title: W303 Prp19-AID Auxin-treated cells replicate 2

city: Warren

first: Katherine

last: Dwyer

address: 29529 Cunningham Drive

institute: Wayne State University

description: Library name: Prp19_2_AUX

data_processing:
genome alignment: STAR (v2.7.11a) maximum intron size of 2,000 nucleotides, a minimum overlap for paired-end alignment of 10 and sorted BAM alignment format
alignment quality evalution: SAM tools (v1.9) BAM files were indexed for downstream analysis
count matrix: featuresCount; countReadPairs -s2 -T6 -texon (subread-2.06)
Differential expression analysis: DESeq2; differentially expressed genes with an adjusted p-value < 0.05 and a log₂ fold-change of greater than 2
Bamcoverage was used to generate bigwig files with computeMatrix function and plotHeatmap function using Galaxy Version 3.3.0.0.1
Assembly: S288C sacCer3 R64-1-1
Supplementary files format and content: File types include .txt and .bw files from raw counts and normalized counts, respectively
Library strategy: GRO-Seq

organism: Saccharomyces cerevisiae

source_name: yeast cells

growth_protocol: The library was prepared from mid-logarithmically growing yeast cells (Saccharomyces cerevisiase W303) of Prp19-AID tagged strains. Cells were grown in YPD media at 30 °C overnight with 1:100 dilution the following morning. Cultures were then grown to mid-log phase to an OD600 of 0.6-0.8 to continue the experiment.

extract_protocol:
Cell lysis and affinity purification of nascent 4-thiouracil labelled RNA was performed as described in O-Brien and Ansari (2023).
The nascent, purified RNA samples were depleted of rRNA. GRO-seq libraries were constructed from the rRNA-depleted samples following the NEBNext Ultra II Directional RNA Library protocol. cDNA library quality was assessed to verify the library size (200-700 bp peak), purity and quantity using Agilent Tape Station 2200 gDNA-QG protocol following the manufacturer’s protocol. Sequencing was performed on a NovaSeq 6000 (2 x 50bp, 10 M reads per sample). To perform sequencing of libraries using Illumina Sequencing system, libraries were normalized to a 1.5 nM pool for a final loading concentration of 300 pM on a NovaSeq 6000 following the Denature and Dilute Libraries Guide. GRO-seq reads were trimmed for quality before alignment to the Saccharomyces genome (Saccer3, downloaded from UCSC database) using hisat2 (version 2.1.0) with the rna-strandness parameter.

treatment_protocol: TRO reaction was performed with permeabilized yeast cells using 4-thiouracil and following the method as described in O-Brien and Ansari (2023).

value: yeast cells

value: W303 WT; Prp19-AID

value: W303 Prp19-AID

value: Auxin


title: W303 Prp19-AID Auxin-treated cells replicate 3

status: Public on Feb 11 2026

type: SRA

state: MI

country: USA

description: Library name: Prp19_3_AUX

library_source: transcriptomic

molecule: total RNA

tag: tissue

tag: cell line

tag: cell type

tag: genotype

tag: treatment


title: ALL, no protein, replicate 3

city: Berlin

first: Markus

last: Schuelke

state: Berlin

address: Augustenburger Platz 1

department: Neuropediatrics

country: Germany

institute: Charite

description:
Library name: ALL_NP_rep3
These samples were sequenced twice (once on NovaSeq X Plus and again on NovaSeq 6000 as read depth was too low). The data files include both sequencing runs merged

library_source: other

data_processing:
Sequence reads were fist trimmed to 40 nucleotides in length (total fragment length)
Cutadapt was used to ensure the reverse primer occured at the 3' end (this is to filter out any sequences where the sequencing adapter bound in the randomised region rather than at the 3' end as intended)
Sequence reads were filtered for a phred score of 30 or above with cutadapt
Sequence reads were trimmed to 14 nucleotides in length (possible randomised region) with cutadapt
cutadapt was used to remove contaminating sequences from FLANK and ALL libraries (they contained high counts of sequences matching the CORE library so these were all removed to prevent skewing of the data).
DESeq2 v1.50.0 was used to determine enrichment or depletion of DNA sequences in the mutant library upon protein binding. Prior to DESeq2 analysis, samples were filtered to remove sequences with less than five occurrences in the unbound library to avoid inaccurate log fold change (LFC) calculations due to low sequence counts.
Supplementary files format and content: *_counts.txt fragment counts (tab delimited text format)
Supplementary files format and content: *_deseq2.csv log fold change statistics between the bound library and the unbound source library for each DNA fragment

molecule: other

organism: synthetic construct

source_name: Artificial

extract_protocol:
DNA was purified from the electrophoresis gel using electroelution and isopropanol purification
Approximately 1 ng of extracted DNA was used in a low cycle number (12-14) PCR reaction (NEBNext Hi Fidelity 2X Mastermix) with Illumina sequencing adapters. Size selection was then performed using 2.5X AMPure XP beads (Beckman Coulter). The quality of samples was measured using the Agilent BioAnalyzer and concentration was measured using the HS dsDNA Qubit assay (Thermo Fisher Scientific).

treatment_protocol: EMSA-seq was performed either without NKX2.1 protein (control for sequence bias), or with DNA:protein ratios of 1:0.1 or 1:0.2

value: Binding reactions were performed in a tube - no cells used


title: HeLa, 1 ng/ml TcdB2 24 h, antibody isotype control (CUT&RUN), rep 1

city: Oklahoma City

first: Jason

last: Larabee

state: OK

address: 940 Stanton L. Young Blvd, BMSB 1035

department: Microbiology and Immunology

institute: The University of Oklahoma Health Science Center

description: Library name: T1

library_source: genomic

data_processing:
The FASTQ files were trimmed with Trim Galore and aligned to the human hg38 canonical genome using bowtie2 (v2.5.3) with the very sensitive end-to-end (--very-sensitive) parameter.  Resulting BAM files were converted to bigWig files and normalized to counts per million (CPM) using bamCoverage.
Assembly: Human GRCh38 genome
Supplementary files format and content: BigWig files
Library strategy: CUT&Run

molecule: genomic DNA

organism: Homo sapiens

source_name: HeLa

growth_protocol: HeLa cell cultures

extract_protocol:
DNA sites binding for CTCF were isolated using CUT&RUN reagents obtained from Cell Signaling Technology (product # 86652).  Antibodies used for this assay include a rabbit monoclonal antibody against CTCF (Cell Signaling Technology; product # 3418); a rabbit monoclonal antibody against H3K4me3 (Cell Signaling Technology; product # 9751); and a rabbit monoclonal isotype control (Cell Signaling Technology; product # 66362).
Sequencing libraries were constructed using XGen DNA Library Prep EZ Kit (IDT), and each library was indexed in order to multiplex for sequencing.

treatment_protocol: Treated with 1 ng/ml TcdB2 for 24 h.

value: HeLa


title: Subject2_exp2_vehicle

city: Dallas

first: Jonathan

last: Rios

state: TX

address: 2222 Welborn Street

institute: Scottish Rite for Children

description:
Library name: Subject2_exp2_vehicle
OFD_vehicle_mirdametinib_DESeq2_normalization.txt
OFD_vehicle_mirdametinib_FPKM_normalization.txt

data_processing:
Sequence reads were mapped to the human reference genome (hg38) using STAR (v2.7.8). Alignments were filtered to remove duplicates, singleton reads, and unaligned reads. Gene expression was quantified using the Ensembl database (release 109). Expression levels of protein-coding transcripts were normalized using the Median Ratio method for DESeq2 analysis. Processed files with FPKM normalization are also included.
Assembly: hg38
Supplementary files format and content: Gene expression matrix normalized by Median Ratio method for DESeq2 analysis
Supplementary files format and content: Gene expression matrix normalized by FPKM

source_name: lesion

growth_protocol: MEM-alpha with 10% FBS and 1% penicillin/streptomycin

extract_protocol:
RNA was extracted using the Rneasy Plus minikit (Qiagen)
Transcriptome sequencing libraries were prepared using the TruSeq Stranded Total RNA LT sample preparation kit to deplete of ribosomal RNA prior to cDNA synthesis. After, indexed adaptors are ligated to cDNA fragments and purified using AMPureXP beads (Beckman Coulter Life Sciences, USA). Sequencing libraries are normalized prior to sequencing using the 150bp paired-end protocol on an Illumina NextSeq2000 instrument.

treatment_protocol: vehicle (DMSO), capmatinib (10uM, Catalog# S2788, Selleck Chemicals, USA), selumetinib (5uM, Catalog# S1008, Selleck Chemicals, USA), mirdametinib (100nM, Catalog# S1036, Selleck Chemicals, USA)

value: lesion

tag: individual

value: primary cell

value: vehicle

tag: batch


title: Wildtype cells, replicate 2 (ChIP)

city: Ann Arbor

first: Vivian

last: Ramirez

address: 1150 W. Medical Center Dr

department: Department of Computational Medicine and Bioinformatics

institute: University of Michigan

description:
Library name: WT rep 2, ChIP
Mean enrichment scores across replicates included in *_mean.bedgraph files

data_processing:
Read pre-processing: Raw NGS reads were processed using a combination of read quality filtering, adapter trimming, alignment and mapping, and protein occupancy using the IPOD-HR pipeline (version 2.8.1) via a singularity container (accessible at https://github.com/freddolino-lab/ipod). Briefly, sequencing adapters were removed using Cutadapt (version 4.0), poor quality reads were trimmed with Trimmomatic (version 0.39) using the parameters “TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:10”.
Alignment and protein occupancy calling: Remaining reads were aligned to the S. venezuelae NRRL B-65442 NZ_CP018074.1 reference genome using bowtie2 [53](version 2.4.4) with “very sensitive, end to end” presets and dovetail alignments allowed. Only concordant pairs were used for downstream quantitation. The surviving processed reads were assessed using FASTQC [54] for sequence content, quality, and duplication and bowtie2 outputs. The quality of alignments were evaluated using bowtie2 output metrics, including the overall alignment rates. Read densities were quantile normalized, and the log ratios of IPOD-HR and RNApol ChIP-seq were calculated at a 5 bp resolution relative to the corresponding input sample. To isolate occupancy scores attributable to DNA-binding proteins distinct from RNApol, the IPOD-HR pipeline subtracts RNApol ChIP-seq normalized robust z-scores (RNApol ChIP-seq vs. input) from the IPOD normalized robust z-scores. The resulting bedgraph files, containing enrichment scores for RNApol ChIP-seq and IPOD-HR, were used for downstream analyses.
Assembly: GCF_001886595.1
Supplementary files format and content: bedgraph

organism: Streptomyces venezuelae

source_name: S. venezuelae NRRL B-65442

growth_protocol: The S. venezuelae NRRL B-65442 strain served as the WT and base strain for all experiments conducted in this study. Spores of each strain (WT, Δlsr2, ΔlsrL, and Δlsr2ΔlsrL) were inoculated in 20 mL of MYM liquid medium and grown overnight with shaking at 30°C. Subsequently, each strain was subcultured into six independent flasks containing 100 mL of liquid MYM to a final OD600 of 0.1 and grown with shaking at 30°C. After 16 h, three 15 mL aliquots were removed from each culture, centrifuged at 7,500 rpm at 4°C for 5 minutes, and the supernatant discarded. . One of the six replicates served as the test sample for determining optimal DNase treatment conditions for the IPOD-HR and RNApol ChIP-seq experiments. For the remaining five replicates, the three replicates that yielded a complete set of the highest quality DNA for IPOD-HR and RNApol ChIP-seq were sent for next-generation sequencing (described below). This approach ensured that data was collected from the same replicates across each experiment.

