title: CROP-seq, transcription factor derived iPSC-microglia (iTF-MG), sgRNA enrichment

city: San Francisco

first: Martin

last: Kampmann

address: 675 Nelson Rising Lane

institute: University of California, San Francisco

description:
Library name: iTF_crop-sgRNA
iTF_processed_merged_crop.h5ad

data_processing:
Single-cell RNA Sequence Alignment The raw single-cell sequencing reads of gene expression libraries were processed using kb-python (version: v0.29.1) from kallisto-bustools133. First, using the GRCh38 human reference transcriptome with Ensembl annotations (release 113), a kallisto index was generated with the default k-mer size (k=31). The 10x reads were then pseudo-aligned to this index using ‘kb count’ from kallisto-bustools with the ‘standard’ workflow and the cell barcode whitelist for Single Cell 3’ v3.1 chemistry.  Single-cell ADT Protein Assignment The raw single-cell sequencing reads of antibody-derived tag (ADT) libraries were processed using the Cell Ranger software from 10x Genomics (version 9.0.0). The computational analysis was conducted on a high-performance computing (HPC) cluster configured with 48 GB of virtual memory and eight local cores. A custom feature reference file83 was used for detecting unique molecular identifiers (UMIs) associated with antibody-derived tags.  CRISPRi knockdown demultiplexing (sgRNA assignment) The raw sequencing reads of the PCR amplified sgRNA library were processed using kallisto-bustools133. First, using ‘kb ref’, a kallisto index of the sgRNA library was generated with k-mer size of 9. The reads were then pseudo-aligned to this index using ‘kb count’ from kallisto-bustools with the ‘standard’ workflow and the cell barcode whitelist for Single Cell 3’ v3.1 chemistry. sgRNA assignment was then conducted using geomux (version: v0.2.5)132, which conducts a hypergeometric test to calculate the significance of observing each sgRNA in each barcode. Cells were assigned to the majority sgRNA if they had a log-odds ratio above 1, Benjamini-Hochberg corrected P-value below 0.05, minimum UMI count to consider a barcode was 3, and minimum number of barcodes to consider a sgRNA was the default, 100. Once cells were assigned to a sgRNA, the barcodes were used to intersect the gene expression and ADT count matrices to store both the RNA and protein expression counts for each sgRNA-assigned cell in MuData format from muon (version: v0.1.7)134. Cells with no barcodes or multiple barcodes were not included for further analysis. Both the iTF-MG and iMG samples were processed this way. Of the two biological replicates from iTF-MG samples, only one includes the CITE-seq library and was included for this additional analysis. All QC was conducted using scanpy (version: v1.11.3)135. For both, the iTF-MG and iMG dataset with RNA and ADT counts, cells were filtered to include those with a minimum count of 200, and a mitochondrial count of less than or equal to 8%. Genes were filtered to include those with minimum cells of 10. For the iTF-MG experiment with only RNA counts, cells were filtered to include those with a minimum count of 1000. All other QC was conducted in the same way as the other datasets. For protein expression levels, four different normalization methods were initially tested and log1p-normalize was chosen as it had the highest median correlation to RNA expression as described in Haage et al. 202583. Thus, for both RNA and protein expression levels, cells in each dataset were log1p-normalized to a target sum of 10,000. The top 5,000 highly variable genes were selected to conduct principal component analysis (PCA). Nearest neighbors were then calculated using the default 15 nearest neighbors, to then build UMAPs. Clustering was performed using different resolutions of the Leiden (version: v0.10.2)136 algorithm, and after setting the resolution to 1. Clusters with low AIF1 expression (non-microglia cells) and low total counts were removed. 6 out of 18 clusters were removed from the iTF-MG replicate with only RNA data, whereas only 2 out of 13 clusters were removed from the iTF-MG replicate with both RNA and ADT data. Similarly, only 2 out of 9 clusters were removed from the iMG dataset. The two iTF-MG replicates were then concatenated and batch corrected using Harmony (version: v0.0.10)137, to get 3 final datasets used in analysis the iTF-MG and iMG MuData with both RNA and ADT counts, and the merged iTF-MG AnnData with the batch-corrected RNA counts. Two sgRNAs, FOXK1 sgRNA 2 (FOXK1_g2) and non-targeting control sgRNA 5 (non-targeting_g5) were also removed from analysis due to functional validation of FOXK1_g2 not producing knockdown (Figure S3H), and rigorous testing of non-targeting_g5 showing a perturbation effect through the Mixscale87 analysis described below. After QC, the resulting iTF-MG had 23,826 cells with RNA expression for 16,398 genes, and 9,040 cells with ADT expression for 180 proteins (166 proteins + 14 antibody controls). The iMG had 8,247 cells with RNA expression for 13,939 genes, and 8,247 cells with ADT expression for 180 proteins with 14 of those proteins included in the panel as controls.
Assembly: hg38
Supplementary files format and content: iTF_processed_cite_crop.h5mu
Supplementary files format and content: iMG_processed_cite_crop.h5mu
Supplementary files format and content: iTF_processed_merged_crop.h5ad
Library strategy: CRISPR Screen

growth_protocol: iMG: iPSCs were passaged in small colonies ~50-100 cells onto Matrigel-coated plates using ReLeSR (Stemcell Technologies, 100-0483) at a density suitable to achieve ~40 colonies per well the next day. Wells with the correct size and density of iPSCs were differentiated to hematopoietic progenitor cells (HPCs) using StemDiff Hematopoietic kit (Stemcell Technologies, 05310). HPCs were collected on days 10, 12, and 14 and frozen in BamBanker (Bulldog Bio, BB05). To generate iMG, HPCs were thawed at 7-10K cells/cm2 into microglia differentiation media: DMEM/F12, 2X insulin-transferrin-selenite, 2X B27, 0.5X N2, 1X glutamax, 1X non-essential amino acids, 400 μM monothioglycerol, 5 μg/mL insulin. Immediately before use, microglial medium was supplemented with 100 ng/mL IL-34, 50 ng/mL TGF-β1, and 25 ng/mL M-CSF (Peprotech) taken from single-use frozen aliquots. Media with fresh cytokines was supplemented 1 mL/well of a 6-well plate every 48 hours for 28 days. iTF-MG: Induced pluripotent stem cells containing six doxycycline-inducible transcription factors: CEPBA, CEBPB, IRF5, IRF8, MAFB, PU.1, were passaged as a single-cell suspension using accutase (Thermo Fisher Scientific, A11105-01) for 7 minutes at 37 ºC. Cells were plated to dual-coated Matrigel (Corning, 356231) and PDL plates (Corning, 356470) and cultured for 48 hours in Essential 8 (Gibco, A1517001) stem cell maintenance media supplemented with 2 µg/mL doxycycline (Clontech, 631311) and 10 µM Y-27632 (Tocris 1254). Two days post passage, cells were moved to microglia differentiation media: BrainPhys (StemCell Technologies, 05790), 0.5x N2 Supplement (Gibco, 17502048), 0.5x B27 (Gibco, 17504044), 10 ng/mL NT-3 (Peprotech, 450-03), 10 ng/mL BDNF (Peprotech, 450-02), 1 µg/mL mouse laminin (Thermo Fisher Scientific, 23017-015) supplemented with 2 ug/mL doxycycline, 100 ng/mL IL-34 (Peprotech 200-34), and 10 ng/mL GM-CSF (Peprotech 300-03). On day 4, day 8, and day 12, media was replaced with microglia differentiation media supplemented with 2 µg/mL doxycycline, 100 ng/mL IL-34, 10 ng/mL GM-CSF, 50 ng/mL M-CSF (Peprotech 300-25), and 50 ng/mL TGF-β1 (Peprotech100-21C). For experiments with CRISPRi, cells were supplemented with 50 nM trimethoprim (TMP) (MP Biomedical, 195527) every other day to maintain strong knockdown.

extract_protocol:
iTF-MG are lifted with TrypLE. iMG are lifted by scraping.
The CROP-seq library was generated targeting 31 genes that had interesting phenotypes in the original six FACS-based screens. The two sgRNAs with the strongest phenotypes in the FACS-based screens were selected for each gene for inclusion in the CROP-seq library. In total, 31 genes with 2 sgRNAs each were pooled alongside 5 non-targeting sgRNAs that did not influence phenotypes in any of the original FACS-based screens, resulting in a final library size of 65 elements. The crop-seq library was prepared as previously described in Drager et al. Nat Neuro 2022 from arrayed oligonucleotides ordered from Integrated DNA Technologies. Top and bottom oligonucleotides for each sgRNA were annealed and then pooled. This pool was cloned into pMK1334115. The pooled plasmid library was cloned into Stellar Competent cells such that each plasmid was estimated to have been transformed into >500 bacteria. We performed sgRNA enrichment PCR and sequenced this plasmid pool using MiSeq to confirm normal distribution of all sgRNAs.

value: iTF iPSC-microglia


title: FGSCs, 917NC-1

status: Public on Jun 18 2026

type: SRA

city: Shanghai

first: Ji

last: Wu

address: No.800, Dongchuan Road

country: China

institute: Shanghai Jiao Tong University

description: Library name: 917NC-1

library_source: transcriptomic

data_processing:
The reads were first mapped to the GENCODE(mouse-mm10) or Ensembl gene set using Bowtie2 version 2.1.0
the gene expression level was estimated using RSEM v1.2.28.
TMM (trimmed mean of M-values) was used to normalize the gene expression. Differentially expressed genes were identified using the edgeR program
Assembly: MM10
Supplementary files format and content: Tab-delimited text files include the number of reads, relative RPKM values, and P-values for control and experimental group Sample.

molecule: total RNA

organism: Mus musculus

source_name: Ovary

extract_protocol:
Total RNA was extracted from shnc and shGm26917 FGSCs using Takara RNAiso Plus.
Next, 1000 ng of the digested RNA was used for the  GENESEED®RNA-seq Library Prep Kit for Illumina, and sequencing libraries were created according to the manufacturer's protocol.

tag: tissue

value: Ovary

tag: cell line

value: FGSCs

tag: cell type

value: germ stem cells

tag: genotype

value: shnc group


title: BACH1_rep3

city: Bethesda

first: Matthew

middle: J

last: Brooks

state: MD

address: 6 Center Dr Bldg 6, Rm 303

department: NEI

country: USA

institute: NIH

description: Library name: BACH1_rep3

library_source: genomic

data_processing:
Fastq files were analyzed using nf-core/cutandrun v3.1 protocol (https://nf-co.re/cutandrun/3.1) and GRCm38 assembly.
SEACR peaks were used to generate consensus peaks by requiring peak to be present in at least two replicates and overlapped with gene promoters using Bedtools mutiinter and intersect tools.
Annotation was performed in R using GenomicRanges with Ensembl v102 annotation. BigWig files were calcualetd by CPM in 10bp bins.
Assembly: GRCm38.p6/ENSEMBL v102
Supplementary files format and content: BigWig files
Supplementary files format and content: text file of annotated consensus promoter peaks
Library strategy: CUT&Tag

molecule: genomic DNA

source_name: Retina

extract_protocol:
NRL-GFP mouse retinas at 1.8-months old were dissected and retina dissociated as previously described (Fadl, et al. Mol Vis 2020). Rod cells containing GFP were flow-sorted and 900,000 cells used as input for CUT&Tag (Active Motif, Carlsbad, CA, Cat No. 53165) with anti-AFT4 or BACH1 antibodies.
Libraries were performed according to the manufacter’s instructions.

