 - rep3

description: Library name: 7357-CH-0105


title: Maf-flox brain macrophages (pool of two samples stained with TotalSeq-B Hashtag reagent B0301 or B0302) GEX

description:
Library name: WT_GEX
10x Genomics


title: 1hr acute infected, PMNs, no acyclovir, rep1

description:
Library name: Inf_1hr_1
the samples were run paired end, but only the L1_1 was processed to match the quiescent samples which were all run as single-end


title: MW32

city: Little Rock

first: ryan

last: allen

state: Arkansas

address: 4301 W. Markham St

department: Physiology and Cell Biology

institute: University of Arkansas for Medical Sciences

description:
Library name: MW32_S32_L001_R1_001.fastq.gz
DMW_30

data_processing:
Bioinformatic processing was performed using the TIGER data analysis pipeline (v20220205), previously described in Allen and Zhao et al. 2018 (Journal of Extracellular Vesicles).
Cutadapt v4.5
Alignment with Bowtie1
spcount (https://github.com/shengqh/spcount)
Assembly: GENCODE GRCm38.p6
Supplementary files format and content: .txt files are all normalized by reads per million (RPM) of total reads

source_name: Biofluid

growth_protocol: Mice were transduced with AAV-GFP or AAV-PCSK9-D377Y and fed a chow-diet or western-diet, respectively, for 14 weeks.

extract_protocol:
Plasma was fractionated by size exclusion chromatography on two Superose-6 Increase columns in tandem. Fractions corresponding with APOB were pooled concentrated by ultrafiltration to ~100 uL. Total RNA was then extracted using Trizol LS and purified with Zymo RNA Clean and Concentrate spin columns.
Approximately 15% of the extracted total RNA was aliquoted for small RNA sequencing using the randomized splint ligation method previously developed by New England Biolabs, Inc54. Briefly, 7 microliters of isolated RNA were ligated to 0.25 pmol of annealed 3’ splint adapter using 200 units of T4 RNA Ligase, truncated KQ (New England Biolabs). The ligation reaction was then treated with 2.5U of lambda exonuclease (New England Biolabs) and 25U of 5’ deadenylase (New England Biolabs), followed by 5U of Uracil-DNA Glycosylase (New England Biolabs) and 20U of Endonuclease IV (New England Biolabs). Ligation of 0.5 pmol of 5’ splinted adapter was performed using 20U of truncated T4 RNA Ligase 2 (New England Biolabs). Reverse transcription of adapter-ligated RNA was performed using Protoscript II (New England Biolabs), and cDNA were purified using CleanNGS DNA Cleanup Magnetic Beads (Bulldog Bio) according to manufacturer instructions. Individual libraries were then amplified by PCR using NEB Next 2X High-Fidelity PCR Master mix (New England Biolabs). Following PCR amplification, individual libraries were size-selected (136-196 bp product) using pre-cast 3% agarose gel cassettes with a Pippin Prep (Sage Sciences) and quantified on a Qubit 4 Fluorometer (Invitrogen) with the Qubit 1X dsDNA HS Assay (Invitrogen).

treatment_protocol: Mice received a surgical unicortical defect in the right femur that was inoculated with PBS (Sham) or 1x10^6 CFU of USA300 LAC methicillin-resistant Staphylococcus aureus (MRSA)

value: Biofluid

value: LDL

value: lipoprotein

value: AAV-PCSK9/Western Diet

value: MRSA


title: HEK293T pSG5-miR-133b 3

data_processing:
Sample primary processing was performed with miRMaster (Fehlmann et al., Nucleic Acids Res, 2021) using standard parameters. Mapping to the genome was performed allowing up to 5 mulit-mapping hits with Bowtie. One mismatch was allowed for the quantification of isomiRs.  As output, miRMaster generated a list with the expression of 80,668 RNAs from 10 RNA classes. The data were normalized to expression in one million reads and further processed with R (R 4.0.4 GUI 1.74 Catalina build (7936)). To limit the effect of low-abundance miRNAs, we considered miRNA isoforms as expressed if at least five in 10 million mapped miRNA reads were found for at least three independent samples.
Supplementary files format and content: Reads per million mapped miRNA normalzed counts for each sample and human miRNA from miRBase v22.

extract_protocol:
0, 4, 8, 24, 48 h after transfections for miR-7 and 48 h for miRNAs miR-34a, -129, -133b, 873, cells were lysed by QIAzol Lysis Reagent (Qiagen, Hilden, Germany), and total RNA was isolated using the miRNeasy Mini Kit (Qiagen, Hilden, Germany).
Library preparation for small RNA sequencing was performed using the MGIEasy Small RNA Library Prep Kit according to the manufacturer’s recommendations. In detail, after 3’ and 5’ adapter ligation, RNAs were reverse transcribed and PCR amplified (21 cycles). PCR products were purified using 6% TBE gels, excising only the 110 bp band corresponding to the PCR product of miRNAs. Subsequently, twelve samples were pooled equimolar into one library, and the library was further circularized and purified. Circularized libraries were sent to BGI Hong Kong for sequencing using the standard MPS protocol as described previously (Li et al., Nucleic Acids Res, 2021).