extract_protocol:
Lysis of S. venezuelae cell pellets: Each of the washed cell pellets from above were resuspended in 1.5 mL lysis buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 14 mg/ml lysozyme, 0.8% Triton X-100, 1× protease inhibitor) and incubated at 37°C for 30 min. The samples were incubated on ice for 2 min, then sonicated for six cycles of 15 s continuous sonication followed by 45 s on ice (output 40%, 4). Cell debris was removed by centrifugation for 20 min at 13,000 rpm at 4°C and the supernatant was transferred to a 15 mL Falcon tube (~1.8 mL).  Determination of optimal DNase treatment conditions: One of the six replicates was used to determine the appropriate conditions for DNase treatment that would yield DNA fragments between 100-200 bp, as we found that required treatment was different for each sample. To determine the appropriate time for DNase treatment, 200 µL of 10× Turbo DNase Buffer, 10 µL Turbo DNase, and RNase A to a final concentration of 15 µg/mL were added to this sample, and incubated for 90 min at 37°C. Every 10 min during the 90-min time course, a 50 µL aliquot was collected from each sample, mixed with 10 µL of 0.5 M EDTA (pH 8), and incubated on ice to inhibit further DNase digestion. Once all aliquots had been collected, they were subjected to phenol-chloroform extraction, and 5-10 µL of the upper phase was run on a 2% agarose gel. The time that yielded the most DNA between 100-200 bp was used to digest the remaining five replicates.  DNase treatment: The remaining five replicates were DNase-treated using the experimentally determined optimal conditions. After DNase digestion, 500 µL of 0.5 M EDTA (pH 8.0) was added to quench the reaction. The samples were clarified by centrifuging at 7,500 rpm for 10 min at 4°C. A 50 µL aliquot (IPOD input DNA) was removed and diluted 1:9 in elution buffer (50 mM Tris, pH 8.0; 10 mM EDTA; 1% SDS) and kept on ice until the reverse crosslinking step. A 100 µL aliquot of the clarified lysate was phenol-chloroform extracted and 5 µL of the upper (DNA) phase was run on a 2% agarose gel to confirm that the DNase treatment was successful and had yielded DNA with a maxima centered between 100-200 bp.  RNA polymerase chromatin immunoprecipitation: Protein A-Sepharose beads were prepared in 1.5 mL tubes by resuspending 125 mg of the beads in 1.2 mL of Buffer A (0.02 M NaH2PO4, 150 mM NaCl, pH 8.0) and incubating at room temperature for 30 min. The beads were then aliquoted into separate 1.5 mL tubes for each sample (100 µL slurry per sample). The beads were equilibrated by removing the storage buffer and adding 1 mL of IP buffer with BSA (100 mM Tris pH 8.0, 300 mM NaCl, 2% Triton X-100, 0.1 mg/mL Bovine Serum Albumin). Beads were centrifuged at 3,000 rpm for 1 min at 4°C and allowed to settle on ice for 2 min. This wash step was repeated a second time, and the beads were resuspended in 100 µL IP buffer with BSA. To pre-clear the lysate, the remaining clarified lysate from after DNase treatment (~1 mL) was added to the equilibrated beads and incubated with gentle shaking at 4°C for 2 h. The beads were pelleted as above, the supernatant (pre-cleared lysate) was transferred to a clean tube, and 10 µL of anti-RNA polymerase antibody (developed in mouse) was added to each sample. The samples were incubated overnight with gentle shaking at 4°C. After removing the lysate, the beads were washed three times with 1 mL of Buffer A and stored at 4°C. The next day, the beads were equilibrated as before in IP buffer with BSA. The antibody-lysate mixture was added to the beads and incubated with gentle shaking at 4°C for 4 h. The beads were centrifuged as above and most of the supernatant discarded by removing with a pipette. The beads were washed at room temperature with 1 mL of each of the following solutions in order: ·       Wash buffer A (100 mM Tris, pH 8.0; 250 mM LiCl; 2% Triton X-100; 1 mM EDTA) ·       Wash buffer B (100 mM Tris, pH 8.0; 500 mM NaCl; 1% Triton X-100; 0.1% sodium deoxycholate; 1 mM EDTA) ·       Wash buffer C (10 mM Tris, pH 8.0; 500 mM NaCl; 1% Triton X-100; 1 mM EDTA) ·       IP buffer with EDTA (100 mM Tris, pH 8.0; 300 mM NaCl; 2% Triton X-100; 1 mM EDTA) ·       TE buffer (10 mM Tris, pH 8.0; 1 mM EDTA) After washing, the beads were resuspended in 500 µL elution buffer and protein-DNA crosslinking was reversed by incubating the samples at 65°C overnight. Samples were treated with RNase A and proteinase K, and the DNA precipitated.
All purified DNA samples were run on an Agilent TapeStation to assess DNA quality prior to library preparation. Libraries were prepared for Illumina sequencing using the NEBNext Ultra II DNA kit (New England Biolabs). The samples were pooled into libraries and sequenced on an Illumina NextSeq 2000, in a P3, 2x50bp configuration at the McMaster Genomics Facility.

treatment_protocol: To the remaining 50 mL of culture, rifampicin was added to a final concentration of 500 mg/mL and the cultures were incubated with shaking at room temperature for 10 min (rifampicin was used to trap RNA polymerase at the promoter and prevent further transcription initiation). Formaldehyde was then added to a final concentration of 1% (v/v) to induce protein and DNA crosslinking, and the cultures were incubated for an additional 30 min at room temperature. Next, glycine was added to a final concentration of 125 mM to quench the crosslinking, after which the cultures were incubated for another 5 min. The cultures were then centrifuged in 50 mL Falcon tubes at 7,500 rpm at 4°C for 5 min, after which the supernatant was discarded. The resulting cell pellets were washed three times with phosphate-buffered saline (PBS), by resuspending the pellets in 20-40 mL cold PBS and centrifuging as above.

tag: strain

value: S. venezuelae NRRL B-65442

value: WT

tag: developmental stage

value: Pre-sporulation phase

tag: media

value: MYM liquid media

value: rifampicin

tag: chip antibody

value: anti-RNA polymerase antibody


title: W303 Prp19-AID Auxin-treated cells replicate 1

description: Library name: Prp19_1_AUX


title: Wildtype cells, replicate 3 (ChIP)

description:
Library name: WT rep 3, ChIP
Mean enrichment scores across replicates included in *_mean.bedgraph files


title: HeLa, untreated, antibody CTCF (CUT&RUN), rep 1

description: Library name: C4


title: Wildtype cells, replicate 1 input

description:
Library name: WT rep 1, input
Mean enrichment scores across replicates included in *_mean.bedgraph files


title: PETER001_HEPG2_24H_X1_B46:F06

type: RNA

city: New York

first: Avi

last: Ma'ayan

state: NY

address: One Gustave L. Levy Place, Box 1603

department: Pharmacological Sciences

institute: Icahn School of Medicine at Mount Sinai

description: L1000 inferred profile; pert_id: Dolutegravir|Tenofovir Alafenamide Fumarate

scan_protocol: Following treatment and lysis, gene expression was profiled using the L1000 platform, a high-throughput assay that measures the expression of ∼1,000 landmark genes and computationally infers the expression of nonmeasured transcripts.

data_processing: The data was normalized relative to the expression of 80 invariant landmark control genes. The expression of additional genes was inferred via a linear model trained on thousands of Affymetrix gene expression datasets from the gene expression omnibus (GEO). In this study, we utilized the L1000 data with the 12,328 measured and inferred mRNAs.

label: biotin

source_name: HEPG2 treated with Dolutegravir|Tenofovir Alafenamide Fumarate 0.1|0.5 uM for 24.0 h

label_protocol: biotin labeling. Processing was done by th Broad Institute as previously described (10.1016/j.cell.2017.10.049)

growth_protocol:
HepG2 cells (5000 cells/well; ATCC, HB-8065) were cultured in Eagle's Minimum Essential Medium (EMEM; ATCC, cat. no.  30-2003), supplemented with 10% FBS and 1% P/S  at 37°C, 5% CO2.
HK-2 cells (5000 cells/well; ATCC, cat. no. CRL-2190) were cultured in Keratinocyte Serum Free Medium (K-SFM), Kit (Gibco, cat. no. 17005-042)  at 37°C, 5% CO2.
THP-1 cells (10,000 cells/well; ATCC, cat. no. TIB-202) were cultured in RPMI-40 medium (Gibco, cat. no. 11875-093) supplemented with 10% fetal bovine serum (FBS, Gibco, cat. no. 10438026) and 1% penicillin/streptomycin (P/S, Corning, cat. no. 30-002) at 37°C, 5% CO2. Macrophage differentiation was induced in vitro with phorbol-12-myristate-13-acetate (PMA, 50 ng/mL; Sigma) for 72 hours at 37°C in 5% CO2

extract_protocol: After treatment, cells were lysed using the TCL Buffer (Qiagen, cat. no. 1031576) and incubated for 30 minutes at 37°C. The lysates were then stored at -80°C. RNA was extracted by the Broad Institute, according to their protocol.

treatment_protocol: After 24 hours of culture for HepG2 and HK-2 cells, and 72 hours for THP-1 macrophages, the cells were placed in their respected culture media supplemented with 2% FBS for 16 hours. Subsequently, they were treated for 6 or 24 hours with either vehicle (DMSO) or with ART medications at a concentration of 0.1, or 0.5 µM.

tag: duration

tag: plate

tag: well


title: HeLa, untreated, antibody H3K4me3 (CUT&RUN), rep 2

description: Library name: C3


title: Wildtype cells, replicate 2 input

description:
Library name: WT rep 2, input
Mean enrichment scores across replicates included in *_mean.bedgraph files


title: Wildtype cells, replicate 1 (ChIP)

description:
Library name: WT rep 1, ChIP
Mean enrichment scores across replicates included in *_mean.bedgraph files


title: PETER001_HEPG2_24H_X1_B46:F05

value: Dolutegravir|Tenofovir Alafenamide Fumarate 0.1|0.5 uM


title: HeLa, untreated, antibody H3K4me3 (CUT&RUN), rep 1

description: Library name: C2


title: Δlsr2ΔlsrL mutant cells, replicate 3 (input)

description:
Library name: Δlsr2ΔlsrL rep 3, input
Mean enrichment scores across replicates included in *_mean.bedgraph files

extract_protocol:
Lysis of S. venezuelae cell pellets: Each of the washed cell pellets from above were resuspended in 1.5 mL lysis buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 14 mg/ml lysozyme, 0.8% Triton X-100, 1× protease inhibitor) and incubated at 37°C for 30 min. The samples were incubated on ice for 2 min, then sonicated for six cycles of 15 s continuous sonication followed by 45 s on ice (output 40%, 4). Cell debris was removed by centrifugation for 20 min at 13,000 rpm at 4°C and the supernatant was transferred to a 15 mL Falcon tube (~1.8 mL).  Determination of optimal DNase treatment conditions: One of the six replicates was used to determine the appropriate conditions for DNase treatment that would yield DNA fragments between 100-200 bp, as we found that required treatment was different for each sample. To determine the appropriate time for DNase treatment, 200 µL of 10× Turbo DNase Buffer, 10 µL Turbo DNase, and RNase A to a final concentration of 15 µg/mL were added to this sample, and incubated for 90 min at 37°C. Every 10 min during the 90-min time course, a 50 µL aliquot was collected from each sample, mixed with 10 µL of 0.5 M EDTA (pH 8), and incubated on ice to inhibit further DNase digestion. Once all aliquots had been collected, they were subjected to phenol-chloroform extraction, and 5-10 µL of the upper phase was run on a 2% agarose gel. The time that yielded the most DNA between 100-200 bp was used to digest the remaining five replicates.  DNase treatment: The remaining five replicates were DNase-treated using the experimentally determined optimal conditions. After DNase digestion, 500 µL of 0.5 M EDTA (pH 8.0) was added to quench the reaction. The samples were clarified by centrifuging at 7,500 rpm for 10 min at 4°C. A 50 µL aliquot (IPOD input DNA) was removed and diluted 1:9 in elution buffer (50 mM Tris, pH 8.0; 10 mM EDTA; 1% SDS) and kept on ice until the reverse crosslinking step. A 100 µL aliquot of the clarified lysate was phenol-chloroform extracted and 5 µL of the upper (DNA) phase was run on a 2% agarose gel to confirm that the DNase treatment was successful and had yielded DNA with a maxima centered between 100-200 bp.  IPOD-HR: One milliliter of the resulting clarified lysate was transferred to a 15 mL Falcon tube and mixed with an equal volume of 100 mM Tris base. An equal volume of 25:24:1 phenol:chloroform:isoamyl alcohol was added, the sample vortexed, and incubated for 10 min at room temperature. After incubation, the sample was centrifuged at 7,500 rpm for 5 min at 4°C, allowing the formation of a white disc at the aqueous–organic interface enriched for protein–DNA complexes. Both the upper and lower aqueous phases were removed with a pipette and discarded. The remaining interface disc was washed with 350 µL TE buffer (10 mM Tris, pH 8.0; 1 mM EDTA), 350 µL 100 mM Tris base, and 700 µL 24:1 chloroform:isoamyl alcohol. The sample was vortexed and centrifuged as above. The upper and lower aqueous phases were removed again, and the interface disc was washed with 1 mL TE buffer and 1 mL 24:1 chloroform:isoamyl alcohol. The sample was vortexed and centrifuged as above, and both upper and lower aqueous phases were discarded. The interface disc was resuspended in 700 µL of elution buffer by vortexing. To reverse protein-DNA crosslinking, the input and IPOD-HR samples were incubated at 65°C overnight. The next day, the samples were allowed to cool, and RNase A was added to each input and IPOD-HR sample to a final concentration of 60 µg/mL. The samples were incubated at 37°C for 2 h. Proteinase K was then added to a final concentration of 120 µg/mL and incubated at 50°C for 2 h. DNA was phenol-chloroform extracted, the upper phase was transferred to a clean tube and precipitated by the addition of 2 volumes of cold 95% ethanol and 0.1 volume of precipitation buffer (5 M CH3COONa/CH3COOH, pH 5.2) and incubation at -20°C overnight. The next day, samples were centrifuged at 13,000 rpm for 30 min at 4°C, and the supernatant discarded. The DNA pellet was washed with ice-cold 70% ethanol and dried by placing the tube up-side-down on a paper towel. The DNA pellet was resuspended in 30 µL 10 mM Tris-HCl, pH 8.0.
All purified DNA samples were run on an Agilent TapeStation to assess DNA quality prior to library preparation. Libraries were prepared for Illumina sequencing using the NEBNext Ultra II DNA kit (New England Biolabs). The samples were pooled into libraries and sequenced on an Illumina NextSeq 2000, in a P3, 2x50bp configuration at the McMaster Genomics Facility.