value: Retina

tag: age

value: 1.8 month

value: Rod photoreceptor

value: NRL-EGFP

tag: Sex

value: male

tag: antibody

tag: replicate

value: replicate 3


title: CROP-seq, transcription factor derived iPSC-microglia (iTF-MG), GEX

state: CA

description:
Library name: iTF_crop-GEX
iTF_processed_merged_crop.h5ad

library_source: transcriptomic single cell

data_processing:
Single-cell RNA Sequence Alignment The raw single-cell sequencing reads of gene expression libraries were processed using kb-python (version: v0.29.1) from kallisto-bustools133. First, using the GRCh38 human reference transcriptome with Ensembl annotations (release 113), a kallisto index was generated with the default k-mer size (k=31). The 10x reads were then pseudo-aligned to this index using ‘kb count’ from kallisto-bustools with the ‘standard’ workflow and the cell barcode whitelist for Single Cell 3’ v3.1 chemistry.  Single-cell ADT Protein Assignment The raw single-cell sequencing reads of antibody-derived tag (ADT) libraries were processed using the Cell Ranger software from 10x Genomics (version 9.0.0). The computational analysis was conducted on a high-performance computing (HPC) cluster configured with 48 GB of virtual memory and eight local cores. A custom feature reference file83 was used for detecting unique molecular identifiers (UMIs) associated with antibody-derived tags.  CRISPRi knockdown demultiplexing (sgRNA assignment) The raw sequencing reads of the PCR amplified sgRNA library were processed using kallisto-bustools133. First, using ‘kb ref’, a kallisto index of the sgRNA library was generated with k-mer size of 9. The reads were then pseudo-aligned to this index using ‘kb count’ from kallisto-bustools with the ‘standard’ workflow and the cell barcode whitelist for Single Cell 3’ v3.1 chemistry. sgRNA assignment was then conducted using geomux (version: v0.2.5)132, which conducts a hypergeometric test to calculate the significance of observing each sgRNA in each barcode. Cells were assigned to the majority sgRNA if they had a log-odds ratio above 1, Benjamini-Hochberg corrected P-value below 0.05, minimum UMI count to consider a barcode was 3, and minimum number of barcodes to consider a sgRNA was the default, 100. Once cells were assigned to a sgRNA, the barcodes were used to intersect the gene expression and ADT count matrices to store both the RNA and protein expression counts for each sgRNA-assigned cell in MuData format from muon (version: v0.1.7)134. Cells with no barcodes or multiple barcodes were not included for further analysis. Both the iTF-MG and iMG samples were processed this way. Of the two biological replicates from iTF-MG samples, only one includes the CITE-seq library and was included for this additional analysis. All QC was conducted using scanpy (version: v1.11.3)135. For both, the iTF-MG and iMG dataset with RNA and ADT counts, cells were filtered to include those with a minimum count of 200, and a mitochondrial count of less than or equal to 8%. Genes were filtered to include those with minimum cells of 10. For the iTF-MG experiment with only RNA counts, cells were filtered to include those with a minimum count of 1000. All other QC was conducted in the same way as the other datasets. For protein expression levels, four different normalization methods were initially tested and log1p-normalize was chosen as it had the highest median correlation to RNA expression as described in Haage et al. 202583. Thus, for both RNA and protein expression levels, cells in each dataset were log1p-normalized to a target sum of 10,000. The top 5,000 highly variable genes were selected to conduct principal component analysis (PCA). Nearest neighbors were then calculated using the default 15 nearest neighbors, to then build UMAPs. Clustering was performed using different resolutions of the Leiden (version: v0.10.2)136 algorithm, and after setting the resolution to 1. Clusters with low AIF1 expression (non-microglia cells) and low total counts were removed. 6 out of 18 clusters were removed from the iTF-MG replicate with only RNA data, whereas only 2 out of 13 clusters were removed from the iTF-MG replicate with both RNA and ADT data. Similarly, only 2 out of 9 clusters were removed from the iMG dataset. The two iTF-MG replicates were then concatenated and batch corrected using Harmony (version: v0.0.10)137, to get 3 final datasets used in analysis the iTF-MG and iMG MuData with both RNA and ADT counts, and the merged iTF-MG AnnData with the batch-corrected RNA counts. Two sgRNAs, FOXK1 sgRNA 2 (FOXK1_g2) and non-targeting control sgRNA 5 (non-targeting_g5) were also removed from analysis due to functional validation of FOXK1_g2 not producing knockdown (Figure S3H), and rigorous testing of non-targeting_g5 showing a perturbation effect through the Mixscale87 analysis described below. After QC, the resulting iTF-MG had 23,826 cells with RNA expression for 16,398 genes, and 9,040 cells with ADT expression for 180 proteins (166 proteins + 14 antibody controls). The iMG had 8,247 cells with RNA expression for 13,939 genes, and 8,247 cells with ADT expression for 180 proteins with 14 of those proteins included in the panel as controls.
Assembly: hg38
Supplementary files format and content: iTF_processed_cite_crop.h5mu
Supplementary files format and content: iMG_processed_cite_crop.h5mu
Supplementary files format and content: iTF_processed_merged_crop.h5ad

molecule: polyA RNA

organism: Homo sapiens


title: strep_WT_plus-glucose_6.5h_rep2

city: Chicago

first: Hoang Anh

middle: V

last: Tran

state: IL

address: 5801 S Ellis Ave

institute: University of Chicago

description: Library name: Sample S20

data_processing:
[Sample T1-T16] The methods of the previously published pipeline (PMID 30559359) were used with some modifications. Raw paired-end sequence reads of 75 or 100 nucleotides were processed by Illumina-utils (PMID 23799126). Inserts contained a 7 nucleotide sample barcode and a random 6 nucleotide unique molecular identifier (UMI). Given that tRNA molecules range in length from 74-96 nucleotides, forward and reverse 100 nucleotide reads fully covered some tRNA sequences and partially overlapped for others. We upgraded the Illumina-utils ‘iu-merge-pairs’ command to merge both fully and partially overlapping reads, while trimming overhanging adapter sequences in the case of more than full overlap (the command line flag, ‘--marker-gene-stringent’, enables consideration of full as well as partial overlap). We minimized erroneous base calls, important for our analysis of modification-induced mutations, by retaining reads that matched with zero mismatches in the overlapping region (option ‘--max-num-mismatches 0’)
[Sample T1-T16] The procedure for de novo profiling of full-length and fragmentary tRNA reads introduced in tRNA-seq-tools (PMID 30559359) was modified as follows. tRNA structural features were identified from the 3’ end of the read. The minimum criteria for tRNA identification were the correct distance to the 7 canonically conserved nucleotides in the T arm, of which 5 must be found, and base pairing in of the T stem. A full-length read must contain a base-paired acceptor stem and all features in between.
[Sample S1-S24] Raw sequences reads were demultiplexed using Je to detect encoded barcode reads. Following this, reads were aligned to a curated Streptococcus mutans tRNA, rRNA and reference genome.
[Sample S1-S24] The aligned reads were then used to determine RNA sequence abundance using custom python script. RNA modifications were detected based on aligned reads using samtools sort (http://www.htslib.org/, Li et al., 2009) feature sort the reads in a bam file format. Then IGVtools count (https://software.broadinstitute.org/software/igv/igvtools) count feature was utilized to output a wig files using the following parameters: -z 5 -w 1 -e 250 —bases. The resulting wig files were processed using a custom python script to identify nucleotide mutations as well as coverage of aligned reads.
Assembly: [Sample T1-T16] tRNA sequences were taxonomically annotated by using the Global Alignment for Sequence Taxonomy (GAST) tool (PMID 19023400) to search a set of reference tRNA sequences that tRNAscan-SE (v1.3.1) (PMID 9023104) identified from 4,235 gold-standard bacterial genomes (non-endosymbiont genomes with an assembly level of “chromosome”) stored in the Ensembl Genomes 2016 database (PMID 26553803).
Assembly: [Sample S1-S24] ASM746v2
Supplementary files format and content: [Sample T1-T16] .tsv files with family taxonomy and tRNA anticodon counts per sample
Supplementary files format and content: [Sample S1-S24] .tsv files with gene name and abundance per sample
Supplementary files format and content: [Sample S1-S24] .sam.tsv files with mutation, deletion, coverage for each gene position per sample

organism: Streptococcus mutans

source_name: bacterial cells

growth_protocol: [Sample S1-S24] S. mutans strains were cultured in Brain Heart Infusion Broth without Dextrose (BHI-D; Alpha Biosciences) at 37°C.

extract_protocol:
[Sample T1-T16] The tongue dorsum scrapings were combined with RNAlater Stabilization solution (Invitrogen) and stored at −80°C. For extraction, RNAlater was first removed by centrifugation. Pelleted material was lysed in 0.3M NaOAc/HOAc,10mM EDTA, pH 4.8 with an equal volume of acetate-saturated phenol:chloroform pH 4.5 (Invitrogen). After addition of 1.0 mm glass lysing beads (Bio-Spec Products) in a 1:1 ratio (bead:sample weight), samples were placed in a reciprocating bead beater (Mini-Beadbeater-16, Bio-Spec Products) for two 1-min intervals on maximum intensity. Samples were centrifuged before re-extraction and isopropanol precipitation of total RNA. Pellets were washed with 75% ethanol before resuspension in an acid-buffered elution buffer (10mM NaOAc, 1mM EDTA, pH 4.8). 1 ug of total RNA was used for the construction of sequencing libraries. RNA quality and quantity were assessed using the Agilent bio-analyzer.
[Sample S1-S24] RNA was isolated using the Quick-RNA Fungal/Bacterial Microprep Kit (Zymo) and treated with DNAse I according to the DNAfree kit (Invitrogen). 1 ug of total RNA was used for the construction of sequencing libraries. RNA quality and quantity were assessed using the Agilent bio-analyzer.
MSR-seq (PMID 35513407)
[Sample T1-T16] Standard MSR-seq protocol was used with additional chemical treatment. Specifically, after the first ligation and streptavidin bead binding of all CHO, samples were split in two: one mock-treated, and the other subjected to AlkB demethylase reaction (PMID 35513407).

treatment_protocol: [Sample S1-S24] A starter culture for each strain was thawed, resuspended in BHI-D, and used to inoculate BHI-D or BHI-D + 0.15% glucose before incubation. Cells were harvested by centrifugation at 1 h and at early stationary phase (3 h for BHI-D, or 6.5 h for BHI-D + 0.15% glucose).

value: bacterial cells

value: WT

tag: treatment

value: plus-glucose, 6.5h


title: Target group 1 day after mRNA treatment, #3

city: Osaka

first: Kazuma

last: Handa

state: Not required for this country

address: Yamada-Oka 2-2, Suita

department: Department of Cardiovascular Surgery

country: Japan

institute: The University of Osaka

description: Library name: Factor No3

data_processing:
Adapter trimming and quality filtering were performed using Trimmomatic v0.39 with default parameters. Filtered reads were mapped to the mouse reference genome (GRCm38) using HISAT2 v2.1.0. Gene-level read counts were calculated using featureCounts v2.0.6. Differential gene expression analysis was conducted using Cuffdiff v2.2.1.
Assembly: GRCm38
Supplementary files format and content: raw_count data

source_name: left ventricular

extract_protocol:
The myocardial tissue was sacrificed and the left ventricle was cut into small pieces, immediately sealed in a polypropylene tube containing RNA later, and stored at -80°C.
Total RNA was extracted from frozen myocardial tissue samples using the miRNeasy Mini Kit (QIAGEN, Hilden, Germany), and RNA quality was assessed by Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA sequencing libraries were constructed using the TruSeq Stranded mRNA (Illumina, San Diego, CA, USA) and subjected to a paired-end 101bp sequencing run on the NovaSeq X plus (Illumina).