title: MW31

description:
Library name: MW31_S31_L001_R1_001.fastq.gz
DMW_29


title: HEK293T pSG5-miR-873 3


title: Maf-flox : Crybb1- Cre CD45 negative cells 2

description:
Library name: KO2
10x Genomics

data_processing:
FASTQ files were trimmed, filtered, and aligned using the cellranger count pipeline from 10X genomics.
Filtered counts matrices containing transcript counts for each single cell were imported into Seurat for downstream processing.
Cells were clustered and visualized using Seurat.
Assembly: GRCm39
Supplementary files format and content: Filtered cell counts matrix generated by 10X cellranger count pipeline.

extract_protocol:
To sort brain blood vessels, cortices were homogenized with GentleMACS tissue dissociator (Mylteni Biotech) in prewarmed DMEM supplemented with 2% Bovine Calf Serum (BCS, HyClone), 25µg/ml Liberase DL, 100µg/ml Dispase, 0.5mg/ml Papain, 10µg/ml DNase-I (all enzymes from Sigma) for 30 min at 37°C. After homogenization the cell suspension was filtered through a 70 μm strainer and myelin depletion was performed in 30% isotonic percoll (800g for 20 min at 4°C). Cell pellets were stained with Fc block, CD45-PE (1:200), CD31-AlexaFluor 647 (1:100), and Ly6C-APCCy7 (1:200) for 30 min on ice (all antibodies from Biolegend). For live/dead discrimination, a 1 mg/ml of DAPI (Sigma) at 1:5000 final dilution was added to the sample immediately prior to sorting. The CD45/DAPI double-negative population was sorted on FACS Aria-II (BD Bioscience) using a 100µm nozzle. Cells were collected into a 1.5ml Eppendorf tube containing 400µl of PBS + 0.4% UltraPure BSA (Thermo Fisher) + 1U/µl of Recombinant RNasin Ribonuclease Inhibitor (Promega). Sorted cells were washed in PBS and resuspended into PBS + 0.04% UltraPure BSA buffer at a concentration of approximately 1,000 cells/µl.
Single-cell libraries were prepared using the Chromium GEM-X Single Cell 3' v4 protocol, targeting 20,000 cells/sample. Briefly, cDNA was prepared after the GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps. Purified cDNA was amplified for 11-13 cycles before being cleaned up using SPRIselect beads, and cDNA concentration was determined on the Bioanalyzer. GEX libraries were prepared as recommended by the Chromium GEM-X - Single Cell 3' Reagent Kits v4 (CG000731) with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform, the Chromium GEM-X Single Cell 3' Kit v4 - 16 rxns (PN-1000691), Chromium GEM-X Single Cell 3' Chip Kit v4 - 4 chips (PN-1000690) and Dual Index Kit TT Set A, 96 rxns (PN-1000215) were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer’s protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeq X Plus instrument. Normalized libraries were sequenced on a NovaSeq X Plus Flow Cell using the 151x10x10x151 sequencing recipe according to manufacturer protocol. Read 1 was trimmed to the 10x Genomics recommendation of 28bp. A median sequencing depth of 50,000 reads/cell was targeted for each gene expression library.

value: CD45 negative


title: SUDHL4 - BMS158_1.25_CC282_50 - D1 - rep2

description: Library name: 7357-CH-0104

data_processing:
We used featureCounts 2.0.6 to count the number of reads on gene level.
Assembly: GRCh38
Supplementary files format and content: readcount on gene level

extract_protocol:
Cells for RNAseq were prepared according to instructions from Discovery Life Sciences.
Five million cells per sample were collected, washed, fixed, and stored at -80 C prior to shipping to Discovery Life Sciences where downstream RNAseq was carried out.


title: WSUDLCL2 - BMS158_2.5 - D1 - rep2

description: Library name: 7357-CH-0125


title: Maf-flox : Crybb1- Cre CD45 negative cells 1

description:
Library name: KO1
10x Genomics


title: SUDHL4 - BMS158_1.25_CC282_50 - D1 - rep1

description: Library name: 7357-CH-0103


title: MW30

description:
Library name: MW30_S30_L001_R1_001.fastq.gz
DMW_28


title: Maf-flox CD45 negative cells 2

description:
Library name: WT2
10x Genomics


title: SUDHL4 - CC282_50 - D1 - rep3

description: Library name: 7357-CH-0102


title: MW29

description:
Library name: MW29_S29_L001_R1_001.fastq.gz
DMW_27


title: HEK293T pSG5 2


title: HEK293T pSG5-miR-7 2


title: Maf-flox CD45 negative cells 1

description:
Library name: WT1
10x Genomics


title: SUDHL4 - CC282_50 - D1 - rep2

description: Library name: 7357-CH-0101


title: MW28

description:
Library name: MW28_S28_L001_R1_001.fastq.gz
DMW_26


title: SUDHL4 - CC282_50 - D1 - rep1

description: Library name: 7357-CH-0100


title: HEK293T pSG5-miR-34a 2


title: MW27

description:
Library name: MW27_S27_L001_R1_001.fastq.gz
DMW_25