title: W303 Prp19-AID DMSO-treated cells replicate 3

description: Library name: Prp19_3_DMSO

value: DMSO (mock)


title: Δlsr2ΔlsrL mutant cells, replicate 3 (IPOD-HR)

description:
Library name: Δlsr2ΔlsrL rep 3, ChIP
Mean enrichment scores across replicates included in *_mean.bedgraph files


title: PETER001_HEPG2_24H_X1_B46:F03

description: L1000 inferred profile; pert_id: Cobicistat|Emtricitabine|Tenofovir Alafenamide Fumarate

source_name: HEPG2 treated with Cobicistat|Emtricitabine|Tenofovir Alafenamide Fumarate 0.1|0.1|0.5 uM for 24.0 h


title: W303 Prp19-AID DMSO-treated cells replicate 2

description: Library name: Prp19_2_DMSO

value: yeast cells


title: PETER001_HEPG2_24H_X1_B46:F04


title: W303 Prp19-AID DMSO-treated cells replicate 1

description: Library name: Prp19_1_DMSO

value: W303 WT; Prp19-AID


title: Δlsr2ΔlsrL mutant cells, replicate 2 (IPOD-HR)

description:
Library name: Δlsr2ΔlsrL rep 2, ChIP
Mean enrichment scores across replicates included in *_mean.bedgraph files


title: perictyes, 0.1 ng/ml TcdB2 + 10 µM XMU-MP1 24 h, rep 2

description:
Library name: F2
coloumn name in all_genes_counts.csv: F2_663557998

data_processing:
The FASTQ files were processed and aligned to the Human GRCh38 genome using  DRAGEN RNA v4.2.4 (Basespace Illumina).Differential expression was performed using DRAGEN Differential Expression v4.2.4 (Basespace Illumina).
Assembly: Human GRCh38 genome
Supplementary files format and content: The number of reads aligned to specific genes.

source_name: primary cultured pericytes

growth_protocol: Primary human brain pericytes (ACBRI 498) were purchased from Cell Systems and cultured following the protocol provided by Cell Systems.

extract_protocol:
RNA was isolated in duplicate from cultured pericytes  using Direct-Zol microprep (Zymogen) and suspended in RNase-free water
Stranded RNA-seq libraries were constructed with 1 µg of RNA using the NEBNext poly(A) mRNA isolation kit (New England Biolabs) followed directly by the  XGen Broad Range RNA Library Prep kit (IDT).  Each of the libraries was indexed during library construction in order to multiplex for sequencing. Libraries were quantified using a Qubit 4 fluorometer (Invitrogen) and checked for size and quality on an Agilent Bioanalyzer (Agilent Technology).

treatment_protocol: Treated with vehicle control (0.1 % DMSO), 0.1 ng/ml TcdB2 +  0.1 % DMSO, 10 µM XMU-MP1, or 0.1 ng/ml TcdB2 + 10 µM XMU-MP1

value: primary cultured pericytes


title: perictyes, 0.1 ng/ml TcdB2 + 10 µM XMU-MP1 24 h, rep 1

description:
Library name: F1
coloumn name in all_genes_counts.csv: F1_663557995


title: FLANK, 1:0.1, replicate 2

description:
Library name: FLANK_0.1_rep2
FLANK_0.1_deseq2.csv


title: HeLa, untreated, antibody isotype control (CUT&RUN), rep 1

description: Library name: C1


title: PETER001_HEPG2_24H_X1_B46:F01

value: Cobicistat|Emtricitabine|Tenofovir Alafenamide Fumarate 0.1|0.1|0.5 uM


title: PETER001_HEPG2_24H_X1_B46:F02


title: Δlsr2ΔlsrL mutant cells, replicate 2 (input)

description:
Library name: Δlsr2ΔlsrL rep 2, input
Mean enrichment scores across replicates included in *_mean.bedgraph files


title: perictyes, 10 µM XMU-MP1 24 h, rep 1

description:
Library name: D1
coloumn name in all_genes_counts.csv: D1_663557994


title: non-cardiomyocytes from digested mouse heart harvested 20 days after sham surgery

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

institute: MPI for heart and lung research

description: Library name: Heart-Sham

library_source: transcriptomic single cell

data_processing:
Reads were aligned against the mouse genome (mm10) and counted by CellRanger (10xGenomics).
Preprocessed counts were further analysed using Seurat.
Assembly: mm10
Supplementary files format and content: cellranger outputs

organism: Mus musculus

source_name: heart

extract_protocol:
adult non-cardiomyocytes were isolated using the Langendorff-free method61, with some modifications: After euthanizing mice, the chest was opened to expose and cut the descending aorta. The ascending aorta was clamped with a Reynolds forceps and the right ventricle was injected with 15 ml of EDTA buffer (130 mM NaCl, 5 mM KCl, 0.5 mM NaH2PO4 , 10 mM HEPES, 10 mM Glucose, 10 mM BDM, 10 mM taurine, 5 mM EDTA). The perfused heart was then transferred to a plastic dish containing EDTA buffer and 10 mL perfusion buffer (130 mM NaCl, 5 mM KCl, 0.5 mM NaH2PO4, 10 mM HEPES, 10 mM glucose, 10 mM BDM, 10 mM taurine, 1 mM MgCl2) were injected into the left ventricle. This was followed by repeated (approximately 4) injections of 10 ml digestion buffer (perfusion buffer plus collagenase II 0.5 mg/mL (Worthington Biochemical Corporation, Cat. LS004177), collagenase IV 0.5 mg/Ml (Sigma Aldrich, C5138), protease XIV 0.05 mg/mL) (Sigma Aldrich, P5147)) until the heart became pale and soft. The ventricle was then minced with a forceps and cellular dissociation was completed by gentle trituration of heart fragments. 5 mL stopping buffer (perfusion buffer with 5% FCS) were added to terminate digestion. The cell suspension was passed through a 100-μm filter and cardiomyocytes were allowed to settle by gravity for 20-30 minutes at room temperature. Non-cardiomyocytes were collected from the supernatant, followed by red blood cells lysis using RBC lysis buffer (Thermo Scientific Chemicals, J62150.AP) and debris removal using Debris Removal Solution (Miltenyi, 130-109-398). Dead cells were stained with DAPI and viable single cells were then sorted by a flow cytometer (BD, FACSAria III).
The cell suspensions were counted with Moxi cell counter and diluted according to manufacturer’s protocol to obtain 10.000 single cell data points per sample. Each sample was run separately on a lane in Chromium controller with Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10xGenomics). Single cell RNAseq library preparation was done using a standard protocol.

value: heart

value: non-cardiomyocytes

tag: gender

value: male

value: C57BL6/J wildtype

value: d20 after sham surgery


title: Dis3WT_Naive

city: Warszawa

first: Tomasz

middle: M.

last: Kuliński

state: Mazowieckie

address: ul. Ks. Trojdena 4

country: Poland

institute: International Institute of Molecular and Cell Biology in Warsaw

data_processing:
Library strategy: DamID-seq
saples were mapped with STAR split read alligner
mapped reads were processed, QC and filtered by custom scripts empling elements of SAMtools, bedtools, Bwtool, RSeQC packages
Differential expression analysis was performed using the DESeq2 bioconductor package
genomic structural variants were called using SVDetect
mait-pair reads preprocessed using Nxtrim
Genome_build: human samples were mapped to the GRCh38 reference geneome murine samples were mapped to the GRCm38 reference geneome

source_name: mouse CH12F3 B lymphocyte cell line lentivirally transfected with Dam taged DIS3 WT protein

growth_protocol: The mouse CH12F3 B lymphocyte cell line was acquired from the Ricken BRC cell bank. Cells were cultured in RPMI 1640 ATCC’s modified medium,supplemented with 10% FBS, 5% NCTC-109 medium, 50 μM 2-mercaptoethanol, penicillin/streptomycin.

extract_protocol: RNA was isolated using TRIreagent DNA was extracted

value: CH12F3-2a cell line

tag: dis3 genotype/expressed variant

value: DIS3 WT


title: non-cardiomyocytes from digested mouse heart harvested 20 days after transverse aortic constriction (TAC)

description: Library name: Heart-TAC

value: d20 after TAC surgery


title: Δlsr2ΔlsrL mutant cells, replicate 1 (input)

description:
Library name: Δlsr2ΔlsrL rep 1, input
Mean enrichment scores across replicates included in *_mean.bedgraph files

value: S. venezuelae NRRL B-65442

value: Pre-sporulation phase

value: MYM liquid media


title: PETER001_HEPG2_24H_X1_B46:F07

description: L1000 inferred profile; pert_id: Darunavir|Ritonavir

source_name: HEPG2 treated with Darunavir|Ritonavir 0.1|0.5 uM for 24.0 h

value: Darunavir|Ritonavir 0.1|0.5 uM


title: perictyes, 10 µM XMU-MP1 24 h, rep 2

description:
Library name: D2
coloumn name in all_genes_counts.csv: D2_663557997


title: Δlsr2ΔlsrL mutant cells, replicate 1 (IPOD-HR)

description:
Library name: Δlsr2ΔlsrL rep 1, ChIP
Mean enrichment scores across replicates included in *_mean.bedgraph files

value: rifampicin


title: PETER001_HEPG2_24H_X1_B46:E21

description: L1000 inferred profile; pert_id: Rapamycin

source_name: HEPG2 treated with Rapamycin 5 uM for 24.0 h

value: Rapamycin 5 uM


title: PETER001_HEPG2_24H_X1_B46:E20


title: PETER001_HEPG2_24H_X1_B46:E19


title: perictyes, 0.1 ng/ml TcdB2 +  0.1 % DMSO 24 h, rep 2

description:
Library name: C2
coloumn name in all_genes_counts.csv: C2_663558060


title: ΔlsrL mutant cells, replicate 2 (input)

description:
Library name: ΔlsrL rep 2, input
Mean enrichment scores across replicates included in *_mean.bedgraph files

value: dlsrL


title: Dis3WT_Act2

growth_protocol: The mouse CH12F3 B lymphocyte cell line was acquired from the Ricken BRC cell bank. Cells were cultured in RPMI 1640 ATCC’s modified medium,supplemented with 10% FBS, 5% NCTC-109 medium, 50 μM 2-mercaptoethanol, penicillin/streptomycin. CH12F3 cells were activated for 3 days by supplementing the standard culture medium with 1 ng/ml TGF-𝛃1, IL-4 5 ng/ml, 1 μg/ml 𝛂CD40.


title: PETER001_HEPG2_24H_X1_B46:E18

description: L1000 inferred profile; pert_id: Tenofovir Disoproxil Fumarate

source_name: HEPG2 treated with Tenofovir Disoproxil Fumarate 0.1 uM for 24.0 h

value: Tenofovir Disoproxil Fumarate 0.1 uM


title: perictyes, 0.1 ng/ml TcdB2 +  0.1 % DMSO 24 h, rep 1

description:
Library name: C1
coloumn name in all_genes_counts.csv: C1_663558059

value: primary cultured pericytes


title: ΔlsrL mutant cells, replicate 2 (IPOD-HR)

description:
Library name: ΔlsrL rep 2, ChIP
Mean enrichment scores across replicates included in *_mean.bedgraph files


title: pericytes, vehicle control (0.1 % DMSO) 24 h, rep 2

description:
Library name: A2
coloumn name in all_genes_counts.csv: A2_663557996


title: PETER001_HEPG2_24H_X1_B46:E16


title: Dis3WT_Act3


title: pericytes, vehicle control (0.1 % DMSO) 24 h, rep 1

description:
Library name: A1
coloumn name in all_genes_counts.csv: A1_663557993


title: ΔlsrL mutant cells, replicate 1 (input)

description:
Library name: ΔlsrL rep 1, input
Mean enrichment scores across replicates included in *_mean.bedgraph files


title: Dis3G766R_Naive

source_name: mouse CH12F3 B lymphocyte cell line lentivirally transfected with Dam taged DIS3G766R protein

value: DIS3 G766R


title: PETER001_HEPG2_24H_X1_B46:E17


title: ΔlsrL mutant cells, replicate 1 (IPOD-HR)

description:
Library name: ΔlsrL rep 1, ChIP
Mean enrichment scores across replicates included in *_mean.bedgraph files


title: Dis3G766R_Act2


title: Δlsr2 mutant cells, replicate 3 (input)

description:
Library name: Δlsr2 rep 3, input
Mean enrichment scores across replicates included in *_mean.bedgraph files


title: Dis3WT_Act1


title: dKO4M2Dex

city: RTP

first: Thomas

last: Randall

state: NC

address: 101 TW Alexander

institute: NIEHS

description: Library name: Sample 48

data_processing:
Alignment using STAR (V 2.5.1).
Read count was produced with featureCount (subread, Version 1.5.1)
Statistical analysis done with DEseq2 (v 1.38.3)
DEG was obtained using adjusted pvalue <0.05
For Cut&Tag, reads were trimmed using Cutadapt and aligned to both mm10 and hg19 genomes using Bowtie2. Aligned reads were deduplicated with picardtools. Deduplicated read counts were used to calculate the mouse:human ratio, which was then used to calculate scaling factors for samples performed side by side with the same antibody. The scale factor was calculated by dividing the mouse:human read ratio for each sample using a given antibody by the maximum human:mouse read ratio from samples using the same antibody. Scaled and normalized coverage files were generated using deepTools with calculated scale factors and rpkm normalization.
Assembly: mm39/genecode_vM34
Supplementary files format and content: readcounts in txt format

growth_protocol: Bone marrow monocytes (BMM) were purified through negative selection using the EasySepTM mouse monocytes Isolation kit and resuspended in complete medium comprising RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, and 100 U/mL penicillin-streptomycin and 75 ng/mL murine recombinant M-CSF. After 7 days, 1x10^6 BMDM were re-seeded in 6-well plate and polarized to M1 and M2- profiles.