value: left ventricular

value: cardiomyocytes and other cells


title: BACH1_rep2

description: Library name: BACH1_rep2

value: replicate 2


title: strep_WT_plus-glucose_6.5h_rep1

description: Library name: Sample S19


title: BACH1_rep1

description: Library name: BACH1_rep1

value: replicate 1


title: Target group 1 day after mRNA treatment, #2

description: Library name: Factor No2


title: CITE-seq, cytokine derived iPSC-microglia (iMG), GEX

description:
Library name: iMG_cite_crop-GEX
iMG_processed_cite_crop.h5mu

value: iMG iPSC-microglia


title: NON_inj

description: Library name: NON_inj

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v8.0.0 with –force-cells 10k option.
Data matrixes were imported into Seurat v4.3.0 (Satija, et al. 2015) in the R v4.2.3 statistical environment. Initial matrix filtering of >5 cells per gene, >500 features per cell, and cells with mitochindria reads <20% was perfomed.
Sample count matrixes were merged and ambiant RNA was removed using decontX function in the celda v1.14.2 package.
Sample decontX adjusted count matrixes were normalized and variance stabalized using SCTransform v2 (Choudhary and Satija, 2022). PCA (50 dimensions), FindNeighbors (30 dimensions), RunUMAP (30 dimensions), and FindClusters were performed using Seurat.
Cell types were assigned using scType (https://github.com/IanevskiAleksandr/sc-type) with sctype 'eye' database "Astrocytes", "Fibroblasts", "Extracellular matrix cells, "Retinal pigment epithelial cells", and custom cell type gene lists (https://github.com/MBrooks313/scRNA-seq/blob/main/Retina_Cell_type_MB_v4.xlsx). High resolution clustering was perfomed to assess and remove clusters exhibiting expression of canonical cell type markers from more than one cell type.
Assembly: GRCm38.p6/ENSEMBL v98
Supplementary files format and content: HDF5 file of filtered CellRanger output
Supplementary files format and content: Seurat object of final data

extract_protocol:
CD-1 mouse retinas were dissected, GFP-positive punches extracted, and retina dissociated as previously described (Fadl, et al. Mol Vis 2020). One sample from each condition was used. Dissociate was used as input for 10X Genomics Chromium controller using v3.1 (Chromium Next GEM Single Cell 3ʹ GEM, Library and Gel Bead Kit v3.1) chemistry. Approximately 17,000 live cells per sample were loaded into the Chromium chip following the Cell Suspension Volume Calculator Table to capture transcripts from approximately 10,000 cells.
Libraries were performed according to the manufacter’s instructions (Single Cell 3’ v3.1 protocol, 10x Genomics)

value: whole retina

tag: strain


title: strep_JCC341_minus-glucose_3h_rep3

description: Library name: Sample S18

value: minus-glucose, 3h


title: ATF4_rep3

description: Library name: ATF4_rep3


title: Target group 1 day after mRNA treatment, #1

description: Library name: Factor No1


title: CITE-seq, cytokine derived iPSC-microglia (iMG), ADT

description:
Library name: iMG_cite_crop-ADT
iMG_processed_cite_crop.h5mu

library_source: other

data_processing:
Single-cell RNA Sequence Alignment The raw single-cell sequencing reads of gene expression libraries were processed using kb-python (version: v0.29.1) from kallisto-bustools133. First, using the GRCh38 human reference transcriptome with Ensembl annotations (release 113), a kallisto index was generated with the default k-mer size (k=31). The 10x reads were then pseudo-aligned to this index using ‘kb count’ from kallisto-bustools with the ‘standard’ workflow and the cell barcode whitelist for Single Cell 3’ v3.1 chemistry.  Single-cell ADT Protein Assignment The raw single-cell sequencing reads of antibody-derived tag (ADT) libraries were processed using the Cell Ranger software from 10x Genomics (version 9.0.0). The computational analysis was conducted on a high-performance computing (HPC) cluster configured with 48 GB of virtual memory and eight local cores. A custom feature reference file83 was used for detecting unique molecular identifiers (UMIs) associated with antibody-derived tags.  CRISPRi knockdown demultiplexing (sgRNA assignment) The raw sequencing reads of the PCR amplified sgRNA library were processed using kallisto-bustools133. First, using ‘kb ref’, a kallisto index of the sgRNA library was generated with k-mer size of 9. The reads were then pseudo-aligned to this index using ‘kb count’ from kallisto-bustools with the ‘standard’ workflow and the cell barcode whitelist for Single Cell 3’ v3.1 chemistry. sgRNA assignment was then conducted using geomux (version: v0.2.5)132, which conducts a hypergeometric test to calculate the significance of observing each sgRNA in each barcode. Cells were assigned to the majority sgRNA if they had a log-odds ratio above 1, Benjamini-Hochberg corrected P-value below 0.05, minimum UMI count to consider a barcode was 3, and minimum number of barcodes to consider a sgRNA was the default, 100. Once cells were assigned to a sgRNA, the barcodes were used to intersect the gene expression and ADT count matrices to store both the RNA and protein expression counts for each sgRNA-assigned cell in MuData format from muon (version: v0.1.7)134. Cells with no barcodes or multiple barcodes were not included for further analysis. Both the iTF-MG and iMG samples were processed this way. Of the two biological replicates from iTF-MG samples, only one includes the CITE-seq library and was included for this additional analysis. All QC was conducted using scanpy (version: v1.11.3)135. For both, the iTF-MG and iMG dataset with RNA and ADT counts, cells were filtered to include those with a minimum count of 200, and a mitochondrial count of less than or equal to 8%. Genes were filtered to include those with minimum cells of 10. For the iTF-MG experiment with only RNA counts, cells were filtered to include those with a minimum count of 1000. All other QC was conducted in the same way as the other datasets. For protein expression levels, four different normalization methods were initially tested and log1p-normalize was chosen as it had the highest median correlation to RNA expression as described in Haage et al. 202583. Thus, for both RNA and protein expression levels, cells in each dataset were log1p-normalized to a target sum of 10,000. The top 5,000 highly variable genes were selected to conduct principal component analysis (PCA). Nearest neighbors were then calculated using the default 15 nearest neighbors, to then build UMAPs. Clustering was performed using different resolutions of the Leiden (version: v0.10.2)136 algorithm, and after setting the resolution to 1. Clusters with low AIF1 expression (non-microglia cells) and low total counts were removed. 6 out of 18 clusters were removed from the iTF-MG replicate with only RNA data, whereas only 2 out of 13 clusters were removed from the iTF-MG replicate with both RNA and ADT data. Similarly, only 2 out of 9 clusters were removed from the iMG dataset. The two iTF-MG replicates were then concatenated and batch corrected using Harmony (version: v0.0.10)137, to get 3 final datasets used in analysis the iTF-MG and iMG MuData with both RNA and ADT counts, and the merged iTF-MG AnnData with the batch-corrected RNA counts. Two sgRNAs, FOXK1 sgRNA 2 (FOXK1_g2) and non-targeting control sgRNA 5 (non-targeting_g5) were also removed from analysis due to functional validation of FOXK1_g2 not producing knockdown (Figure S3H), and rigorous testing of non-targeting_g5 showing a perturbation effect through the Mixscale87 analysis described below. After QC, the resulting iTF-MG had 23,826 cells with RNA expression for 16,398 genes, and 9,040 cells with ADT expression for 180 proteins (166 proteins + 14 antibody controls). The iMG had 8,247 cells with RNA expression for 13,939 genes, and 8,247 cells with ADT expression for 180 proteins with 14 of those proteins included in the panel as controls.
Assembly: hg38
Supplementary files format and content: iTF_processed_cite_crop.h5mu
Supplementary files format and content: iMG_processed_cite_crop.h5mu
Supplementary files format and content: iTF_processed_merged_crop.h5ad
Library strategy: CITE-seq

molecule: protein


title: strep_JCC341_minus-glucose_3h_rep2

description: Library name: Sample S17


title: Control group 1 day after mRNA treatment, #3

description: Library name: Control No3


title: WT parental, untreated, rep1

city: Cambridge

first: Whitney

last: Henry

state: MA

address: 500 Main St

department: Biology

institute: MIT

description: Library name: 1099_1

data_processing:
Mapping was performed with STAR (2.7.1a) using the canonical chromosomes from GRCm38/mm10, with an index built with genes from Ensembl 96 and '--sjdbOverhang 49'.
Gene counts were obtained using featureCounts (v1.6.2) and Ensembl 96 gene annotations and '-s 2'.
Gene counts were normalized and differential expression assayed with DESeq2 (v1.26.0), using 'independentFiltering=FALSE'.
Assembly: GRCm38/mm10
Supplementary files format and content: Ensembl gene ID, raw counts
Supplementary files format and content: Ensembl gene ID, matrix of DESeq2-normalized counts

source_name: Breast

growth_protocol: Cells were cultured in DMEM high glucose supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 1% glutamine.

extract_protocol:
Total RNA was extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol.
Libraries were prepared for RNA-Seq using Roche Diagnostics KAPA mRNA HyperPrep Kit, according to manufacturer’s directions.

treatment_protocol: Cells were treated with 2 ng/ml TGF- β treatment for 10 days

value: Breast

value: PyMT-1099 parental

value: Untreated


title: eq_OA_Ctrl_GJA10

city: Fort Collins

first: Steven

last: Dow

state: CO

address: 300 W Drake Rd

department: Clinical Sciences

institute: Colorado State University

description: Library name: sample34

data_processing:
All paired end RNA-seq reads were assessed for quality using Partek® Flow® software (Illumina, San Diego, CA). Low quality reads (Q ≤ 20) were filtered and adapter sequences were trimmed with CUTadapt (version 1.12)
Filtered reads were mapped to the Equcab 3.0 reference genome (GCA_002863925.1; https://www.ensembl.org/Equus_caballus/Info/Index) using STAR (version 2.7.3a)
HTSeq (version 0.11.0)[54] was used to count the number of reads aligning to annotated genes. Genes with read counts greater than 10 and expressed in ≥ 10 horses (~50% of OA group) were retained for downstream analysi
Reads were normalized using Remove Unwanted Variation from RNA-Seq Data (RUVSeq) accounting for residuals (RUVr)
Differential expression analysis was performed with limma-voom (version 3.64.1) in RStudio (version 2024.04.2) [59,60] with correction for multiple testing (Padj ≤ 0.05, 1.5 ≤ Log2Fold Change ≤ -1.5)
Assembly: Equcab 3.0
Supplementary files format and content: csv file includes raw counts from each sample

organism: Equus caballus

source_name: PBMC

growth_protocol: peripheral blood mononuclear cells (PBMC) isolated using ficol gradient.

extract_protocol:
Ficol gradiant seperation from whole blood
Library construction was performed with a polyA capture protocol with the ABclonal mRNA-seq Lib Prep Kit for Illumina (ABclonal Technology, Woburn MA).

treatment_protocol: healthy horse PBMC compared to horses with naturally occuring OA

value: PBMC

value: Ctrl


title: ATF4_rep2

description: Library name: ATF4_rep2

value: Retina

value: 1.8 month

value: Rod photoreceptor

value: NRL-EGFP

value: male


title: CITE-seq, transcription factor derived iPSC-microglia (iTF-MG), sgRNA enrichment

description:
Library name: iTF_cite_crop-sgRNA
iTF_processed_cite_crop.h5mu

value: iTF iPSC-microglia


title: eq_OA_Ctrl_GJA29

description: Library name: sample39


title: strep_JCC341_minus-glucose_3h_rep1

description: Library name: Sample S16

value: bacterial cells


title: Control group 1 day after mRNA treatment, #2

description: Library name: Control No2

value: left ventricular

value: cardiomyocytes and other cells


title: ATF4_rep1

description: Library name: ATF4_rep1

value: replicate 1


title: strep_WT_plus-glucose_6.5h_rep3

description: Library name: Sample S21

value: WT

value: plus-glucose, 6.5h


title: CITE-seq, transcription factor derived iPSC-microglia (iTF-MG), GEX

description:
Library name: iTF_cite_crop-GEX
iTF_processed_cite_crop.h5mu


title: strep_WT_minus-glucose_3h_rep3

description: Library name: Sample S15

value: minus-glucose, 3h


title: Day 28 G1 clone 1

first: Laura

last: Biggins

address: Babraham Research Campus

department: Bioinformatics

country: United Kingdom

institute: The Babraham Institute

description: Library name: Day 28 G1 clone 1

data_processing:
Trimmed reads were aligned with HISAT2 (v2.1.0). Gene-level counts preformed in SeqMonk (v1.48.1) were imported into DESeq2 for differential expression analysis (Benjamini–Hochberg FDR < 0.05). Principal component analysis confirmed replicate clustering by condition. Gene ontology enrichment was performed using Gorilla (41).
Assembly: GRCh38
Supplementary files format and content: Reads on opposite strand to gene annotations were quantified and datasets size factor normalised.