extract_protocol:
The cell lysates were collected after the 4 hour Vehicle or Dex treatment. Total RNA was isolated from the primary macrophages using Qiagen RNeasy mini kit.
The integrity of the total RNA was determined using the TapeStation (Agilent Technologies), and RNA concentration was assessed using a Qubit (ThermoFisher Scientific).  A TruSeq RNA kit (Illumina, San Diego, CA) was utilized to prepare the poly(A)-enriched RNA-seq libraries that were sequenced on the Illumina NovaSeq 6000 in a 75-base paired-end mode according to the manufacturer’s protocol.

treatment_protocol: M0, M1-like and M2-like macrophages from WT and GRKO were stimulated with vehicle or 100nM Dex for 4 hours for gene expression.

tag: Sex

value: GRKO

value: Dex


title: Δlsr2 mutant cells, replicate 3 (IPOD-HR)

description:
Library name: Δlsr2 rep 3, ChIP
Mean enrichment scores across replicates included in *_mean.bedgraph files


title: PETER001_HEPG2_24H_X1_B46:E14

description: L1000 inferred profile; pert_id: Cobicistat

source_name: HEPG2 treated with Cobicistat 0.1 uM for 24.0 h

value: Cobicistat 0.1 uM


title: dKO4M2Veh

description: Library name: Sample 47

tag: tissue

value: male

value: Vehicle


title: PETER001_HEPG2_24H_X1_B46:E15

tag: cell line


title: Dis3G766R_Act3

tag: cell type

value: CH12F3-2a cell line


title: DamOnly_Naive

source_name: mouse CH12F3 B lymphocyte cell line lentivirally transfected with Dam


title: Δlsr2 mutant cells, replicate 2 (input)

description:
Library name: Δlsr2 rep 2, input
Mean enrichment scores across replicates included in *_mean.bedgraph files

tag: genotype


title: PETER001_HEPG2_24H_X1_B46:E13

tag: treatment


title: dKO4M1Dex

description: Library name: Sample 46


title: dKO4M1Veh

description: Library name: Sample 45


title: Dis3G766R_Act1


title: PETER001_HEPG2_24H_X1_B46:E11

description: L1000 inferred profile; pert_id: Bictegravir|Tenofovir Alafenamide Fumarate

source_name: HEPG2 treated with Bictegravir|Tenofovir Alafenamide Fumarate 0.5|0.5 uM for 24.0 h


title: Δlsr2 mutant cells, replicate 1 (input)

description:
Library name: Δlsr2 rep 1, input
Mean enrichment scores across replicates included in *_mean.bedgraph files


title: DamOnly_Act3


title: dKO4M0Dex

description: Library name: Sample 44


title: dKO4M0Veh

description: Library name: Sample 43


title: dKO3M2Dex

description: Library name: Sample 42


title: Δlsr2 mutant cells, replicate 1 (IPOD-HR)

description:
Library name: Δlsr2 rep 1, ChIP
Mean enrichment scores across replicates included in *_mean.bedgraph files


title: PETER001_HEPG2_24H_X1_B46:E12

value: Bictegravir|Tenofovir Alafenamide Fumarate 0.5|0.5 uM


title: TotalAscites_57p

data_processing:
saples were mapped with STAR split read alligner
mapped reads were processed, QC and filtered by custom scripts empling elements of SAMtools, bedtools, Bwtool, RSeQC packages
Differential expression analysis was performed using the DESeq2 bioconductor package
genomic structural variants were called using SVDetect
mait-pair reads preprocessed using Nxtrim
Genome_build: human samples were mapped to the GRCh38 reference geneome murine samples were mapped to the GRCm38 reference geneome

source_name: murine Pristane induced peritoneal plasmocytoma

growth_protocol: Mice were injected 3 times intraperitoneally, in 2-month intervals, with 0.5 ml of pristane oil (2,6,10,12-tetramethylpentadecane) to induce the development of mouse plasmacytoma (MPC), an established murine model of multiple myeloma. The ascit was collected by paracentesis.

value: Peritoneal plasma cells


title: PETER001_HEPG2_24H_X1_B46:E08

description: L1000 inferred profile; pert_id: Bictegravir|Emtricitabine

source_name: HEPG2 treated with Bictegravir|Emtricitabine 0.5|0.1 uM for 24.0 h

value: Bictegravir|Emtricitabine 0.5|0.1 uM


title: Δlsr2 mutant cells, replicate 2 (IPOD-HR)

description:
Library name: Δlsr2 rep 2, ChIP
Mean enrichment scores across replicates included in *_mean.bedgraph files

value: S. venezuelae NRRL B-65442

value: Pre-sporulation phase

value: MYM liquid media


title: PETER001_HEPG2_24H_X1_B46:E07


title: PETER001_HEPG2_24H_X1_B46:E10


title: dKO3M1Dex

description: Library name: Sample 40


title: Wildtype cells, replicate 2 (input)

description:
Library name: WT rep 2, input
Mean enrichment scores across replicates included in *_mean.bedgraph files

value: WT

value: rifampicin


title: dKO3M2Veh

description: Library name: Sample 41


title: TotalAscites_10p


title: DamOnly_Act1


title: DamOnly_Act2


title: WT_43_MEF766

source_name: primary Mouse Embryonic Fibroblasts WT DIS3 replicat_4,3

growth_protocol: Primary cell cultures of mouse embryonic fibroblasts (MEF) will be isolated from E13.5 embryos as defined by the date of the vaginal plug and confirmed by characteristic features of the embryo morphology. The corpses was be gutted and mechanically disintegrated by chopping and passing the tissue fragments suspended in culture media (DMEM supplemented with 10% FBS, 50 μM 2-mercaptoethanol, penicillin/streptomycin) with a syringe through the gauge 19 needle. Cells were passaged for at least 3 passages before RNA was collected.

value: Mouse embryonic fibroblast


title: Wildtype cells, replicate 2 (IPOD-HR)

description:
Library name: WT rep 2, ChIP
Mean enrichment scores across replicates included in *_mean.bedgraph files


title: PETER001_HEPG2_24H_X1_B46:E06

description: L1000 inferred profile; pert_id: Emtricitabine|Tenofovir Alafenamide Fumarate

source_name: HEPG2 treated with Emtricitabine|Tenofovir Alafenamide Fumarate 0.1|0.5 uM for 24.0 h


title: Wildtype cells, replicate 3 (IPOD-HR)

description:
Library name: WT rep 3, ChIP
Mean enrichment scores across replicates included in *_mean.bedgraph files

source_name: S. venezuelae NRRL B-65442


title: dKO3M1Veh

description: Library name: Sample 39


title: PETER001_HEPG2_24H_X1_B46:E05

value: Emtricitabine|Tenofovir Alafenamide Fumarate 0.1|0.5 uM


title: dKO3M0Dex

description: Library name: Sample 38


title: Wildtype cells, replicate 1 (input)

description:
Library name: WT rep 1, input
Mean enrichment scores across replicates included in *_mean.bedgraph files


title: PETER001_HEPG2_24H_X1_B46:E04


title: Wildtype cells, replicate 3 (input)

description:
Library name: WT rep 3, input
Mean enrichment scores across replicates included in *_mean.bedgraph files


title: HET_44_MEF766

source_name: primary Mouse Embryonic Fibroblasts hetetozygous for the DIS3 G766R mutattion from replicat_4.4


title: Wildtype cells, replicate 1 (IPOD-HR)

description:
Library name: WT rep 1, ChIP
Mean enrichment scores across replicates included in *_mean.bedgraph files


title: WT_15_MEF766

source_name: primary Mouse Embryonic Fibroblasts WT DIS3 replicat_1,5


title: PETER001_HEPG2_24H_X1_B46:E01

description: L1000 inferred profile; pert_id: Cobicistat|Elvitegravir|Emtricitabine

source_name: HEPG2 treated with Cobicistat|Elvitegravir|Emtricitabine 0.1|0.1|0.1 uM for 24.0 h


title: WT_22_MEF766

source_name: primary Mouse Embryonic Fibroblasts WT DIS3 replicat_2,2


title: dKO2M2Dex

description: Library name: Sample 36


title: dKO3M0Veh

description: Library name: Sample 37


title: dKO2M2Veh

description: Library name: Sample 35


title: PETER001_HEPG2_24H_X1_B46:E03


title: Δlsr2ΔlsrL mutant cells, replicate 2 (RNA-seq)

description: Library name: Δlsr2ΔlsrL rep 2, RNA-seq

data_processing:
The raw RNA-sequencing reads were processed using the IPOD-HR pipeline, beginning with the removal of Illumina adapter sequences with cutadapt (version 4.0) and trimming of low quality reads with trimmomatic (version 0.39), as described above. High quality reads were evaluated using FASTQC for quality, sequence duplication, and sequence content.
Transcript abundance from processed RNA-seq reads was estimated in transcripts per million (TPM) units using Kallisto (version 0.44.0). Although rRNA depletion was performed experimentally, rRNA and tRNA gene annotations were also removed from the gene-level annotation FASTA file of the S. venezuelae NRRL B-65442 NZ_CP018074.1 reference genome to further ensure that transcript abundance estimates were not biased by residual rRNA or tRNA. Additionally, each gene sequence was extended by 50 bp upstream and downstream in a strand-aware manner to account for potential reads overlapping gene boundaries. The modified annotation fasta file was then used to construct the Kallisto index with a kmer size of 17 (“-k 17”). RNA-seq reads were pseudoaligned and quantified with Kallisto (version 0.44.0) with default settings save for a change in number of bootstraps: “-b 100, -rf-stranded”. The quality of pseudoalignment was evaluated across all samples using Kallisto metric outputs, including percent pseudoaligned and percent unique. To assess replicate-to-replicate consistency, Spearman’s rank correlation coefficients were calculated in Python 3.10.10 based on the quantified transcript abundances. Due to the inconsistency and poor quality of replicate 3 in the ΔlsrL mutant, based on Spearman’s rank correlation coefficient, and pseudoalignment metrics, replicate 3 was removed, leaving three replicates for WT, three replicates for Δlsr2, two replicates for ΔlsrL, and three replicates for Δlsr2ΔlsrL for gene expression analysis. Differential gene expression calling of quantified reads was performed using the sleuth  (version 0.30.1) R package, employing the Sleuth response error measurement (full) model with the formula “~genotype” where genotype represents the four conditions (WT, Δlsr2, ΔlsrL, and Δlsr2ΔlsrL). The Wald test was used to quantify differential expression values between each genotype and the WT.
Assembly: GCF_001886595.1
Supplementary files format and content: Tab separated files of the normalized abundance estimates and transcript length information.

growth_protocol: The S. venezuelae NRRL B-65442 strain served as the WT and base strain for all experiments conducted in this study. Spores of each strain (WT, Δlsr2, ΔlsrL, and Δlsr2ΔlsrL) were inoculated in 20 mL of MYM liquid medium and grown overnight with shaking at 30°C. Subsequently, each strain was subcultured into six independent flasks containing 100 mL of liquid MYM to a final OD600 of 0.1 and grown with shaking at 30°C. After 16 h, three 15 mL aliquots were removed from each culture, centrifuged at 7,500 rpm at 4°C for 5 minutes, and the supernatant discarded. These aliquots were stored at -80°C and subsequently used for RNA extraction.

extract_protocol:
Using the cell pellets that were set aside from the cultures used for IPOD-HR and RNApol ChIP-seq, RNA was extracted from three to five replicates each of WT, Δlsr2, ΔlsrL, and Δlsr2/ΔlsrL mutant strains. RNA was isolated as described in Moody et al. (2013), using a modified guanidium thiocyanate protocol (Chomczynski and Sacchi, 1987). Primers amplifying a 144 bp region of rpoB (vnz_21470) were used for PCR checks, alongside a quantified chromosomal DNA control, to confirm the absence of DNA contamination. RNA quality was assessed first by agarose gel electrophoresis, and subsequently using an Agilent 2100 Bioanalyzer. In the case where RNA was successfully extracted from more than three replicates, those with the highest quality were used for sequencing. Purified RNA was depleted of rRNA using the Bacterial NEBNext rRNA Depletion Kit (New England Biolabs).
Library preparation was conducted using the NEBNext Ultra II Directional RNA kit (New England Biolabs). The samples were pooled into libraries and were sequenced on an Illumina NextSeq 2000, P3, in a 2×50bp configuration at the McMaster Genomics Facility.