growth_protocol: PC9 cells were engineered to stably express the FUCCI4 reporter (Addgene #83841) containing mKO2-CDT1 or Clover-GMNN marker proteins. Cells were cultured in RPMI-1640 medium supplemented with GlutaMAX (Gibco, 61870-010), 10% fetal bovine serum (Gibco, A5256701), and 100 IU/mL penicillin–streptomycin (Sigma-Aldrich, P4333-100ML). Cells were maintained at 37°C in a humidified incubator with 5% CO₂ and passaged at 70–80% confluence using 0.25% trypsin–EDTA (Gibco, 25200056). Cells were thawed from authenticated master stocks and used within 10–12 passages. Mycoplasma contamination was excluded by routine PCR-based testing. All experiments were performed using cells in logarithmic growth phase unless otherwise stated

extract_protocol:
Cells were fixed with glyoxal as described to preserve RNA integrity during sorting (39). Following fixation cells were saponin-permeabilized and using FACS were sorted into G1, S, and G2 populations. Cells were first gated based on DAPI content, followed by refinement using mKO2-CDT1 and Clover-GMNN marker expression, and were collected directly into TRI Reagent for RNA extraction. Total RNA was extracted using TRI Reagent (Sigma, T9424) followed by organic phase separation and isopropanol precipitation. RNA purity was assessed by spectrophotometry.
Poly(A)+ RNA was isolated using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490S). Strand-specific libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB, E7760S). Libraries were amplified for 10–18 PCR cycles based on test amplifications carried out on a small portion of the sample. Following AMPure XP bead purification, libraries were quality assessed by Agilent Bioanalyzer, quantified by KAPA Library Quantification Kit. Sequencing was performed (75 bp single-end) to an average depth of ~24 million reads per sample.

treatment_protocol: Cells were continuously exposed to 500 nM osimertinib dissolved in DMSO for 28 days. Media containing drug or vehicle was replaced every 3–4 days. Final DMSO concentration did not exceed 0.1% (v/v).

value: NSCLC

value: 500nM Osimertinib

tag: time point

value: Day 28 G1


title: EO771 tumor_Gq DREADD4

city: Cold Spring Harbor

first: Yue

state: NY

address: One Bungtown Rd, Demerec RM 120

department: Cancer and Neuroscience

institute: Cold Spring Harbor Laboratory

description: Library name: Sample8

data_processing:
JupyterHub platform
Quality control: FastQC, Trimming: cutadapt wrapper; Alignment: STAR; featureCounts;
Quantification: Differential expression: DESeq2 and visulization in JupyterHub with R kernel
Assembly: mm39
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample

source_name: Tumor

growth_protocol: EO771 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37°C.

extract_protocol:
Total RNA was harvested using Trizol. 1 µg of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using NextSeq2000 P1 PE150 mRNA-Seq Library Prep Kit following manufacturer’s protocols.

value: Tumor

value: Breast cancer cell

value: DCZ


title: Control group 1 day after mRNA treatment, #1

description: Library name: Control No1


title: CITE-seq, transcription factor derived iPSC-microglia (iTF-MG), ADT

description:
Library name: iTF_cite_crop-ADT
iTF_processed_cite_crop.h5mu


title: strep_WT_minus-glucose_3h_rep2

description: Library name: Sample S14


title: Exercised4

city: Newark

first: Alexander

last: Lemenze

state: New Jersey

address: 185 South Orange Ave

institute: Rutgers

description: Library name: Sample465

data_processing:
Barcode deconvolution and alignment via cellranger 9.0.1
Assembly: mRatBN7
Supplementary files format and content: .h5 raw matrix file

organism: Rattus norvegicus

source_name: Basal Ganglia

extract_protocol:
10X Genomics scRNA
10X Single Cell 3’ v4

value: Basal Ganglia

value: Sprague Dawley blank male

value: Exercised


title: EO771 tumor_Gq DREADD3

description: Library name: Sample7


title: Human DC 18h post-infection

first: EnJun

last: Yang

state: California

address: 1 Letterman Drive, Suite D3500

institute: Parker Institute for Cancer Immunotherapy

description:
Library name: Rhapsody_DC_18h
BD Rhapsody WTA + abseq + SMK

data_processing:
SevenBridges Genomics pipelines as per BD's recommendations were used to analyze each timepoint separately to generate per-cell molecule counts (RSEC_MolsPerCell.csv) and SMK-based cell identity calls (Sample_Tag_Calls.csv).
Assembly: GRCh38 (hg38)
Supplementary files format and content: SevenBridges Genomics pipelines as per BD's recommendations were used to analyze each timepoint separately to generate per-cell molecule counts (*_RSEC_MolsPerCell.csv) and SMK-based cell identity calls (*_Sample_Tag_Calls.csv). Donor and condition identity for each SMK tag is provided in 20220507_Rhapsody_metadata.csv.

source_name: blood

extract_protocol:
Library extraction was performed as per BD Rhapsody's protocols (Doc ID: 23-24120 Rev. 1.0) for their WTA + SMK + Abseq kit
At each timepoint, a separate BD Rhapsody cartridge was used. For each cartridge, Abseq + SMK- stained cells from each donor and condition were pooled in equal numbers before processing via the BD Rhapsody Express system. A total count of 40,000 cells was attempted for each cartridge. Library preparation was performed as per manufacturer’s protocols (Doc ID: 23-24120 Rev. 1.0). Final sequencing libraries were pooled and sequenced on a single lane of Novaseq X, together with 20% PhiX spike in as per BD’s recommendations.

value: blood

value: Dendritic Cells

value: Mock, RSV, RSV (heat killed), Flu


title: strep_WT_minus-glucose_3h_rep1

description: Library name: Sample S13

value: bacterial cells


title: CITE-seq, cytokine derived iPSC-microglia (iMG), sgRNA enrichment

description:
Library name: iMG_cite_crop-sgRNA
iMG_processed_cite_crop.h5mu

value: iMG iPSC-microglia


title: Exercised3

description: Library name: Sample464


title: EO771 tumor_Gq DREADD2

description: Library name: Sample6

value: WT


title: Human DC 8h post-infection

description:
Library name: Rhapsody_DC_8h
BD Rhapsody WTA + abseq + SMK


title: Exercised2

description: Library name: Sample463


title: strep_JCC341_plus-glucose_1h_rep3

description: Library name: Sample S12

value: plus-glucose, 1h


title: Cortical organoids. MECP2[R306C] mutation, rep1

first: Tatsuya

last: Osaki

address: 43 Vassar street, 46-6227

department: Picower Institute for Learning and Memory

institute: Massachusetts Institute of Technology

description:
Library name: CO_Rett_patient-derived_R306C_1
Feature_count_matirx

data_processing:
Fastq files were combined and aligned with STAR 2.0.4b Gene counts derived with Subread:featureCount 1.45. RPKM and CPM calculated from gene counts with custom R scripting.
Assembly: GRCh37/hg19
Supplementary files format and content: Feature counts matrix
Supplementary files format and content: Differential_gene_expression_data

growth_protocol: All hiPS cells were maintained on Matrigel-coated 6-well plates in mTeSR Plus medium (STEMCELL Technologies) with 10 μM Y-23632 (Rock inhibiter, STEMCELL Technologies) for the first day after each passage and subcultured every 5–7 days using ReLeSR reagent (STEMCELL Technologies). To generate cerebral organoids, two mutant hiPS cell lines and isogenic control iPS cells on 6-well plates were dissociated into single cells with TrypLE express and then plated at 30,000 cells per well on U-bottom ultra-low attachment 96-well plates (Corning) with mTeSR Plus supplemented with 20 μM of Y-23632. After 24 h, the culture medium was replaced with neural induction medium (knockout DMEM-F12, 15% (v/v) knockout serum replacement, 1% (v/v) MEM-NEAA, 1% (v/v) GlutaMAX, 100 nM LDN-193189, 10 μM SB431542, and 10 μM XAV939) and changed every other day. After 10 days of culture, the culture medium was replaced with 1:1 mixture of knockout DMEM/F12 and neurobasal medium supplemented with 0.5% (v/v) N2 supplement, 1% (v/v) B27 plus supplement with vitamin A, 1% (v/v) GlutaMAX, 1% (v/v) MEM-NEAA, and 2.5 ng/ml (v/v) human insulin solution. This medium was changed every 2 days until 18 days of culture had elapsed. After 18 days, the organoids were transferred to flat-bottom ultra-low attachment 24-well plates (Corning) on a rocking shaker (120 rpm) and switched to a maintenance medium (Neurobasal medium supplemented with 1% (v/v) N2 supplement, 2% (v/v) B27 supplement with vitamin A, 1% (v/v) GlutaMAX, 1% (v/v) MEM-NEAA, 0.25 mg/ml (v/v) human insulin solution, 20 ng/ml BDNF, 200 μM ascorbic acid, 100 μM dibutyryl-cAMP (Sigma-Aldrich), and 1% (v/v) Penicillin/Streptomycin). After 30 days from differentiation, the culture medium was changed twice per week with BrainPhys medium supplemented with 2% (v/v) B27 supplement with vitamin A, 1% (v/v) GlutaMAX, 20 ng/ml BDNF, 20 ng/ml GDNF, and 1%(v/v) Penicillin/Streptomycin until the time of imaging.

extract_protocol:
Total RNA was isolated from the cells from 2D culture using RNAeasy Mini Kit (Qiagen) following manufacture protocol.
NA libraries for RNA sequencing were prepared using the TruSeq Stranded Total RNA with Ribo-Zero Globin library prep kit according to the manufacturer’s instructions.

value: Female

value: Cortical organoids

value: biol rep1


title: Mulitome, Farrerol, SCNT, E12.5, ATAC

first: Shanshan

last: Yi

state: Shanghai

address: 1239 Siping Road, Yangpu District

department: College of Life Science and Technology

institute: Tongji University

description:
Library name: F-SCNT125_ATAC
10x Genomics Multiome ATAC: Read1 contains ATAC sequences; Read2 contains cell barcode; Read3 contains ATAC sequences
F-SCNT125_filtered_feature_bc_matrix.h5
scMutiomics_Metadata.txt.gz

library_source: genomic single cell

data_processing:
The 10X Genomics CellRanger-ARC (v2.0.2) pipeline were used to process multiome data. The raw fastq files of RNA-seq and ATAC-seq libraries from the sample were aligned to UCSC mouse genome (mm10) and quantified using 'cellranger-arc count'.
Assembly: mm10
Supplementary files format and content: h5 file: contains both RNA-seq counts and ATAC-seq counts; tsv.gz: fragments file for ATAC-seq; tsv.gz.tbi: fragment file index for ATAC-seq

source_name: placenta

growth_protocol: For natural fertilization, female C57BL/6n mice were mated with male PWK/PhJ mice, followed by placental tissue collection at specified developmental stages. For nuclear transfer, cumulus cells from BPF1 (female C57BL/6n×male PWK/PhJ) female mice were injected into enucleated oocytes. The reconstructed oocytes were then incubated in CZB medium for 1 h before activation. Activation was performed by incubation in Ca2+-free CZB medium containing 1 mM SrCl2 and 5 mg/mL CB for 6 h, and then the oocytes were thoroughly washed and cultured in G-1 PLUS medium (Vitrolife) at 37℃ under 5% CO2. The SCNT embryos were transplanted into the uterus of pseudopregnant recipients at the stage of 2-cell development, and the samples were collected at the appropriate day.

extract_protocol:
Placental samples were collected from SCNT and naturally fertilized pregnant mice. Pregnant ICR mice at E12.5, E14.5 and E18.5 were humanely sacrificed and their placenta were dissected. Placentas were washed with PBS to remove blood and blotted dry with absorbent paper. The treated placentas were snap-frozen in liquid nitrogen and stored to a liquid nitrogen tank. Placental tissue was thoroughly washed in ice-cold PBS to eliminate blood contaminants and precisely weighed (2-3 g). The tissue was then carefully minced into ~1 mm³ fragments using sterile scissors before enzymatic digestion in pre-warmed DMEM/F12 medium (Gibco) containing 0.5 mg/mL collagenase IV (Thermo Fisher Scientific, 17104019) and 0.2 mg/mL DNase I (Roche 10104159001). The digestion was performed at 37°C for 20 minutes in a shaking water bath with gentle manual inversion every 10 minutes.
Libraries were constructed by following the 10X Genomics Chromiun Next GEM Sing Cell Multiome ATAC + Gene Expression protocol (CG000338).

treatment_protocol: Small molecule treatments were performed with 50nM Farrerol for 16h starting after activation.