treatment_protocol: (see growth protocol)


title: HET_24_MEF766

source_name: primary Mouse Embryonic Fibroblasts hetetozygous for the DIS3 G766R mutattion from replicat_2.4


title: Δlsr2ΔlsrL mutant cells, replicate 3 (RNA-seq)

description: Library name: Δlsr2ΔlsrL rep 3, RNA-seq


title: PETER001_HEPG2_24H_X1_B46:D21

description: L1000 inferred profile; pert_id: Vorinostat

source_name: HEPG2 treated with Vorinostat 5 uM for 24.0 h

value: Vorinostat 5 uM


title: PETER001_HEPG2_24H_X1_B46:D20


title: dKO2M1Dex

description: Library name: Sample 34

value: GRKO


title: Δlsr2ΔlsrL mutant cells, replicate 1 (RNA-seq)

description: Library name: Δlsr2ΔlsrL rep 1, RNA-seq


title: dKO2M1Veh

description: Library name: Sample 33


title: ΔlsrL mutant cells, replicate 2 (RNA-seq)

description: Library name: ΔlsrL rep 2, RNA-seq

value: dlsrL


title: ALL, 1:0.1, replicate 3

description:
Library name: ALL_0.1_rep3
ALL_0.2_deseq2.csv

source_name: Artificial

value: Binding reactions were performed in a tube - no cells used


title: ALL, 1:0.2, replicate 3

description:
Library name: ALL_0.2_rep3
ALL_0.1_deseq2.csv


title: PETER001_HEPG2_24H_X1_B46:D19


title: ΔlsrL mutant cells, replicate 1 (RNA-seq)

description: Library name: ΔlsrL rep 1, RNA-seq


title: dKO2M0Veh

description: Library name: Sample 31

value: male


title: HET_14_MEF766

source_name: primary Mouse Embryonic Fibroblasts hetetozygous for the DIS3 G766R mutattion from replicat_1.4


title: ALL, 1:0.1, replicate 2

description:
Library name: ALL_0.1_rep2
ALL_0.2_deseq2.csv


title: dKO2M0Dex

description: Library name: Sample 32

value: Dex


title: PETER001_HEPG2_24H_X1_B46:D18

description: L1000 inferred profile; pert_id: Efavirenz

source_name: HEPG2 treated with Efavirenz 0.5 uM for 24.0 h

value: Efavirenz 0.5 uM


title: Δlsr2 mutant cells, replicate 3 (RNA-seq)

description: Library name: Δlsr2 rep 3, RNA-seq


title: PETER001_HEPG2_24H_X1_B46:D17


title: Δlsr2 mutant cells, replicate 2 (RNA-seq)

description: Library name: Δlsr2 rep 2, RNA-seq


title: ALL, 1:0.2, replicate 2

description:
Library name: ALL_0.2_rep2
ALL_0.1_deseq2.csv


title: PETER001_HEPG2_24H_X1_B46:D16


title: ALL, 1:0.1, replicate 1

description:
Library name: ALL_0.1_rep1
ALL_0.2_deseq2.csv


title: PETER001_HEPG2_24H_X1_B46:D15

description: L1000 inferred profile; pert_id: Cobicistat|Tenofovir Alafenamide Fumarate

source_name: HEPG2 treated with Cobicistat|Tenofovir Alafenamide Fumarate 0.1|0.5 uM for 24.0 h


title: Δlsr2 mutant cells, replicate 1 (RNA-seq)

description: Library name: Δlsr2 rep 1, RNA-seq


title: ALL, 1:0.2, replicate 1

description:
Library name: ALL_0.2_rep1
ALL_0.1_deseq2.csv


title: dKO1M2Veh

description: Library name: Sample 29

value: Vehicle


title: Wildtype cells, replicate 3 (RNA-seq)

description: Library name: WT rep 3, RNA-seq


title: PETER001_HEPG2_24H_X1_B46:D14

value: Cobicistat|Tenofovir Alafenamide Fumarate 0.1|0.5 uM


title: PETER001_HEPG2_24H_X1_B46:D12

description: L1000 inferred profile; pert_id: Efavirenz|Tenofovir Disoproxil Fumarate

source_name: HEPG2 treated with Efavirenz|Tenofovir Disoproxil Fumarate 0.5|0.1 uM for 24.0 h


title: FLANK, 1:0.1, replicate 3

description:
Library name: FLANK_0.1_rep3
FLANK_0.1_deseq2.csv


title: Wildtype cells, replicate 2 (RNA-seq)

description: Library name: WT rep 2, RNA-seq


title: Wildtype cells, replicate 1 (RNA-seq)

description: Library name: WT rep 1, RNA-seq


title: dKO1M2Dex

description: Library name: Sample 30


title: FLANK, no protein, replicate 3

description: Library name: FLANK_NP_rep3


title: dKO1M1Veh

description: Library name: Sample 27


title: dKO1M1Dex

description: Library name: Sample 28


title: dKO1M0Dex

description: Library name: Sample 26


title: PETER001_HEPG2_24H_X1_B46:D11

value: Efavirenz|Tenofovir Disoproxil Fumarate 0.5|0.1 uM


title: PETER001_HEPG2_24H_X1_B46:D10


title: Human_Tumor_C09_HPV_Positive

type: genomic

city: Campinas

first: Felipe

last: Silva

address: R. Márcia Mendes, 506

country: Brazil

institute: Boldrini

hyb_protocol: DNA from frozen tissues was converted using the EZ DNA Methylation kit (Zymo Research, USA) according to the manufacturerâ€™s recommendations. Bisulfite-converted DNA was hybridized in the Human Methylation 450 BeadChip microarrays (HM450K, Illumina, USA), following the Illumina Infinium HD methylation protocol.

scan_protocol: Acquisition was perfomed on the Illumina iScan SQ scanner (Illumina, USA)

data_processing: Minfi R package (Aryee et al. 2014) was used to examine IDAT files, and the functional normalization (FunNorm) method (Fortin et al. 2014) was used to normalize them. 428,062 probes remained for additional analysis after XYS and low-quality probes were eliminated.

source_name: tissue

label_protocol: Standard Illumina Protocol

extract_protocol: Genomic DNA was extracted from tumor tissue using DNeasy Blood & Tissue Kit (Qiagen) following the manufacturerâ€™s instructions

value: tissue


title: Human_Tumor_C05_HPV_Positive


title: FLANK, 1:0.2, replicate 3

description:
Library name: FLANK_0.2_rep3
FLANK_0.2_deseq2.csv


title: FLANK, 1:0.2, replicate 2

description:
Library name: FLANK_0.2_rep2
FLANK_0.2_deseq2.csv


title: dFlox4M2Dex

description: Library name: Sample 24

value: wild-type


title: PETER001_HEPG2_24H_X1_B46:D09

description: L1000 inferred profile; pert_id: Emtricitabine|Tenofovir Disoproxil Fumarate

source_name: HEPG2 treated with Emtricitabine|Tenofovir Disoproxil Fumarate 0.1|0.1 uM for 24.0 h


title: dKO1M0Veh

description: Library name: Sample 25


title: FLANK, no protein, replicate 2

description: Library name: FLANK_NP_rep2


title: dFlox4M2Veh

description: Library name: Sample 23


title: dFlox4M1Dex

description: Library name: Sample 22


title: PETER001_HEPG2_24H_X1_B46:D05

description: L1000 inferred profile; pert_id: Efavirenz|Emtricitabine

source_name: HEPG2 treated with Efavirenz|Emtricitabine 0.5|0.1 uM for 24.0 h

value: Efavirenz|Emtricitabine 0.5|0.1 uM


title: Human_Tumor_C022_HPV_Positive


title: Human_Tumor_C03_HPV_Positive


title: FLANK, no protein, replicate 1

description: Library name: FLANK_NP_rep1


title: PETER001_HEPG2_24H_X1_B46:D03


title: PETER001_HEPG2_24H_X1_B46:D08

value: Emtricitabine|Tenofovir Disoproxil Fumarate 0.1|0.1 uM


title: Human_Tumor_C023_HPV_Positive


title: FLANK, 1:0.1, replicate 1

description:
Library name: FLANK_0.1_rep1
FLANK_0.1_deseq2.csv


title: PETER001_HEPG2_24H_X1_B46:D06


title: FLANK, 1:0.2, replicate 1

description:
Library name: FLANK_0.2_rep1
FLANK_0.2_deseq2.csv


title: CORE, 1:0.2, replicate 3

description:
Library name: CORE_0.2_rep3
CORE_0.2_deseq2.csv


title: PETER001_HEPG2_24H_X1_B46:D02

description: L1000 inferred profile; pert_id: Cobicistat|Elvitegravir|Emtricitabine


title: Human_Tumor_C012_HPV_Positive


title: dFlox4M0Dex

description: Library name: Sample 20


title: PETER001_HEPG2_24H_X1_B46:D04


title: dFlox3M1Dex

description: Library name: Sample 16


title: Human_Tumor_C021_HPV_Positive


title: CORE, 1:0.2, replicate 1

description:
Library name: CORE_0.2_rep1
CORE_0.2_deseq2.csv


title: dFlox3M2Dex

description: Library name: Sample 18


title: CORE, no protein, replicate 3

description: Library name: CORE_NP_rep3


title: dFlox4M0Veh

description: Library name: Sample 19


title: Human_Tumor_C013_HPV_Positive


title: Human_Tumor_C016_HPV_Positive


title: dFlox3M2Veh

description: Library name: Sample 17


title: dFlox4M1Veh

description: Library name: Sample 21


title: PETER001_HEPG2_24H_X1_B46:D01


title: CORE, 1:0.1, replicate 3

description:
Library name: CORE_0.1_rep3
CORE_0.1_deseq2.csv


title: Human_Tumor_C08_HPV_Negative


title: PETER001_HEPG2_24H_X1_B46:C21

description: L1000 inferred profile; pert_id: Genistein

source_name: HEPG2 treated with Genistein 5 uM for 24.0 h

value: Genistein 5 uM


title: delsarA3

city: Eugene

first: Caitlin

last: Kowalski

state: OR

address: 1585 E 13th Ave

institute: Univeristy of Oregon

description: Library name: Sample 9

data_processing:
Adapter trimming and quality control were performed using Illumina’s bcl-convert (version 4.2.4) with default parameters
Read mapping was performed with HISAT2 (version 2.2.1) using default parameters +’--very-sensitive’
Read quantification was performed using Subread’s featureCounts (version 2.0.6) using default parameters + ‘-Q 20’
Reads were normalized in R (version 4.0.2) using edgeR’s Trimmed Mean of M values (TMM) algorithm with default parameters and values were converted to counts per million (CPM)
Analysis of differential expression was performed using edgeR’s glmQLFTest and a subset of genes with |log2FC| > 1 and p < .05 were determined to have significant differential expression between compared groups
Assembly: Reference genome is SAMN41193238
Supplementary files format and content: tab deliminated text file with counts per each sample
Supplementary files format and content: raw CDS counts, columns labeled with sample title, SAM for reference genome provided

organism: Staphylococcus aureus

source_name: bacteria

growth_protocol: Tryotic soy broth, 37C, 250 rpm

extract_protocol:
Qiagen Rneasy Plus Mini Kit, extration from 5mL of bacterial culture
rRNA depletion using Illumina Standard Total RNA prep with Ribo-Zero plus Microbiome kit. Performed by Seqcenter (Pittsburgh, PA)

treatment_protocol: no treatment

value: bacteria

value: EVOL-A {delta}sarA (delsarA)


title: Human_Tumor_C06_HPV_Negative


title: dFlox3M1Veh

description: Library name: Sample 15


title: delsarA2

description: Library name: Sample 8


title: PETER001_HEPG2_24H_X1_B46:C20


title: Human_Tumor_C04_HPV_Negative


title: PETER001_HEPG2_24H_X1_B46:C19


title: Human_Tumor_C024_HPV_Negative


title: dFlox3M0Veh

description: Library name: Sample 13


title: WT3

description: Library name: Sample 6

value: WT NRS193 (WT)


title: dFlox3M0Dex

description: Library name: Sample 14


title: PETER001_HEPG2_24H_X1_B46:C18

description: L1000 inferred profile; pert_id: Emtricitabine

source_name: HEPG2 treated with Emtricitabine 0.1 uM for 24.0 h

value: Emtricitabine 0.1 uM


title: Human_Tumor_C020_HPV_Negative


title: PETER001_HEPG2_24H_X1_B46:C16


title: delsarA1

description: Library name: Sample 7


title: PETER001_HEPG2_24H_X1_B46:C17


title: dFlox2M2Dex

description: Library name: Sample 12


title: dFlox2M2Veh

description: Library name: Sample 11


title: WT1

description: Library name: Sample 4


title: Human_Tumor_C02_HPV_Negative


title: PETER001_HEPG2_24H_X1_B46:C15

description: L1000 inferred profile; pert_id: Elvitegravir|Emtricitabine

source_name: HEPG2 treated with Elvitegravir|Emtricitabine 0.1|0.1 uM for 24.0 h

value: Elvitegravir|Emtricitabine 0.1|0.1 uM


title: WT2

description: Library name: Sample 5


title: dFlox2M1Veh

description: Library name: Sample 9


title: Human_Tumor_C017_HPV_Negative


title: dFlox2M1Dex

description: Library name: Sample 10


title: EV3

description: Library name: Sample 3

value: EVOL-A (EV)


title: Human_Tumor_C018_HPV_Negative


title: EV2

description: Library name: Sample 2


title: PETER001_HEPG2_24H_X1_B46:C14


title: Human_Tumor_C015_HPV_Negative


title: dFlox2M0Dex

description: Library name: Sample 8

value: male


title: EV1

description: Library name: Sample 1


title: PETER001_HEPG2_24H_X1_B46:C13


title: Human_Tumor_C014_HPV_Negative


title: Tg_4

city: Bethesda

first: Mark

last: Cookson

state: MD

address: Building 35, Room 1A116, MSC3707 35, Convent Drive

institute: National Institute on Aging

hyb_protocol: There were 348 total genes in our Xenium panel. 248 came from the base mouse brain panel, and we created a custom panel of 100 genes which are known to be involved in α-synuclein processing and toxicity. Probe hybridization occurred overnight at 50ºC, followed by washes the following day to remove unbound probes. Probes were ligated for 2 hours at 37ºC, followed by enzymatic rolling circle amplification for 2 hours at 30ºC. Finally, autofluorescence quenching was performed followed by nuclei staining, and slides were loaded onto the Xenium Analyzer instrument. This process followed the 10x Genomics demonstrated protocol CG000582. Slides were then stored in PBST at 4ºC until use for immunofluorescence. We used standard IF protocols to stain for NeuN (neuronal nuclei) and phosphorylated α-synuclein (clone EP1536Y). Immunofluorescence images can be found archived on Zenodo: https://doi.org/10.5281/zenodo.12626000

scan_protocol: Xenium Analyzer is fully automated. On the instrument, samples underwent successive rounds of fluorescent probe hybridization, imaging, and probe removal. Data in this paper was acquired with Xenium Analyzer software v1.7.6.0.