value: placenta

tag: developmental stage

value: embryonic day (E) 12.5

tag: origin

value: Somatic cell nuclear transfer

value: Farrerol

value: C57BL/6n x PWK/PhJ

tag: library type

value: ATAC


title: EO771 tumor_Gq DREADD1

description: Library name: Sample5

value: Tumor

value: Breast cancer cell

value: DCZ


title: Human DC 4h post-infection

description:
Library name: Rhapsody_DC_4h
BD Rhapsody WTA + abseq + SMK

value: blood

value: Dendritic Cells

value: Mock, RSV, RSV (heat killed), Flu


title: strep_JCC341_plus-glucose_1h_rep2

description: Library name: Sample S11

value: bacterial cells


title: Exercised1

description: Library name: Sample461

value: Basal Ganglia

value: Sprague Dawley blank male

value: Exercised


title: EO771 tumor_Ctr4

description: Library name: Sample4

value: WT

value: PBS


title: Human DC 0h post-infection

description:
Library name: Rhapsody_DC_0h
BD Rhapsody WTA + abseq + SMK

value: Mock


title: strep_JCC341_plus-glucose_1h_rep1

description: Library name: Sample S10

value: plus-glucose, 1h


title: Sedentary5

description: Library name: Sample460

value: Sedentary


title: EO771 tumor_Ctr3

description: Library name: Sample3


title: elav[WDd]_rep1

city: New York

first: David

middle: W. J.

last: McQuarrie

address: 1300 York Avenue

department: Pharmacology

institute: Weill Cornell Medicine, Cornell University

description: Library name: Sample10

data_processing:
Sequencing quality was assessed using a standard quality-control pipeline. Adapter sequences were removed with Trim Galore (v0.6.7), and reads were aligned to the D. melanogaster dm6 reference genome using STAR (v2.7.11a). Gene-level counts were generated with FeatureCounts (v2.0.8) and used for differential gene expression analysis with DESeq2 (v1.40.2) under default parameters.
Assembly: dm6
Supplementary files format and content: tab-delimited text file with counts for each sample replicate

organism: Drosophila melanogaster

source_name: Embryo

extract_protocol:
RNA was extracted with Trizol followed by phenol chloroform extraction.
mRNA was enriched from total RNA by poly(A) selection. Sequencing libraries were constructed by Novogene (UK) Company Limited using their standard Illumina-compatible mRNA library preparation protocol (Plant and Animal Eukaryotic mRNA, WOBI) and sequenced on an Illumina NovaSeq X Plus system.

value: Embryo

value: elav[WDd] rep1


title: Cerebellum, Exercise Wt, 197

city: Albany

first: J

middle: Andrew

last: Berglund

address: 1400 Washington Avenue

institute: University at Albany, State University of New York

description: Library name: 197

data_processing:
After sequencing the quality of the fastq files were checked using the program FastQC (version 0.11.9).
FASTQ files with >20% adapter content were trimmed using fastp (version 0.24.0) and quality re-confirmed using FastQC
STAR (version 2.7.10a) was used to align the fastq files to the GRcm39/mm39 genome with the --quantModeGeneCounts option added to count the reads per gene.
Assembly: GRCm39/mm39
Supplementary files format and content: Tab-delimited text files contain the raw read count abundances for each sample.

source_name: Cerebellum

extract_protocol:
Total RNA was extracted from the cerebellum using TRIzol reagent (Invitrogen). The tissues were first homogenized with RNase-free beads and then processed using the RNeasy kit (Qiagen), followed by DNase treatment.
The NEBNext Ultra II Directional RNA Library Prep Kit (Illumina) with NEBNext rRNA Depletion Kit (New England Biolabs) was used to prepare RNA-seq libraries, with a total of 250 ng input RNA from each sample. The manufacturer’s protocols were followed, with the following exceptions: 40× adaptor dilutions were used, and 13 cycles of library amplification were performed. The resulting libraries were pooled in equimolar amounts, quantified using the NEBNext Library Quant Kit for Illumina (New England Biolabs), quality checked via capillary electrophoresis on the 5300 fragment analyzer (Agilent Bioanalzyer) using the DNF-474 HS NGS kit (Agilent). Libraries with sufficient quantity were loaded into P2 flow cell 1000/2000 by Illumina and were sequenced using paired-end, 151 base pair sequencing on the Illumina NextSeq 2000 sequencer at the University at Albany RNA Institute. All datasets had total read depths greater than 90 million paired-end reads.

value: Cerebellum

value: Wt

value: Exercise


title: Sedentary4

description: Library name: Sample459


title: S23_ChP_Ctrl

city: Boston

first: Maria

last: Lehtinen

address: 300 Longwood Ave, Mailcode Bch3400

institute: Boston Children's Hospital

description: Library name: S23

data_processing:
Novogene aligned with STAR2
Reads were quantified with featurecounts 2.0.8
Assembly: GRCh39
Supplementary files format and content: tab delimited text file with each sample as a column

source_name: Choroid Plexus

extract_protocol:
Qiagen RNAEasy miniprep
, total RNA extracted from ChP tissue was submitted to Novogene for library preparation with poly A selection and mRNA directional sequencing

value: Choroid Plexus


title: strep_WT_plus-glucose_1h_rep3

description: Library name: Sample S9


title: S22_ChP_BD

description: Library name: S22


title: Cerebellum, Exercise Wt, 201

description: Library name: 201


title: strep_WT_plus-glucose_1h_rep2

description: Library name: Sample S8


title: EO771 tumor_Ctr2

description: Library name: Sample2

value: Tumor

value: Breast cancer cell


title: Sedentary3

description: Library name: Sample458


title: S21_ChP_BD

description: Library name: S21


title: EO771 tumor_Ctr1

description: Library name: Sample1


title: strep_WT_plus-glucose_1h_rep1

description: Library name: Sample S7

value: bacterial cells


title: Sedentary2

description: Library name: Sample457

value: Basal Ganglia


title: CRH neuron_Tumor2

description: Library name: Sample6

source_name: Brain neuron

growth_protocol: Primay CRH neurons were freshly isolated from mouse brain and FACSed

value: Brain neuron

value: CRH neuron

value: FACSed CRH neuron


title: S20_ChP_BD

description: Library name: S20


title: Sedentary1

description: Library name: Sample456

value: Sprague Dawley blank male


title: CRH neuron_Tumor1

description: Library name: Sample5

value: WT


title: strep_JCC341_minus-glucose_1h_rep3

description: Library name: Sample S6

value: minus-glucose, 1h


title: S19_ChP_Ctrl

description: Library name: S19


title: strep_JCC341_minus-glucose_1h_rep2

description: Library name: Sample S5


title: RS2Z36-1 mutant, Replicate 3

city: Frankfurt am Main

first: Remus

middle: Richard Elrik

last: Rosenkranz

address: Max-von-Laue-Str. 9

country: Germany

institute: Goethe University Frankfurt

description: Library name: 20103R-01-06

data_processing:
Raw reads were mapped against the tomato reference genome (S. lycopersicum cv. Heinz 1706, genome build 4.0; ITAG 4.0) in a splice-aware manner using Star (Galaxy version 2.7.8a) with default settings, except for the following changes: --genomeSAindexNbases 14, --sjdbGTFfile ITAG4.0_gene_models.gff, -- sjdbOverhang 149, --chimOutType don’t report, --outFilterType yes, --outFilterMultimapNmax 1, -- outFilterMismatchNmax 999, --outFilterMismatchNoverLmax 0.05.
Reads per gene were counted using htseq-count (Galaxy version 0.9.1) with the following settings: --mode union. –stranded no, --minaqual 10, --type gene, --idattr ID
Read counts per gene (ITAG 4.0 annotation) are deposited as processed data.
Assembly: Tomato Genome version SL4.0
Supplementary files format and content: tab-delimited text files contain read counts for each sample

organism: Solanum lycopersicum

extract_protocol:
Total RNA was isolated using TriZol (Invitrogen) followed by DirectZol (Zymo Research)
RNA libraries for RNA-seq were prepared using the NEBNext Ultra II non-directional RNA Library Kit with polyA selection

value: Ovary

value: RS2Z36 KO

value: control


title: S18_ChP_BD

description: Library name: S18


title: strep_JCC341_minus-glucose_1h_rep1

description: Library name: Sample S4


title: eq_OA_OA_GJA74

description: Library name: sample31

value: PBMC

value: OA


title: CRH neuron_Ctr1

description: Library name: Sample1

value: PBS


title: S17_ChP_Ctrl

description: Library name: S17

value: Choroid Plexus


title: RS2Z36-1 mutant, Replicate 1

description: Library name: 20103R-01-04


title: S16_ChP_BD

description: Library name: S16


title: C_11

city: Pamplona

first: Josune

last: Orbe

address: Avenida Pio XII 55

country: Spain

institute: CIMA

description: Library name: 9_S9

data_processing:
The quality of the samples was verified using FastQC v0.11.8 software.
FASTQ files were mapped to GRCh38 human assembly using STAR 2.7.9a with default parameters.
UMI deduplication was performed using UMI-tools v1.0.0
featureCounts v1.6.0 software and Gencode v47 release were used to obtain read counts at gene level considering only those genes aligned to exonic regions
Obtained gene counts were normalized using edgeR and voom function of limma
Assembly: GRCh38
Supplementary files format and content: tab-delimited text files include gene counts obtained with featureCounts for each sample.

source_name: Plasma EVs

extract_protocol:
EVs were isolated by centrifugation at 20.000 xg during 70 min from frozen  citrate  plasma samples at -80°C after double centrifugation (10 min 2000 xg and 15 min 2500 xg)
Plasma EVs were resuspended in 100 μL of lysis/binding Buffer (Invitrogen). RNA transcripts were captured with dynabeads oligo (dT) (Invitrogen) and reverse transcribed. RNA samples underwent a linear amplification by in vitro transcription followed by a fragmentation into 250–350 bp. Partial Illumina adaptor sequences were used, and a second reverse transcription reaction was performed. Full Illumina adaptor sequences were successfully added and the libraries sequenced (NextSeq 2000, Illumina)

value: Plasma EVs

tag: batch


title: S15_ChP_Ctrl

description: Library name: S15


title: strep_WT_minus-glucose_1h_rep2

description: Library name: Sample S2

data_processing:
[Sample T1-T16] The methods of the previously published pipeline (PMID 30559359) were used with some modifications. Raw paired-end sequence reads of 75 or 100 nucleotides were processed by Illumina-utils (PMID 23799126). Inserts contained a 7 nucleotide sample barcode and a random 6 nucleotide unique molecular identifier (UMI). Given that tRNA molecules range in length from 74-96 nucleotides, forward and reverse 100 nucleotide reads fully covered some tRNA sequences and partially overlapped for others. We upgraded the Illumina-utils ‘iu-merge-pairs’ command to merge both fully and partially overlapping reads, while trimming overhanging adapter sequences in the case of more than full overlap (the command line flag, ‘--marker-gene-stringent’, enables consideration of full as well as partial overlap). We minimized erroneous base calls, important for our analysis of modification-induced mutations, by retaining reads that matched with zero mismatches in the overlapping region (option ‘--max-num-mismatches 0’)
[Sample T1-T16] The procedure for de novo profiling of full-length and fragmentary tRNA reads introduced in tRNA-seq-tools (PMID 30559359) was modified as follows. tRNA structural features were identified from the 3’ end of the read. The minimum criteria for tRNA identification were the correct distance to the 7 canonically conserved nucleotides in the T arm, of which 5 must be found, and base pairing in of the T stem. A full-length read must contain a base-paired acceptor stem and all features in between.
[Sample S1-S24] Raw sequences reads were demultiplexed using Je to detect encoded barcode reads. Following this, reads were aligned to a curated Streptococcus mutans tRNA, rRNA and reference genome.
[Sample S1-S24] The aligned reads were then used to determine RNA sequence abundance using custom python script. RNA modifications were detected based on aligned reads using samtools sort (http://www.htslib.org/, Li et al., 2009) feature sort the reads in a bam file format. Then IGVtools count (https://software.broadinstitute.org/software/igv/igvtools) count feature was utilized to output a wig files using the following parameters: -z 5 -w 1 -e 250 —bases. The resulting wig files were processed using a custom python script to identify nucleotide mutations as well as coverage of aligned reads.
Assembly: [Sample T1-T16] tRNA sequences were taxonomically annotated by using the Global Alignment for Sequence Taxonomy (GAST) tool (PMID 19023400) to search a set of reference tRNA sequences that tRNAscan-SE (v1.3.1) (PMID 9023104) identified from 4,235 gold-standard bacterial genomes (non-endosymbiont genomes with an assembly level of “chromosome”) stored in the Ensembl Genomes 2016 database (PMID 26553803).
Assembly: [Sample S1-S24] ASM746v2
Supplementary files format and content: [Sample T1-T16] .tsv files with family taxonomy and tRNA anticodon counts per sample
Supplementary files format and content: [Sample S1-S24] .tsv files with gene name and abundance per sample
Supplementary files format and content: [Sample S1-S24] .sam.tsv files with mutation, deletion, coverage for each gene position per sample