data_processing: We re-segmented the data from a 15 to 5 um nuclear expansion method using Xenium Ranger v1.7.1.1 followed by loading into Seurat (v5.0+) for post-processing. Information on Xenium outputs can be found here: https://www.10xgenomics.com/support/software/xenium-onboard-analysis/latest/analysis/xoa-output-understanding-outputs. Code used to process this data can be found here: https://github.com/neurogenetics/Xenium_Line61_spatial.

label: Xenium Mouse Probe Set

source_name: Coronal brain section, Line 61 α-synuclein transgenic mouse

label_protocol: Deparaffinization and decrosslinking were performed to facilitate availability of RNA targets following 10x Genomics demonstrated protocol CG000580.

extract_protocol: Mice were harvested via trans-cardiac perfusion with phosphate-buffered saline (PBS) after injection of pentobarbital. Brains were removed from the skull, and the right hemispheres were fixed in 10% neutral buffered formalin (NBF) overnight at 4ºC, cut into 4 mm thickness coronally, dehydrated, then embedded in paraffin. 4 hemispheres of non-tg or α-syn-tg were embedded together in one block, then sections were cut at a thickness of 6 μm on a microtome and mounted onto Xenium slides or normal glass slides. We followed guidelines in 10x Genomics demonstrated protocol CG000578.

value: Coronal brain section

value: Male

tag: age (months)

tag: molecule type

value: In situ RNA


title: PETER001_HEPG2_24H_X1_B46:C12

description: L1000 inferred profile; pert_id: Abacavir|Emtricitabine

source_name: HEPG2 treated with Abacavir|Emtricitabine 0.5|0.1 uM for 24.0 h

value: Abacavir|Emtricitabine 0.5|0.1 uM


title: PETER001_HEPG2_24H_X1_B46:C11


title: Human_Tumor_C010_HPV_Negative


title: Tg_3


title: dFlox2M0Veh

description: Library name: Sample 7

tag: strain

value: Vehicle


title: PETER001_HEPG2_24H_X1_B46:C10


title: dFlox1M2Dex

description: Library name: Sample 6

value: Dex


title: fASht_3

city: Los Angeles

first: Charmi

last: Dedhia

state: CA

address: 4661 W Sunset Blvd, MS#35,

institute: Children's Hospital Los Angeles

description:
Library name: Sample 12
AS hetero female mouse glomeruli, 3.5m, fASht, rep3

data_processing:
Agilent Technologies 4200Tape station and Qubit 3.0 (Thermo Fisher Scientific Inc) Fluorometer
Raw reads were quality checked with the FASTQC package. Adapters were removed as required for each kit using the cutadapt software and tolerating 10% error.
Raw FASTQ files for each sample were uploaded onto the Partekflow suite for downstream analysis.
Pre-alignment quality assurance/quality control (QA/QC) was performed, and reads were trimmed at the 3-prime (right end) and aligned to the mouse genome using STAR.
The aligned reads were counted and annotated against the latest UCSC mouse transcript set on Partek to determine gene expression levels and counts normalized using the TMM (Trimmed Mean of M-value).
Assembly: mm10 (mouse)
Supplementary files format and content: xlsx file with gene_name, gene_type, mgi_id, count s and Chromosome number

source_name: Col4a5+/- (B6.Cg-Col4a5^tm1Yseg/J), heterozygous females

extract_protocol:
Total RNA was isolated using RNeasy plus Mini Kit (Qiagen, C#74134). RNA integrity was checked by Agilent Bioanalyzer 2100.
Library for RNA-Seq was prepared according to KAPA Stranded mRNA Hyper prep polyA selected kit with 201-300 bp insert size (KAPA Biosystems) using 250ng total RNAs as input.

value: Glomeruli

value: Glomerular cells

value: Col4a5+/- (B6.Cg-Col4a5^tm1Yseg/J)

value: female


title: Tg_1

value: Female


title: Tg_2


title: dFlox1M1Dex

description: Library name: Sample 4

value: wild-type


title: fASht_2

description:
Library name: Sample 11
AS hetero female mouse glomeruli, 3.5m, fASht, rep2


title: PETER001_HEPG2_24H_X1_B46:C08

description: L1000 inferred profile; pert_id: Dolutegravir|Emtricitabine

source_name: HEPG2 treated with Dolutegravir|Emtricitabine 0.1|0.1 uM for 24.0 h

value: Dolutegravir|Emtricitabine 0.1|0.1 uM


title: dFlox1M1Veh

description: Library name: Sample 3


title: dFlox1M2Veh

description: Library name: Sample 5


title: nTg_3

source_name: Coronal brain section, non-transgenic mouse


title: dFlox1M0Dex

description: Library name: Sample 2


title: PETER001_HEPG2_24H_X1_B46:C09


title: Human_Tumor_C01_HPV_Negative

value: tissue


title: PETER001_HEPG2_24H_X1_B46:C07


title: fWT_3

description:
Library name: Sample 9
WT female mouse glomeruli, 3.5m, fWT, rep3

source_name: C57BL/6J mice, female

value: C57BL/6J mice


title: dFlox1M0Veh

description: Library name: Sample 1


title: nTg_4


title: PETER001_HEPG2_24H_X1_B46:C06

description: L1000 inferred profile; pert_id: Abacavir|Dolutegravir

source_name: HEPG2 treated with Abacavir|Dolutegravir 0.5|0.1 uM for 24.0 h

value: Abacavir|Dolutegravir 0.5|0.1 uM


title: nTg_2


title: JNK3-KO, NMDA, rep2

city: Seoul

first: Jung-Woong

last: Kim

address: 84 Heuksuk-ro Dongjak-ku

department: Life Science

country: South Korea

institute: Chung-Ang University

data_processing:
STAR-2.7.1a  was used to align mRNA-Seq reads
Indices were generated from either the genome assembly sequence or representative transcript sequences for alignment to the genome and transcriptome.
The alignment file was used to assemble transcripts, estimate their abundances, and detect gene expression differences using RSEM(v.1.3.3).
Differentially expressed transcripts were identified using EdgeR (v.3.36.0) within R version 4.1.0 (R  development Core Team, 2011) using the R package.
Assembly: GRCm38.p6.101
Supplementary files format and content: tsv, FPKM table (Transcript and Gene)

molecule: polyA RNA

source_name: Retina

growth_protocol: C57BL/6J wild type mice and Jnk3-/- mice was obtained from the Jackson Laboratory in 2018 and kept them strictly inbred in our animal facility's pathogen-free, individually ventilated cage housing. Control male C57Bl/6J mice were also purchased from the Jackson Laboratory

extract_protocol:
Total RNA was extracted using the TRIzol solution. 100 ng of total RNA from retinas (> 3 retineas) were extracted, and then reverse transcription was carried out using an oligo-dT primer that had an Illumina-compatible sequence at its 5' end.
RNA libraries for RNA-seq were prepared using the TruSeq mRNA Library Prep Kit (Illumina, Inc., USA) following manufacturer's instructions.

treatment_protocol: Intravitreal injections were performed under a microscope with a microsyringe and a 30-gauge needle. In control experiments, eyes (n = 6 retinas each; 3 independent experiments) were pretreated by injecting 1 μL of 1X PBS (pH 7.4), and in experimental groups, eyes (n = 6 retinas each; 3 independent experiments) were treated by injecting 1 μL of 40 mM NMDA (Sigma; corresponding to 40 nmoles) prepared in PBS. We injected 2 μl of control AAV (pU6-shScramble-GFP: 3.87× 1011 GC/ml) and knockdown AAV (pU6-shPax6-GFP: 3.93 × 1011 GC/ml).

value: Retina

value: NMDA

tag: read length


title: fASht_1

description:
Library name: Sample 10
AS hetero female mouse glomeruli, 3.5m, fASht, rep1


title: fWT_2

description:
Library name: Sample 8
WT female mouse glomeruli, 3.5m, fWT, rep2


title: nTg_1

value: Coronal brain section


title: PETER001_HEPG2_24H_X1_B46:C05


title: JNK3-KO, NMDA, rep1


title: 2r_S6

city: Tübingen

first: Kay

last: Nieselt

state: DE

address: Sand 14

department: Department for Computer Science

institute: Institute for Bioinformatics and Medical Informatics

description:
Library name: Q4WOL016A2
Pilot2CountsWolbachia.txt
Pilot2CountsAedes.txt

data_processing:
All read mappings were performed independently against the reference strain of Wolbachia pipientis wAlbB (RefSeq ID: NZ_CP031221.1) and the reference strain of Aedes albopictus (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_006496715.1/).
The resulting mapping files in SAM format were converted to BAM format using SAMtools v1.9
Gene counts for all samples were computed with featureCounts v1.6.4 (Liao et al., 2014) based on the annotation of the respective reference genome, where the selected feature type was set to transcript records (featureCounts parameter -t transcript).
A quality check for rRNA was performed with a self-written script based on the absolute counts of annotated rRNAs
All read mappings were performed independently against the reference strain of Wolbachia pipientis wAlbB (RefSeq ID: NZ_CP031221.1) and the reference strain of Aedes albopictus (RefSeq ID: NC_006817.1).
Assembly: NZ_CP031221.1
Supplementary files format and content: Pilot1: a tab-delimited count table from the feature counts step against Wolbachia
Supplementary files format and content: Pilot2: two tab-delimited count table from the feature counts step, one against Wolbachia and one against Aedes albopictus.

organism: Aedes albopictus

source_name: whole cell

growth_protocol: Wolbachia-infected C6/36 cells were incubated in plug-sealed 75 cm2 culture flasks (Greiner, Kremsmünster, Austria) at 26 °C in 15 ml Leibovitz’s L15 medium (Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented with 20% heat-inactivated FCS (PAN-Biotech, Aidenbach, Germany), 1% MEM nonessential amino acids (Thermo Fisher Scientific), 2% tryptose phosphate broth (Sigma-Aldrich, St. Louis, Missouri, USA)  and 1% penicillin/streptomycin (Thermo Fisher Scientific). They were passaged every 7 days, with 3 ml of the passage added to a new flask with 12 ml fresh medium. Medium exchange (10 ml) was performed 3 days after passaging. For the assay with custom-designed riboPOOLs, 2 ml per sample were harvested from cell culture flasks.

extract_protocol:
Total RNA was extracted from wAlbB-infected C6/36 cells using the miRNeasy Mini Kit (Qiagen, Hilden, Germany), with protocol adaptations. Samples were incubated at 37 °C in a heating block until completely thawed, ensuring that all salts were fully dissolved. After an additional 5 min incubation at room temperature, samples were mixed with 100 µl 1-Bromo-3-chloro-2-propanol (BCP, Tokyo Chemical Industry, Tokyo, Japan). The sample-BCP mixture was vigorously shaken for 15 seconds, followed by a 2–3 min incubation at room temperature. Phase separation was achieved through a 15 min centrifugation at 12,000 g, 4 °C. The upper aqueous phase, containing the nucleic acids, was retained, with 350 µl applied to the Qiagen column. The extract was also subject to DNase I treatment to eliminate contaminating DNA.
Pilot 1: Three samples were subjected to rRNA depletion using Illumina TruSeq Stranded Total RNA with Ribo-Zero Plus rRNA Depletion according to the manufacturer’s instructions, using the maximum input of 5 µg RNA. Pilot 2: Four libraries corresponding to two technical replicates.