title: WT, Replicate 3

description: Library name: 20103R-01-03


title: strep_WT_minus-glucose_1h_rep1

description: Library name: Sample S1


title: WT, Replicate 2

description: Library name: 20103R-01-02


title: C_10

description: Library name: 7_S7


title: S14_ChP_BD

description: Library name: S14


title: Muscle cells, in vitro cultured, differentiated, Male, Rep.2

city: Hwaseong

first: Kwang-Hwan

last: Choi

address: 10, Dongtan-daero 21-gil

department: R&D center

country: South Korea

institute: Space F corporation

description: Library name: Sample 8

data_processing:
Low-quality reads were filtered according to the following criteria: > 10% skipped bases (marked as ‘N’s), > 40% of bases with a quality score < 20, and reads with an average quality score of < 20. The filtering was performed using Cutadapt v. 1.6. Filtered reads were mapped to the reference genome using TopHat v. 2.0.12.1. Gene expression levels were measured with Cufflinks v. 2.2.1 using the gene annotation database. Non-coding gene regions were excluded from gene expression measurement using the –mask option. To improve the accuracy of the measurement, the multi-read-correction and frag-bias-correct options were applied. All other options were set to default values.
Assembly: Sscrofa10.2
Supplementary files format and content: tab-delimited text file includes FPKM values for each Sample

organism: Sus scrofa

source_name: Skeletal Muscle

extract_protocol:
Total RNA was extracted from undifferentiated and differentiated MuSCs using TRIzol reagent. The quality of RNA samples was examined using the Agilent 2100 BioAnalyzer (Agilent, Santa Clara, CA, USA). The extracted RNA samples were used for construction of cDNA libraries.
The cDNA library preparation and RNA-Seq analysis were performed by Theragen Etex (www.theragenetex.com, Korea). The libraries were prepared for 100-bp paired-end sequencing using the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara Bio, Kusatsu, Japan) according to manufacturer’s instruction. The quality of the cDNA libraries was evaluated using the Agilent 2100 BioAnalyzer (Agilent). The cDNA libraries were quantified using the KAPA Library Quantification Kit (Kapa Biosystems, Wilmington, MA, USA) according to the manufacturer’s instructions.

value: Skeletal Muscle

value: primary cells

value: Male

value: LYD


title: T_9

description: Library name: 6_S6


title: S13_ChP_Ctrl

description: Library name: S13


title: Muscle cells, in vitro cultured, differentiated, Male, Rep.1

description: Library name: Sample 7


title: C_9

description: Library name: 5_S5


title: S12_ChP_Ctrl

description: Library name: S12


title: Muscle cells, in vitro cultured, differentiated, Female, Rep.2

description: Library name: Sample 6

value: Female


title: Mock_3

city: Nagoya-city

first: Fumiya

last: Sakamoto

address: Furo-cho, Chikusa-ku

institute: Nagoya University

description: Library name: Mock_3

data_processing:
Paired-end 100-bp reads were generated, and approximately 20 million clean reads were obtained per sample. Reads were mapped to the GCF_000001635.26_GRCm38.p6 reference genome, and gene-level count data were generated for downstream analysis by Dr. Tom (Kobe, Japan).
Assembly: GCF_000001635.26_GRCm38.p6
Supplementary files format and content: Matrix table containing gene-level raw read counts for all genes in all samples. Column headers correspond to the library names listed in the metadata spreadsheet.

source_name: B16 melanoma

extract_protocol:
Total RNA was extracted from various organs from B16-enpty vector stable cell line using Neucleospin RNA according to the  NucleoSpin RNA kit (Takara Bio, Japan)’s instructions.
RNA-seq libraries were prepared by BGI using their standard eukaryotic transcriptome library preparation protocol.

value: B16 melanoma

value: mouse melanoma


title: S11_ChP_BD

description: Library name: S11


title: Mock_2

description: Library name: Mock_2


title: Muscle cells, in vitro cultured, differentiated, Female, Rep.1

description: Library name: Sample 5


title: C_8

description: Library name: 4_S4


title: Mock_1

description: Library name: Mock_1


title: T_8

description: Library name: 3_S3


title: S10_ChP_Ctrl

description: Library name: S10


title: Muscle cells, in vitro cultured, undifferentiated, Male, Rep.2

description: Library name: Sample 4


title: sample_94

type: genomic

city: Tampere

first: Keijo

last: Viiri

address: Arvo Ylpön katu 34

country: Finland

institute: Tampere University

description: PGCp

hyb_protocol: Aliquots of 1 µg of DNA were subjected to bisulfite conversion, and 4 µl of bisulfite-converted DNA underwent whole-genome application, enzymatic fragmentation, and hybridization onto the MethylationEPIC BeadChip (EPICv2).

scan_protocol: The arrays were scanned using the iScan system (Illumina, San Diego, CA, USA).

data_processing:
The DNA methylation data was pre-processed using the default meffil pipeline, with a p-value threshold of 0.05. Probes that failed quality control (QC) were removed from further analysis. One saple was removed from processed data afte QC
Average Beta values

label: biotin

source_name: Whole blood

label_protocol: DNA methylation profiling was performed using the Illumina Infinium MethylationEPIC version 2 BeadChip at Helmholtz Zentrum, Munich, Germany

extract_protocol: Genomics DNA was extracted from whole blood samples with Chemagic 360 robot with CMG-1091 kit (Revvity, Waltham, MA, USA) in combination with chemagic™ DNA Blood 400 kit H96 (Cat. No. CMG-1091), following the manufacture’s protocol (VD190913.che).

value: Whole blood

value: placebo

value: M

tag: timepoint

value: challenge


title: T_7

description: Library name: 21_S21


title: S9_ChP_Ctrl

description: Library name: S9


title: Muscle cells, in vitro cultured, undifferentiated, Male, Rep.1

description: Library name: Sample 3


title: T_6

description: Library name: 20_S20


title: sample_93

description: GFDp

value: baseline


title: S8_ChP_Ctrl

description: Library name: S8


title: sample_92

description: PGCp

value: F


title: Muscle cells, in vitro cultured, undifferentiated, Female, Rep.2

description: Library name: Sample 2


title: T_5

description: Library name: 2_S2


title: Muscle cells, in vitro cultured, undifferentiated, Female, Rep.1

description: Library name: Sample 1


title: sample_91

description: GFDp


title: C_7

description: Library name: 19_S19


title: S6_ChP_BD

description: Library name: S6


title: sample_90

description: PGCd

value: drug


title: T_4

description: Library name: 18_S18


title: Muscle cells, in vitro cultured

description: Library name: Sample 1

data_processing:
The demultiplexing, barcode processing, and gene counting were performed using the Cell Ranger v7.0.1 pipeline (10x Genomics)
Assembly: Sus scrofa 11.1
Supplementary files format and content: Tab-separated values files and matrix files

extract_protocol:
Cells were washed twice with cold Ca2+ and Mg2+-free PBS at 500 × g for 3 min, resuspended in culture medium. Viability was assessed using acridine orange and propidium iodide staining on a LUNA-FX7™ cell counter. RNA was not extracted separately; poly(A)+ RNA was captured directly from single cells within GEMs using the 10x Genomics Single Cell 3' v3.1 workflow.
Single-cell RNA-seq libraries were prepared according to the 10x Chromium Next GEM Single Cell 3’ v3.1 protocol (10x Genomics, CG000315). Approximately 10,000 cells were loaded per channel into a Next GEM Chip G (10x Genomics, PN-1000120) with reverse transcription master mix, gel beads, and partitioning oil to generate GEMs. Barcoded cDNA was synthesized and amplified, then subjected to fragmentation, end-repair, A-tailing, adaptor ligation, and index PCR. Libraries were quantified using qPCR (KAPA Biosystems) and qualified with an Agilent 4200 TapeStation (Agilent Technologies).

value: Primary cells


title: BE2_OE_3

first: rui

last: Dong

address: 399 Wan Yuan Road

institute: Department of Pediatric Surgery, Children’s Hospital of Fudan University, and Shanghai Key Laboratory of Birth Defect

description:
Library name: BE2_OE_3
BE2_OE_3

data_processing:
Raw sequencing reads were trimmed for adapters using Skewer, and quality control was performed with FastQC (v0.11.2). Clean reads were aligned to the human reference genome (hg38) using STAR. Transcript assembly was conducted with StringTie (v1.3.1c), and expression levels were quantified as fragments per kilobase of transcript per million mapped reads (FPKM) using Perl.
Assembly: hg38
Supplementary files format and content: xls file include FPKM values for each sample

growth_protocol: SK-N-BE(2) and SH-SY5Y cells were cultured Minimum Essential Medium (Gibco) containing 10% fetal bovine serum (Gibco), supplemented with sodium glutamate (Gibco), pyruvate (Gibco), and a mixture of non-essential amino acids (Gibco), and maintained in a humidified incubator at 37 °C with 5% CO₂.

extract_protocol:
Total RNA was extracted using TRIzol reagent (Invitrogen), and RNA concentration was measured with NanoDrop 2000 spectrophotometer (Thermo Scientific).
Strand-specific libraries were prepared from three biological replicates using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA), and sequencing was carried out on the Illumina NovaSeq 6000 platform.

treatment_protocol: Lentiviral vectors containing the full-length human NDUFS6 coding sequence and short hairpin RNAs (shRNAs) targeting NDUFS6 were constructed to generate stable overexpression (OE) and knockdown (KD) cell lines, respectively. Corresponding empty vectors were used as negative controls. Cells were incubated with lentiviral particles in culture medium supplemented with 5 μg/mL polybrene (sigma TR-1003) for 16 hours. Selection with 2 μg/mL puromycin (A1113803) began 48 hours post-infection and continued for approximately one week.

value: Neuroblastoma


title: sample_89

description: GFDd


title: C_6

description: Library name: 17_S17

value: Plasma EVs


title: S4_ChP_BD

description: Library name: S4

value: Choroid Plexus


title: sample_88

description: PGCp


title: C_5

description: Library name: 16_S16


title: Pig ESC-derived cells, at day 12, directed myogenic differentiation

city: Seoul

first: Jinsol

last: Jeong

address: 1 Gwanak-ro, Gwanak-gu

institute: Seoul National University

description:
Library name: Sample 3
Cell viability: 93.0%, Loaded cell number: 30,000, Estimated cell number: 26,231, Library QC (Average yield: 131.25 nM, Fragment size peak: 560 bp), Sequencing depth (Mean reads/cell: 18,105, Median UMI/cell: 8,361, Median genes/cell: 3,256, Total gene detected: 19,555)

data_processing:
Data processing:  Raw scRNA-seq reads were de-multiplexed into FASTQ files using cellranger mkfastq (Cell Ranger v7.0.1, 10x Genomics). Read alignment and gene counting were performed with the cellranger count pipeline (v7.0.1) against the pig reference genome Sus scrofa 11.1 (Ensembl release 108), with the option “--include-introns” enabled. Feature-barcode and gene-barcode expression matrices were generated following cellular barcode and UMI processing.
Data filtering:  Processed data were imported into R (v4.3.3) and analyzed using Seurat (v4.3.0). Quality control was performed at the single-cell level, and low-quality cells were excluded based on the following thresholds: <200 detected genes, <1,000 UMI counts, or >20% mitochondrial reads.
Assembly: Sus scrofa 11.1
Supplementary files format and content: Tab-separated values files and matrix files.