value: whole cell

value: Aedes albopictus C6/36 insect cells infected with Wolbachia

value: insect cell


title: PETER001_HEPG2_24H_X1_B46:C04


title: fWT_1

description:
Library name: Sample 7
WT female mouse glomeruli, 3.5m, fWT, rep1


title: mAS_3

description:
Library name: Sample 6
AS male mouse glomeruli, 3.5m, mAS, rep3

source_name: Col4a5-/- mice (B6.Cg-Col4a5 tm1Yseg/J), male

value: Col4a5-/- mice (B6.Cg-Col4a5 tm1Yseg/J)

value: male


title: 1r_S5

description:
Library name: Q4WOL015AS
Pilot2CountsWolbachia.txt
Pilot2CountsAedes.txt


title: JNK3-KO, PBS, rep1

value: PBS


title: 7m_S2

description:
Library name: Q4WOL014AK
Pilot2CountsWolbachia.txt
Pilot2CountsAedes.txt


title: mAS_2

description:
Library name: Sample 5
AS male mouse glomeruli, 3.5m, mAS, rep2

value: Glomeruli

value: Glomerular cells


title: JNK3-KO, PBS, rep2


title: mAS_1

description:
Library name: Sample 4
AS male mouse glomeruli, 3.5m, mAS, rep1


title: PETER001_HEPG2_24H_X1_B46:C02

description: L1000 inferred profile; pert_id: DMSO

source_name: HEPG2 treated with DMSO 0.5 uM for 24.0 h

value: DMSO 0.5 uM


title: shPax6, NMDA, rep2


title: shPax6, NMDA, rep1


title: 5m_S1

description:
Library name: Q4WOL013AC
Pilot2CountsWolbachia.txt
Pilot2CountsAedes.txt


title: PETER001_HEPG2_24H_X1_B46:C01

description: L1000 inferred profile; pert_id: DMSO


title: mWT_3

description:
Library name: Sample 3
WT male mouse glomeruli, 3.5m, mWT, rep3

source_name: C57BL/6J mice, male


title: PETER001_HEPG2_24H_X1_B46:C03

description: L1000 inferred profile; pert_id: DMSO


title: RNA-seq of zinc-treated MFF-1 cells sample 2

city: Zhuhai

first: Mincong

last: Liang

state: Guangdong

address: No. 2 Daxue Road

department: School of Marine Sciences

country: China

institute: Sun Yat-sen University

description: Library name: Zinc-2

data_processing:
Raw data were preprocessed and quality-controlled using fastp v0.24.0 with the parameters: “-2 --dont_eval_duplication 6 -5 -3 -r --cut_front_window_size 1 --cut_front_mean_quality 5 --cut_tail_window_size 1 --cut_tail_mean_quality 5 --cut_right_window_size 5 --cut_right_mean_quality 20 -l 50 -q 20 -u 30”. The resulting clean data were aligned to the Siniperca chuatsi reference genome  using HISAT2 v2.2.1. Gene expression levels were then quantified using samtools v1.22.1 and StringTie v3.0.0.
Assembly: GCA_020085105.1
Supplementary files format and content: Tab-delimited text files containing raw gene counts and transcript counts for all samples, respectively.

organism: Siniperca chuatsi

source_name: MFF-1 cell line

growth_protocol: MFF-1 cells were cultured in 6-well plates in DMEM supplemented with 10% FBS until reaching 90–100% confluence.

extract_protocol:
After 72 h of treatment, the medium was removed, cells were washed twice with sterile PBS, and total RNA was collected following TRIzol lysis.
After total RNA extraction, sequencing libraries were prepared according to the manufacturer’s instructions.

treatment_protocol: The medium was replaced with either fresh medium (control group, Mock) or fresh medium supplemented with 200 μM zinc sulfate (stimulation group, Zinc), with three parallel replicates per treatment.

value: MFF-1 cell line

value: Epithelial cells

value: 200 μM zinc sulfate for 72 hours


title: PETER001_HEPG2_24H_X1_B46:B21

description: L1000 inferred profile; pert_id: Water

source_name: HEPG2 treated with Water 0.01 uM for 24.0 h

value: Water 0.01 uM


title: shPax6, PBS, rep2


title: mWT_2

description:
Library name: Sample 2
WT male mouse glomeruli,  3.5m, mWT, rep2

value: C57BL/6J mice


title: RNA-seq of zinc-treated MFF-1 cells sample 3

description: Library name: Zinc-3


title: PETER001_HEPG2_24H_X1_B46:B20

description: L1000 inferred profile; pert_id: Water


title: mWT_1

description:
Library name: Sample 1
WT male mouse glomeruli, 3.5m, mWT, rep1


title: RNA-seq of zinc-treated MFF-1 cells sample 1

description: Library name: Zinc-1


title: shPax6, PBS, rep1

value: Retina


title: TC-1 tumor, circRNA vaccine, Day12, rep5

city: Beijing

first: Rong

last: Zhou

state: Beijing

address: No. 9 North Yongteng Road

institute: Therorna

description: Library name: X33_FPKM

data_processing:
Raw sequencing reads in FASTQ format were processed using Cutadapt (v1.9.1) to remove technical sequences and low-quality bases.
Clean reads were aligned to the reference genome (Mum musculus, GRCm39) using HISAT2 (v2.2.1).
Gene-level counts were quantified using HTSeq (v0.6.1). Reads overlapping exons were counted, and multi-mapping reads or reads overlapping multiple genes were excluded.
DEGs were identified using DESeq2 (v1.34.0), which employs a negative binomial distribution model.
Assembly: GRCm39
Supplementary files format and content: tab-delimited text file including FPKM values for each sample

source_name: tumor

extract_protocol:
Frozen tumor tissues were homogenized in 1 ml of Trizol reagent. RNA was extracted with trizol-chloroform and preciptated by isopropanol.
1 μg total RNA was used for the following library preparation. For mRNA enrichment, poly(A) mRNA isolation was performed using Oligo(dT) beads. The mRNA fragmentation was performed using divalent cations and high temperature. Priming was performed using Random Primers. First strand cDNA synthesis was carried out using reverse transcriptase, followed by second-strand cDNA synthesis. The purified double-stranded cDNA was then treated to repair both ends and add a dA-tail in a single reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of adaptor-ligated DNA was then performed using DNA Clean Beads. Each sample was then amplified by PCR using P5 and P7 primers, and the PCR products were validated.

value: tumor

value: murine lung epithelial cells

value: circRNA vaccine, Day12


title: PETER001_HEPG2_24H_X1_B46:B18

description: L1000 inferred profile; pert_id: Dolutegravir

source_name: HEPG2 treated with Dolutegravir 0.1 uM for 24.0 h

value: Dolutegravir 0.1 uM


title: RNA-seq of control MFF-1 cells sample 3

description: Library name: Mock-3

value: Control


title: RNA-seq of control MFF-1 cells sample 2

description: Library name: Mock-2


title: Wild type, NMDA, rep1

value: Wild type

value: NMDA


title: TC-1 tumor, circRNA vaccine, Day12, rep4

description: Library name: X3_FPKM


title: PETER001_HEPG2_24H_X1_B46:B17


title: TC-1 tumor, circRNA vaccine, Day12, rep3

description: Library name: X29_FPKM


title: Wild type, PBS, rep2

value: PBS


title: PETER001_HEPG2_24H_X1_B46:B16


title: PETER001_HEPG2_24H_X1_B46:B15

description: L1000 inferred profile; pert_id: Cobicistat|Elvitegravir

source_name: HEPG2 treated with Cobicistat|Elvitegravir 0.1|0.1 uM for 24.0 h

value: Cobicistat|Elvitegravir 0.1|0.1 uM


title: TC-1 tumor, circRNA vaccine, Day12, rep1

description: Library name: X1_FPKM


title: PETER001_HEPG2_24H_X1_B46:B14


title: scRNA-seq of ISKNV-infected Siniperca chuatsi

description: Library name: WT

data_processing:
Raw sequencing reads were trimmed to retain the first 28 bp for Read 1 and the first 91 bp for Read 2. The trimmed reads were aligned to the Siniperca chuatsi reference genome and the ISKNV genome using Cell Ranger v9.0.1 to generate gene expression matrices. The resulting matrices were imported into Seurat v5.3.0 for downstream analysis.
Assembly: GCA_020085105.1
Assembly: OP896201.1
Supplementary files format and content: Tab-separated values files and matrix files.

source_name: spleen

growth_protocol: Healthy Siniperca chuatsi (Mandarin fish, 50 ± 10 g) were obtained from a commercial farm and maintained in recirculating aquariums under controlled conditions (27 ± 1 °C, continuous aeration, and a 12-hour light/dark cycle). Prior to the experiment, fish were acclimatized for at least one week and confirmed free of common viral pathogens via PCR testing.

extract_protocol:
At 7 days post-infection (dpi), fish were euthanized using MS-222. Spleen tissues were harvested, washed twice with sterile PBS, and stored in tissue preservation solution. To minimize individual variation, spleen tissues from six individuals per group were pooled into a single sample after dissociation.
Single-cell libraries were constructed following the manufacturer's instructions (10x Genomics).

treatment_protocol: The mandarin fish were randomly divided into two groups: an infection group and a control group. The infection group received an intraperitoneal injection of 100 µL of Infectious spleen and kidney necrosis virus (ISKNV) (strain OP896201.1, 5 × 10^5 TCID50/mL), while the control group was injected with an equal volume of sterile PBS.

value: spleen

value: ISKNV injection (100 µL, 5×10^5 TCID50/mL)


title: TC-1 tumor, circRNA vaccine, Day12, rep2

description: Library name: X24_FPKM


title: Wild type, NMDA, rep2


title: Wild type, PBS, rep1


title: PETER001_HEPG2_24H_X1_B46:B13


title: TC-1 tumor, control, Day12, rep5

description: Library name: X6_FPKM

value: control, Day12


title: PETER001_HEPG2_24H_X1_B46:B19

description: L1000 inferred profile; pert_id: Water


title: RNA-seq of control MFF-1 cells sample 1

description: Library name: Mock-1

value: MFF-1 cell line

value: Epithelial cells


title: scRNA-seq of control Siniperca chuatsi

description: Library name: Mock

value: PBS injection (100 µL)


title: Skin, unenriched, rerioXalbolineatus, rep5

first: John

last: Postlethwait

address: 1210 University Of Oregon

department: Biology

institute: University of Oregon

description: Library name: Dal5S

data_processing:
We mapped each of the 48 libraries against combined references containing both parental genomes  (BBMap v38.57 with parameters: k=13, maxindel=100000, minid=0.76, ambiguous=best) to distinguish parental alleles in hybrid samples. Only a single best mapping location was retained for each read.
The 48 sequencing libraries  were split into two species-specific sublibraries. One sublibrary (denoted "Drerio") contained all reads that aligned best to the maternal D. rerio genome. The other sublibrary ("Dother" to denote the other Danio species) contained all reads that aligned best to the genome of the paternal species used to generate the hybrid.
We remapped each sublibrary to the D. rerio genome (BBMap v38.57 with parameters: k=13, maxindel=100000, minid=0.76, ambiguous=best) to utilize the available high quality GRCz11 gene annotations.
Gene expression was quantified using featureCounts (Rsubread v2.18). To mitigate the possibility of mapping bias toward the D. rerio reference in regions with poor sequence conservation, we excluded reads mapping to 3’ and 5’ untranslated regions. These feature counts are included in the processed data files DrerioAlleleCounts.csv and PaternalAllelelCounts.csv.
Statistical analysis was performed using DESeq2 (v1.44.0), filtering genes with less than 1 normalized count per sample (retaining 24,672 of 29,005 genes).
Assembly: GRCz11
Supplementary files format and content: DrerioAlleleCounts.csv quantifies the expression of the maternal D. rerio alleles for each gene.
Supplementary files format and content: PaternalAlleleCounts.csv quantifies the expression of the alleles from the paternal species used to generate each hybrid.

organism: Danio rerio x Danio albolineatus

source_name: Whole skin

extract_protocol:
Takara Bio SMART-seq2 RNA-seq kit
Nextera paired-end library preps

value: Whole skin

value: Unenriched

tag: batch


title: TC-1 tumor, control, Day12, rep4

description: Library name: X5_FPKM

value: tumor


title: Skin, unenriched, rerioXalbolineatus, rep4

description: Library name: Dal4S


title: L13968_BCL6LKOFXRKO

city: Chicago

first: Ellen

last: Fruzyna

address: 1349 E Washington St

institute: Northwestern University

description: Library name: sample7

data_processing:
Sequences were aligned to mm10 using STAR version 2.5.2 with ‘--outFilterMultimapNmax 1’ and  ‘--outFilterMismatchNmax 4’.
Tag directories were generated in HOMER v4.10 using 'makeTagDirectory' with '-flip' and 'format sam' parameters. Bedgraphs were generated using 'makeUCSCfile'
Raw counts were generated using Homer's 'analyzeRepeats.pl' with '-noadj' parameter.
Assembly: mm10
Supplementary files format and content: Liver_RNA_RawCounts.txt' contains raw counts for each sample
Supplementary files format and content: UCSC_*.gz ' files contain Bedgraph files for visualization on the UCSC Genome Browser for each sample

source_name: Liver

extract_protocol:
25mg liver was excised, rinsed, and stored in RNA-later (Ambion). Samples were bead-homogenized in Trizol (Ambion). 20% chloroform by volume was added, and the aqueous phase was isolated following centrifugation. RNA was further purified using the RNAeasy kit (Qiagen).
Sequencing libraries were made using the Truseq Stranded mRNA Kit (Illumina) and sequencing was performed on the Illumina NextSeq 2000 using 100 bp single-end reads.