source_name: not applicable (in vitro culture)

growth_protocol: Pig embryonic stem cells derived from in vitro-fertilized embryos were cultured in ESC medium [DMEM/F12 (Gibco; 11320-033) containing 15% (v/v) KnockOut™ Serum Replacement (Gibco; KSR; cat.10828028), 0.1% (v/v) chemically defined lipid concentrate (Gibco; LC; cat.11905031), 1× GlutaMAX™ supplement (Gibco; cat.10565-018), 0.1 mM β-mercaptoethanol (Gibco; cat.21985023), 1× MEM nonessential amino acids (Gibco; cat.11140050), 1× antibiotic–antimycotic (Gibco; cat.15240-062), 20 ng/mL FGF2 (R&D Systems; cat.233-FB), 5 ng/mL activin A (R&D Systems; cat.338-AC), 1.5 µM CHIR99021 (Cayman Chemical; cat.13122), and 2.5 µM IWR-1 (Sigma-Aldrich; cat. I0161)]. Medium was changed every 24 h and cells were maintained at 37°C with 5% CO2 under humidified conditions.

extract_protocol:
The cells pretreated with 10 µM Y-27632 for 24 h were dissociated using TrypLE™ Express and filtered using a 100 µm Cell Strainer (Corning Inc., cat.CLS431752). After centrifugation at 300g for 5 min at RT and removal of the supernatant, cells were washed twice with cold Ca2+ and Mg2+-free 0.04% BSA (Sigma-Aldrich; cat.A6003)/PBS (Welgene; cat.LB 001-02) at 300 × g for 5 min at 4°C, resuspended in the same buffer, and filtered through a 40 µm Flowmi Cell Strainer (SP Bel-Art; cat.H13680-0040). Viability was assessed using acridine orange and propidium iodide staining (Logos Biosystems; cat.F23001) on a LUNA-FX7™ cell counter (Logos Biosystems). RNA was not extracted separately; poly(A)+ RNA was captured directly from single cells within GEMs using the 10x Genomics Single Cell 3' v3.1 workflow.
Single-cell RNA-seq libraries were prepared using the 10x Chromium iX Controller and Next Gem Single cell 3’ Kit v3.1 (10x Genomics; PN-1000268) according to the 10x Chromium Next GEM Single Cell 3’ v3.1 protocol (10x Genomics; doc.CG000315). Cells were loaded per channel into a Next GEM Chip G (10x Genomics; PN-1000120) with reverse transcription reagents (PN-1000123), partitioning oil (PN-1000190), and Next GEM Single Cell 3ʹ Gel Bead (10x Genomics; PN-1000122) to generate GEMs. Barcoded cDNA was synthesized and amplified according to the Next GEM Single Cell 3’ Kit v3.1 protocols (doc.CG000315). Library preparation was performed using the Single Cell 3’ Library Kit v3.1 (PN-1000190), including fragmentation (Fragmentation Enzyme, PN-2000090; Fragmentation Buffer PN-2000091), end-repair/A-tailing with P5/P7 sequences, adaptor ligation (Ligation Buffer, PN-2000092; Adaptor Oligos, PN-2000094; DNA Ligase, PN-220110), and index PCR with i5/i7 sequences, according to the manufacturer’s protocol (10x Genomics; doc.CG000315). Libraries were quantified using qPCR (LightCycler Instrument) according to the qPCR Quantification Protocol Guide (KAPA Biosystems) and qualified with an Agilent 4200 TapeStation (Agilent Technologies).

treatment_protocol:
To induce myogenic differentiation, pig ESCs were pretreated with 10 µM Y-27632 (Santa Cruz Biotechnology; cat. sc-281642) for 24 h, dissociated into small colonies using TrypLE Express (Gibco; cat.12605010), and seeded at a 1:4 ratio onto Geltrex-coated dishes. From the following day, cells were cultured in DMEM/F12 with 15% KSR, 0.1% LC, 1× GlutaMAX, 0.1 mM β-mercaptoethanol, 1× MEM nonessential amino acids, and 1× antibiotic–antimycotic, supplemented sequentially as follows:
- Day 0–4: 3 µM CHIR99021 + 0.05 µM LDN193189  (Cayman Chemical; cat.11802)
- Day 5–6: 3 µM CHIR99021 + 0.05 µM LDN193189 + 20 ng/mL FGF2
- Day 7–8: 10 ng/mL HGF (R&D Systems; cat.294-HG) + 2 ng/mL IGF1(R&D Systems; cat.291-G1) + 20 ng/mL FGF2 + 0.05 µM LDN193189
- Day 9–12: 2 ng/mL IGF1
- Day 13–25: 2 ng/mL IGF1 + 10 ng/mL HGF
Medium was replaced every 24 h throughout differentiation.

value: not applicable (in vitro culture)

value: pig ESC line, IVF-ES-11

value: differentiated pig embryonic stem cells

value: myogenic differentiation for 12 days


title: T_3

description: Library name: 15_S15


title: S2_ChP_Ctrl

description: Library name: S2


title: sample_84

description: PGCd


title: Pig ESC-derived cells, at day 8, directed myogenic differentiation

description:
Library name: Sample 2
Cell viability: 83.1%, Loaded cell number: 30,000, Estimated cell number: 23,029, Library QC (Average yield: 218.35 nM, Fragment size peak: 544 bp), Sequencing depth (Mean reads/cell: 18,870, Median UMI/cell: 9,628, Median genes/cell: 3,382, Total gene detected: 19,361)

value: myogenic differentiation for 8 days


title: T_2

description: Library name: 14_S14


title: S1_ChP_Ctrl

description: Library name: S1


title: SY-OENC-2

city: Shanghai

description:
Library name: SY-OENC-2
SY-OENC-2

value: Control for NDUFS6-overexpression


title: sample_83

description: GFDd

value: Whole blood


title: CMK_KO2

city: Wilmington

first: Anilkumar

last: Gopalakrishnapillai

state: DE

address: 200 Powder Mill road

institute: Nemours Children's Health

description:
Library name: Sample 5
A5

data_processing:
HiSeq Control Software for base calling.
RNA-Seq reads were aligned to human reference genome hg38 with STAR.
Number of read counts per gene locus was summarized using 'featureCounts' using hg38 annotation.
Read counts normalized using TMM normalization (edgeR).
Assembly: Genome_build: hg38
Supplementary files format and content: csv file with TMM normalized read counts

source_name: CMK

extract_protocol:
Total RNA extracted by RNeasy Plus Micro kit
Libraries were prepared using NEBNext Ultra RNA Library Prep Kit. RNA library preparations, sequencing reactions, and initial bioinformatics analysis were conducted at GENEWIZ, LLC. (South Plainfield, NJ, USA).

value: CMK

value: DS-AML


title: CHyMErA in RPE-1 cells, T27 post infection, DMSO control treatment, replicate C

city: Toronto

first: Ulrich

last: Braunschweig

state: Ontario

address: 160 College Street

department: Donnelly Centre

country: Canada

institute: University of Toronto

description: Library name: RPE1_T27_DMSO_C

data_processing:
Demultiplexing of raw sequencing data was performed using Illumina’s bclfastq.
A library FASTA file was generated by placing the Cas9 and Cas12a guides, extracted from the library annotation file, into the sequence context of hgRNA construct.  Specifically, the following was used:  CGAGGTACCG + <Cas9.sgRNA> + GTTTCAGAGCTACTAAGTGTAGAT + <Cas12a.sgRNA> + TTTTTTTTGAATTCTCT. The FASTA file was then indexed by bowtie-build. Reads in FASTQ format were pre-processing using a bespoke Perl script designed to identify the 5’ or 3’ end of the Cas12a direct repeat sequence, and extract the adjacent 23bp upstream sequence (Cas12a guide) from read1 and 20bp downstream sequence (Cas12a) from read2 for mapping. The extracted sequences were written to a new FASTQ file, and aligned to the prepared FASTA file with bowtie (v0.12.1) using the following parameters: -p 16 -v 3 -l 18 --chunkmbs 256 -t intronKnockoutLibrary. Alignments to each hgRNA construct were enumerated from the resulting SAM file using a bespoke Perl script, written to a text file for each sample, and merged with the library annotations in R.
Read counts were depth normalized by summing the mapped reads for each sample, then dividing the count for each hgRNA construct by the total counts and multiplying by 1x10^6.
Assembly: Custom reference, see data processing.
Supplementary files format and content: IntCHyMErA.library.design.tab.gz is a tab-delimited file containing library design information for the CRISPR guide RNA library used in every sample. Each hybdrid guide RNA contains a Cas9 and a Cas12a part.
Supplementary files format and content: IntCHyMErA_counts_raw.txt.gz is a tab-delimited file containing raw hgRNA counts in each sample.
Supplementary files format and content: IntCHyMErA_counts_norm.txt.gz is a tab-delimited file containing read counts normalized to million reads in the library (RPM).

source_name: RPE-1 retinal pigment epithelium

growth_protocol: All cells were maintained under standard sterile cell culture conditions at subconfluent densities (50-70%) at 37°C and in a humidified incubator containing 5% CO2. HAP1 cells (clone C631; male, with Y chromosome loss; Horizon Genomics, RRID: CVCL_Y019) were cultured in Iscove's Modified Dulbecco's Medium (IMDM; Gibco, #12440053) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, #26140079) and penicillin/streptomycin (Gibco, #15140-122). hTERT-RPE-1 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma-Aldrich, #D5796) supplemented with 10% heat-inactivated FBS, penicillin/streptomycin, and sodium pyruvate (Gibco, #11360070).

extract_protocol:
CRISPR sequencing libraries were prepared as previously described (PMID: 32249828). Briefly, at each timepoint, cells were collected at 100-fold of library coverage, washed in PBS, and stored at -80 until processing. Genomic DNA was extracted using the Wizard™ Genomic DNA Purification Kit (Promega #A1125) according to the manufacturer's protocol. DNA concentration was determined using the Qubit dsDNA Broad Range Assay kit (Invitrogen).
For PCR amplification of hgRNA sequences, the following primers were used to amplify the integrated library from genomic DNA at >95-fold coverage, using NEBNext Ultra II Q5 Master Mix (NEB #M0544L). PCR1_Hybrid_Outer_F2: TGAAGAATCGCAAAACCAGCA PCR1_Hybrid_Outer_R1: GAGGGCCTATTTCCCATGATTC The resulting products were analyzed by gel electrophoresis for size and quality. PCR products were then pooled and subjected to indexing by PCR using Illumina TruSeq i5 and i7 barcoded adapters. Sequencing libraries were analyzed by agarose gel electrophoresis. Gel extraction was performed for the correct PCR product using PureLink™ PCR Purification Kit (Invitrogen, #K310001).Sequencing libraries were submitted for quality control analysis and high-throughput sequencing at the Donnelly Sequencing Centre (DSC). 1 μl top stock of each purified library was run on an Agilent Bioanalyzer dsDNA High Sensitivity chip (cat # 5067-4626, Agilent Technologies Inc., Santa Clara, USA). The libraries were quantified using the Quant-iT dsDNA high-sensitivity (cat # Q33120, Thermo Fisher Scientific Inc., Waltham, USA) and were pooled at equimolar ratios after size-adjustment. The final pool was run on an Agilent Bioanalyzer dsDNA High Sensitivity chip and quantified using NEBNext Library Quant Kit for Illumina (cat # E7630L, New England Biolabs, Ipswich, USA). The quantified pool was hybridized at a final concentration of 350 pM and sequenced at a depth of 50 M reads/sample on the Illumina NovaSeq 6000 platform using a SP flow cell of the following parameters: DC1: 29bp, R1: 33bp, IR1: 8bp, IR2: 8bp, DC2:20bp, R2: 32bp.

treatment_protocol: Cells were transduced at a multiplicity of infection of < 0.3 in media supplemented with 8 µg/ml Hexadimethrine bromide. Cells were infected at >400-fold library coverage. Twenty-four hours post-transduction, media was replaced with fresh media supplemented with 1 μg/ml puromycin for HAP1 and 20 µg/ml puromycin for RPE-1 cells (Gibco, A11138-03). Selection was complete for HAP1 cells after 48 hours. To ensure complete selection, RPE-1 cells were trypsinized and re-seeded after 48 hours of puromycin selection and selected for an additional day. At T0, puromycin was removed and the desired MOI (less than 0.3) was confirmed. Subsequently, each screen was divided into three replicates (replicates A, B and C at 250-fold coverage). Non-treated (NT) cells were grown in culture for >20 doublings while maintaining a 250-fold coverage and up to T18 and T27 for HAP1 and RPE-1 cells respectively and were maintained every 3 days. At T3 for HAP1, two treatment arms were separated from each replicate and were treated with either DMSO or TG003 (Sigma T5575-25MG) from T3 to T18 at IC50 (inhibitory dose by 50%, at 30µM - please note the discrepancy with the naming of the samples). For RPE-1 cells, at T6, each replicate arm was divided into a DMSO or TG003 treatment arms at 172-fold coverage. For RPE-1 cells, TG-003 was performed at IC20 (50µM) up to T27. Biological triplicates were sampled for sequencing at T12 and T18 for HAP1 and T21 and T27 for RPE-1 non-treated condition (NT); DMSO-treated condition (DMSO); IC50/IC20 TG-003 treated cells (C20).