value: Liver

value: Alb-Cre, Bcl6(fl/fl), FXR(homo)


title: L13970_BCL6LKOFXRKO

description: Library name: sample8


title: L13813_BCL6LKOFXRKO

description: Library name: sample5


title: L13942_BCL6LKOFXRKO

description: Library name: sample6


title: TC-1 tumor, control, Day12, rep3

description: Library name: X31_FPKM

value: murine lung epithelial cells


title: PETER001_HEPG2_24H_X1_B46:B11

description: L1000 inferred profile; pert_id: Cobicistat|Emtricitabine

data_processing: The data was normalized relative to the expression of 80 invariant landmark control genes. The expression of additional genes was inferred via a linear model trained on thousands of Affymetrix gene expression datasets from the gene expression omnibus (GEO). In this study, we utilized the L1000 data with the 12,328 measured and inferred mRNAs.

source_name: HEPG2 treated with Cobicistat|Emtricitabine 0.1|0.1 uM for 24.0 h

extract_protocol: After treatment, cells were lysed using the TCL Buffer (Qiagen, cat. no. 1031576) and incubated for 30 minutes at 37°C. The lysates were then stored at -80°C. RNA was extracted by the Broad Institute, according to their protocol.

value: Cobicistat|Emtricitabine 0.1|0.1 uM


title: Skin, unenriched, rerioXalbolineatus, rep3

description: Library name: Dal3S


title: Skin, xanthophore-enriched, rerioXalbolineatus, rep5

description: Library name: Dal5X

value: Xanthophore-enriched


title: L13971_FXRKO

description: Library name: sample4

value: Bcl6(fl/fl), FXR(homo)


title: TC-1 tumor, control, Day12, rep2

description: Library name: X28_FPKM


title: PETER001_HEPG2_24H_X1_B46:B08

description: L1000 inferred profile; pert_id: Cobicistat|Elvitegravir|Emtricitabine|Tenofovir Alafenamide Fumarate

source_name: HEPG2 treated with Cobicistat|Elvitegravir|Emtricitabine|Tenofovir Alafenamide Fumarate 0.1|0.1|0.1|0.5 uM for 24.0 h


title: TC-1 tumor, control, Day12, rep1

description: Library name: X10_FPKM


title: L13969_FXRKO

description: Library name: sample3


title: PETER001_HEPG2_24H_X1_B46:B12


title: PETER001_HEPG2_24H_X1_B46:B09

value: Cobicistat|Elvitegravir|Emtricitabine|Tenofovir Alafenamide Fumarate 0.1|0.1|0.1|0.5 uM


title: Skin, xanthophore-enriched, rerioXalbolineatus, rep3

description: Library name: Dal3X


title: GAL4_E

city: Leeds

first: Michaela

last: Agapiou

address: Woodhouse Lane

country: United Kingdom

institute: University of Leeds

data_processing:
The adaptor sequence (AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC) was trimmed from reads using Cutadapt (version 1.1) via Galaxy.
Reads were filtered out (using ‘Filter by quality’ tool in Galaxy (version 1.0.2)) if the quality score was below 20 for 10% or more of the read.
rRNA and tRNA reads were removed using Subread (version 2.0.0). The tRNA fasta file was from release 6.22 of the D. melanogaster genome. The rRNA fasta file was from RiboGalaxy’s Shared Data library (UUID: 0f0983aa-3afb-4b5f-a417-23593a3df1ef).
Transcriptome indexing was carried out using Salmon (version 0.14.2). A decoy-aware transcriptome was indexed with a pre-computed decoy sequence file provided by the Salmon developers. The k-mer size selected for indexing the transcriptome was 31.
Quasi-mapping was carried out using Salmon (version 0.14.2). The reads remaining after filtering were used, and the library parameter (-l) was set to SR, and the k-mer parameter (-k) was set to 31.
Assembly: dmel_r6.22
Supplementary files format and content: Quantification output file from Salmon with: transcript name, length, effective length, TPM, and estimated number of reads.

organism: Drosophila melanogaster

source_name: testes

growth_protocol: Flies were kept in a 25°C humidified room with a 12:12 hour light:dark cycle and raised on 10 ml standard sugar-yeast-agar medium (Bass et al., 2007)

extract_protocol:
The RIP protocol was adapted from Keene, J.D. et al (2006) using anti-HA beads (Pierce, #88837). RNA was extracted from total lysate samples and from the elutions (i.e. the anti-HA magnetic beads at the end of the pull-down experiment). Both types of samples were treated with proteinase K prior to TRIzol extraction. This was then followed by isopropanol precipitation. RNA was stored at -80 ºC in nuclease free water.
RNA samples were prepared using Illumina’s Ribo-Zero rRNA Removal Kit followed by Illumina’s TruSeq Stranded Total RNA Library Prep. 100 ng from each sample was pooled and sequenced on the same lane on a NextSeq 500 Illumina sequencer using the High Output Kit v2.5 (75 Cycles), i.e. 75 bp single-end sequencing.

value: testes

tag: rip antibody beads

value: anti-HA beads (Pierce, #88837)

tag: molecule subtype

value: pull down elution RNA


title: Skin, xanthophore-enriched, rerioXalbolineatus, rep4

description: Library name: Dal4X


title: L13967_FXRKO

description: Library name: sample2


title: GAL4_L

value: total RNA


title: PETER001_HEPG2_24H_X1_B46:B07

tag: duration

tag: plate


title: L13941_FXRKO

description: Library name: sample1


title: HOW_rep3_E


title: Skin, melanophore-enriched, rerioXalbolineatus, rep5

description: Library name: Dal5M

value: Melanophore-enriched


title: PETER001_HEPG2_24H_X1_B46:B06

description: L1000 inferred profile; pert_id: Darunavir|Emtricitabine|Ritonavir|Tenofovir Disoproxil Fumarate

source_name: HEPG2 treated with Darunavir|Emtricitabine|Ritonavir|Tenofovir Disoproxil Fumarate 0.1|0.1|0.5|0.1 uM for 24.0 h

tag: well


title: Skin, melanophore-enriched, rerioXalbolineatus, rep4

description: Library name: Dal4M


title: HOW_rep3_L


title: multiplexed single cell pool, 3x control, 4x KO [HTO]

description: Library name: Sample2

data_processing:
Reads were aligned against the mouse genome (mm10) and counted by StarSolo.
Preprocessed counts were further analysed using Scanpy.
Basic cell quality control was conducted by taking the number of detected genes and mitochondrial content into consideration. We removed 5945  cells in total that did not express more than 1000 genes or had a mitochondrial content greater than 10%. Furthermore, we filtered 14192  genes if they were detected in less than 30 cells (<0.01%).
Raw counts per cell were normalised to the median count over all cells and transformed into log space to stabilise variance. We initially reduced dimensionality of the dataset using PCA, retaining 50 principal components. Subsequent steps, like low-dimensional UMAP embedding and cell clustering via community detection, were based on the initial PCA.
Final data visualization was done by scanpy and cellxgene packages.
Assembly: mm10
Supplementary files format and content: starsolo outputs

source_name: HEART

extract_protocol:
Mice were euthanized using CO2 inhalation, and a gentle perfusion was performed to remove blood from the heart, after which the heart was collected in ice-cold HBSS. The tissue digestion was performed by utilizing the digestion mixture of dispase (1.2 units/ml), collagenase II (2mg/ml), and elastase (0.02 mg/ml) in a serum-free DMEM. A single cell suspension was produced with the help of debris removal solution (Miltenyi, 130-109-398) and RBC lysis buffer (Miltenyi, 130-094-183). The cell suspension was washed three times with PBS and re-suspended in MACS buffer (D-PBS + 2 mM EDTA + 0.5% BSA; filter sterile). 1 μg of single-cell hashing antibody was added to 100 μL of cell suspension (in staining buffer- 2% BSA/0.02% Tween), and incubated for 30 minutes at 4˚C. This was diluted with 2 mL of complete serum-free DMEM/PBS (v/v 2:1). FACS sorting was setup with live/dead exclusion and doublet discrimination, with a 1.5 mL collection tube containing 0.5 ml serum-free DMEM/PBS (v/v 2:1), Cells were stained with calcein-AM dye (Biolegend, 425201) and DAPI, which was incubated for 30 min on ice. Live single cells from individual mice with different hashtags were sorted into one collection tube, serving as one pooled sample for sequencing. Single live calcein AM-positive, DAPI-negative cells were sorted on the BD FACSMelody cell sorter (BD Bioscience) for single-cell RNA sequencing.
The cell suspensions were counted with a Moxi cell counter and diluted according to the manufacturer’s protocol to obtain 10.000 single-cell data points per sample. Each sample was run separately on a lane in Chromium controller with Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10xGenomics). Single-cell RNAseq library preparation was done using a standard protocol.

value: HEART

value: Non cardiomyocytes

value: 6-8 weeks old WT

value: TAC surgery


title: PETER001_HEPG2_24H_X1_B46:B05

value: Darunavir|Emtricitabine|Ritonavir|Tenofovir Disoproxil Fumarate 0.1|0.1|0.5|0.1 uM


title: Skin, melanophore-enriched, rerioXalbolineatus, rep3

description: Library name: Dal3M

value: Whole skin


title: Single-cell RNA-seq of E14.5 mouse ventricular tissue, ETS1 endothelial knockout replicate 2

city: San Diego

first: Zeyu

last: Chen

address: 9500 Gilman Drive, BFRII 1217

department: School of Medicine

institute: UCSD

description:
Library name: ETS1eKO_2
Ventricle from  E14.5 mouse ventricular tissue, ETS1 endothelial knockout replicate 2

data_processing:
Raw reads were aligned to the mouse mm10 reference transcriptome and quantified using Cell Ranger (v7.1.0) (10X Genomics).
Cells with 200–7000 detected genes were retained. Cells were further filtered based on mitochondrial gene percentage (≤ 20%), ribosomal gene percentage (≤ 35%), and hemoglobin gene percentage (≤ 2%).
CellBender was applied to remove ambient RNA contamination.
DoubletFinder (v2.0.3) was used to identify and remove potential doublets with a score > 0.4.
Cells were filtered based on exon ratio (0.18 < exon ratio ≤ 0.5) and total read count thresholds to remove dead or damaged cells.
Data were normalized using SCTransform (Seurat v4.3.0), and batch effects were corrected using Harmony.
Principal component analysis (PCA) followed by UMAP was performed. Clusters were identified using graph-based clustering methods.
Clusters were annotated based on canonical markers and reference datasets of embryonic mouse heart cells.
Differential expression between control and knockout samples was conducted within each major cell type using Seurat’s FindMarkers function, with adjusted p-value < 0.001.
GO and KEGG pathway enrichment analyses were performed using clusterProfiler, with Benjamini-Hochberg correction (adjusted p-value < 0.05).
Assembly: mm10(Mus musculus)
Supplementary files format and content: Tab-separated values files and matrix files for each sample
Supplementary files format and content: An RDS file (Ets1.eKO.scRNA.All_CellType.2024_12.19.rds) containing a processed Seurat object(v4.3.0) with cell type annotations and quality-controlled single-cell RNA-seq data.

source_name: heart (ventricle)

growth_protocol: Mice were maintained under specific pathogen-free (SPF) conditions with a 12-hour light/dark cycle and free access to food and water. ETS1 flox/flox, Tie2Cre mice were maintained on a C57BL/6 background. Embryonic mice of both sexes at E14.5 were used without prior genotyping for gender.

extract_protocol:
Ventricular tissues were enzymatically dissociated using 0.25% trypsin (Gibco) at 37 °C for 20–30 minutes with gentle pipetting to obtain a single-cell suspension. The digestion was stopped by adding DMEM supplemented with 10% fetal bovine serum (FBS). Cells were filtered through a 40-μm cell strainer, centrifuged at 400 g, and resuspended in DMEM with 2% FBS. Viability was assessed by Trypan Blue staining.
Single-cell suspensions were processed using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10X Genomics) according to the manufacturer’s instructions. Single cells were partitioned into Gel Beads-in-Emulsion (GEMs), followed by reverse transcription, cDNA amplification, fragmentation, end repair, A-tailing, adaptor ligation, and sample index PCR. Final libraries included sample indices, UMIs, barcodes, and Illumina sequencing adapters, and were sequenced on the Illumina NextSeq S4 platform.

treatment_protocol: No treatment was applied prior to tissue collection. Embryonic day 14.5 ventricles were directly harvested for single-cell dissociation and RNA extraction.

value: heart (ventricle)

value: ventricular cells

value: Tie2Cre; Ets1^fl/fl

value: No


title: multiplexed single cell pool, 3x control, 4x KO [GEX]

description: Library name: Sample1


title: PETER001_HEPG2_24H_X1_B46:B04


title: Skin, iridophore-enriched, rerioXalbolineatus, rep5

description: Library name: Dal5I

value: Iridophore-enriched


title: HOW_rep2_E


title: Skin, iridophore-enriched, rerioXalbolineatus, rep4

description: Library name: Dal4I