value: RPE-1 retinal pigment epithelium


title: RPE-1 cycloheximinde treatment, replicate 2

description: Library name: RPE1_CHX_2

data_processing:
Base-calling Q-scores were obtained with RTA 3.4.4
Alternative splicing analysis was performed as follows: RNA-Seq data were processed using the vast-tools pipeline, version 2.5.1 https://github.com/vastgroup/vast-tools (Braunschweig et al., 2014; Irimia et al., 2014; Tapial et al., 2017). From primary output, events with poor coverage or junction balance were filtered out (vast-tools quality column score 3 other than SOK/OK/LOW/VLOW for cassette exon [CE], microexon [MIC], and alternative 5' or 3' splice site [Alt5/3] events or coverage less than 10 reads for intron retention [IR] events; score 4 other than OK/B1/B2/Bl/Bn for CE and MIC events and score 5 of less than 0.05 for IR events). If more than one replicate was present, the point estimate from vast-tools diff was used and if the coverage/balance filtering step failed for an event in more than half of the replicates, its value was set to NA. Differential AS was assessed through the vast-tools diff module available with the main pipeline. Changes were considered significant if they were greater than 10 dPSI/dPIR and the expected minimum change was different from zero at p > 0.95 according to vast-tools’ diff module.
In parallel to vast-tools analysis, alternative splicing was analyzed with rMATS v4.1.2 (Shen, Park et al.,  2014) based on reads from which Illumina adaptors had been trimmed off the 3'-end using Trimmomatic v0.33 (Bolger et al., 2014). rMATs was run using options --lipType fr-firststrand --variable-read-length --anchorLength 8 --novelSS with a GENCODE v43 index and annotation.
Gene expression changes were analyzed by pseudo-aligning pre-trimmed reads to GENCODE v43 transcripts using Salmon v1.10 (Patro et al., 2017) and aggregated per gene using the R package tximport (Soneson et al., 2015). Differential expression was then assessed using the classic mode (exactTest) edgeR (McCarthy et al., 2012), and genes with an FDR < 0.05 and a greater than 2-fold change were considered differential. For each comparison, only genes expressed at a minimum of 5 RPKM in one or both conditions were considered.
Assembly: GENCODE v43
Supplementary files format and content: RNAseq.RPE1_vastTools.tab.gz is a tab-delimited file and contains vast-tools output, i.e. event information and raw percent-spliced-in values and quality scores per sample. See https://github.com/vastgroup/vast-tools#combine-output-format.
Supplementary files format and content: RNAseq.RPE1_altSplicing.tab.gz is a tab-delimited file and contains mean percent-spliced-in values per treatment and significance calls for the difference vs intergenic hgRNA control.
Supplementary files format and content: Files names rMATS.[cell line]_[event type].MATS.JC.txt are tab-delimited files containing primary rMATS output (i.e., splicing event coordinates, read counts and splicing information). Files named rMATS.[cell line]_sigEvents.csv are comma-separated files with combined information from all splicing event types and filtered for a mimium of 10 junction reads, FDR < 0.05 and inclusion level difference >= 0.1.
Supplementary files format and content: Expression_RPE1.tab.gz is a tab-delimited file and contains RPKM, log2 fold-change and p-values per treatment vs intergenic hgRNA control.

growth_protocol: HAP1 cells (clone C631; male, with Y chromosome loss; Horizon Genomics, RRID: CVCL_Y019), and hTERT-RPE-1 cells (ATCC, CRL-4000) were maintained under standard sterile cell culture conditions at subconfluent densities (50-70%) and incubated at 37°C in a humidified incubator containing 5% CO2. HAP1 cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM; Gibco, #12440053) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, #26140079) and penicillin/streptomycin (Gibco, #15140-122). RPE-1 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma-Aldrich, #D5796)  supplemented with 10% heat-inactivated FBS, penicillin/streptomycin, and sodium pyruvate (Gibco, #11360070).

extract_protocol:
Total RNA was extracted from HAP1 and RPE-1 cells using the RNeasy Mini Kit (Qiagen #74106). Briefly, cells were washed twice in PBS and lysed directly in the culture vessel using RLT buffer supplemented with the reducing agent β-mercaptoethanol (Milipore, #444203-250ML). All RNA samples were DNase-treated using RNase-Free DNase Set (Qiagen, #79256) according to the manufacturer's protocol.
Stranded/directional mRNA-Seq libraries were prepared as follows. DNase-treated total RNA was assessed using Qubit RNA BR (cat # Q10211, Thermo Fisher Scientific Inc., Waltham, USA) and RNA integrity was evaluated using the Bioanalyzer RNA 6000 Pico kit (cat # 5067-1513, Agilent Technologies Inc., Santa Clara, USA) with all samples exhibiting high quality (RIN ≥ 9, median RIN = 9.7). For each sample, 500 ng of total RNA was used to prepare libraries with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (cat # E7760L; New England Biolabs; protocol 6.0_7/24) incorporating Poly (A) selection, with 7.5 minutes of fragmentation at 94 °C and 11 cycles of amplification. Library quality was verified by running 1 μl of each final library on an Agilent Bioanalyzer using the High Sensitivity dsDNA chip (cat #5067-4626, Agilent Technologies). The libraries were quantified using the Quant-iT dsDNA high-sensitivity (cat # Q33120, Thermo Fisher Scientific Inc., Waltham, USA), then normalized and pooled at equimolar ratios after size-adjustment. The pooled library had an average library size of 502 bp.

treatment_protocol: To indirectly inhibit nonsense-mediated mRNA decay through translation inhibition, HAP1 and RPE-1 cells were seeded and cultured at sub-confluent densities for 48 hours prior to treatment. Cells were then incubated for 6 hours in media supplemented with either vehicle control (DMSO; Fisher BioReagents™, BP231-100) or 50 µM cycloheximide (Sigma-Aldrich, 01810) solubilized in DMSO. Cells were harvested immediately following treatment at a final confluency of ~70% .


title: TCF7L2 KD, NMP, shRNA2, replicate2

city: San Francisco

first: Faranak

last: Fattahi

department: Cellular and Molecular Pharmacology

institute: University of California San Francisco

description:
Library name: TCF7L2-NMP-2-2
Mercurius High Sensitivity BRB-seq kit

data_processing:
Samples with greater than 2e6 total counts were kept for  analysis. Counts and metadata tables were combined into a single dataset using DESeq2 (v1.36.0). For all-sample comparison using PCA, VST-normalized counts were extracted and batch corrected using the limma (v3.54.2) removeBatchEffect() function. For heatmap plotting, the size-factor normalized counts were extracted and used. For differential expression testing, the counts matrices were subset to only include either NEP or NMP data before generating the DESeq object using a ‘ ~ batch + KD_Gene’ design. The lists of differentially expressed genes were filtered to only include genes that crossed the significance threshold in 1 or 2 samples.
Assembly: human GRCh38
Supplementary files format and content: Processed data files consist of demultiplexed single-sample raw gene counts produced from STAR.

extract_protocol:
RNA was extracted using an RNeasy Plus Miniprep Kit (Qiagen) according to manufacturers directions. RNA was quantified on a nanodrop to confirm quality prior to library creation.
Bulk RNA libraries were generated using a Mercurius High Sensitivity BRB-seq Kit (Alithea Genomics) using ~200 ng of RNA per sample according to the manufacturers directions. All samples were pooled after reverse transcription and final libraries were quantified using a Qubit dsDNA HS kit and BioAnalyzer. Libraries were sequenced on a single lane of an Illumina NovaSeq X using paired end sequencing. Samples were aligned using STAR (v2.7.11b) according to the kit directions. Samples were further demultiplexed using BRB-seqTools (v1.6.1) into single-sample .fastq files.

treatment_protocol: Lentiviral supernatant was added to stem cell cultures for 48 hours, then selected for using puromycin (0.5 ug/mL). NEP progenitors were achieved by treating stem cells with dual SMAD inhibition in mTeSR Plus media for 5 days. NMP progenitors were differentiated by exchanging mTeSR Plus media for E6 for 24 hours, followed by the addition of 20 ng/mL bFGF for 24 hours, and finally 20 ng/mL bFGF, 3 uM CHIR99021, and 10 uM SB431542 for 48 hours. After NEP or NMP treatment, both populations were differentiated under identical conditions in neural induction media (DMEM:F12 supplemented with N2, NEAA, L-glut, and PenStrep at 1x concentration and 2 ug/mL heparin and 0.4 ug/mL ascorbic acid). For the first 48 hours after NEP or NMP treatment, NIM was supplemented with 10 ng/mL BDNF and 100 nM retinoic acid. This was followed by 12 days of NIM supplemented with 10 ng/mL BDNF, 1 uM DAPT, 100 nM retinoic acid, and 300 nM purmorphamine. At the final day 19 time point, neurons were lysed in wells with RLT buffer supplemented with betamercaptoethanol and frozen at -80 until use.

value: hPSC-derived neuron

value: TCF7L2 KD; NMP


title: CHyMErA library cloning pool, step 2

description: Library name: Library_pool_Lig2

molecule: other

organism: synthetic construct

source_name: in vitro

value: in vitro

value: cloning step 2


title: sample_82

description: PGCp

value: placebo

value: F

value: challenge


title: C_3

description: Library name: 12_S12

value: Plasma EVs


title: TCF7L2 KD, NMP, shRNA2, replicate1

description:
Library name: TCF7L2-NMP-2-1
Mercurius High Sensitivity BRB-seq kit


title: CHyMErA library cloning pool, step 1

description: Library name: Library_pool_Lig1

value: cloning step 1


title: T_1

description: Library name: 11_S11


title: sample_78

description: PGCd

value: drug

value: M


title: TCF7L2 KD, NMP, shRNA2, replicate1, reseq

description:
Library name: TCF7L2-NMP-2-1-2
Mercurius High Sensitivity BRB-seq kit


title: C_2

description: Library name: 10_S10


title: TCF7L2 KD, NMP, shRNA1, replicate2

description:
Library name: TCF7L2-NMP-1-2
Mercurius High Sensitivity BRB-seq kit


title: Sprague–Dawley rats, ISO Test2

city: Kunming

first: Kang

last: Luo

state: Yunnan

address: No. 1168, Chunrong West Road, Yuhua Street, Chenggong District, Kunming,

institute: Kunming Medical University

description: Library name: T79

data_processing:
The clustering of the index-coded samples was performed on a cBot Cluster
Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to
the manufacturer’s instructions. After cluster generation, the library preparations were
sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were
generated.
Assembly: mm10
Supplementary files format and content: RNAseq_counts_matrix.csv
Supplementary files format and content: RNAseq_counts_matrix.csv

source_name: Heart

extract_protocol:
RNA was harvested using Rneasy mini plus kit (Qiagen). 1 µg RNA oftotal RNA was used for the construction of sequencing libraries
Sequencing libraries were generated using NEBNext® UltraTM.RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample.

value: Heart

value: WT

value: subcutaneous injection of ISO (1.5 mg/kg/day) for 7 consecutive days


title: sample_77

description: GFDd

value: Whole blood

value: baseline


title: Sprague–Dawley rats, ISO Test1

description: Library name: T77


title: C_1

description: Library name: 1_S1


title: TCF7L2 KD, NMP, shRNA1, replicate1

description:
Library name: TCF7L2-NMP-1-1
Mercurius High Sensitivity BRB-seq kit


title: sample_76

description: PGCd


title: TCF7L2 KD, NEP, shRNA2, replicate2

description:
Library name: TCF7L2-NEP-2-2
Mercurius High Sensitivity BRB-seq kit

value: TCF7L2 KD; NEP


title: Sprague–Dawley rats, Saline Control3

description: Library name: C83

value: subcutaneous injection of equal volume of saline for 7 consecutive days