title: scSTAMP-seq_DropletBasedSequencing_Hela_Transcriptome

description: adapted for 10X Chromium Next GEM droplet based sequencing workflow. Method is detailed in Piscopio et al.

data_processing:
Supplementary files format and content: tab separated values file where rows indicate barcode. tab separated values file where column 1 contains feature ID, column 2 contains gene name, and column 3 identifies the type of feature. matrix file where column 1 refers to the "gene id" index, column 2 refers to "cell id" index, and column 3 represents the total UMI counts per cell and gene combination.
scSTAMP-seq data processing and gene expression analysis. Sequencing FASTQ data files were processed using custom Perl scripts on a Linux high performance computing (HPC) cluster at the University of California, Santa Barbara. Sequencing data was post-processed and analyzed using Seurat (version 4.3.0) in R (version 4.2.2) and RStudio (version 2022.12.0+353). Representative figures were generated in RStudio and compiled in Adobe Illustrator (version 26.5).  scSTAMP-seq barcode processing and analysis. A custom Perl script was written to evaluate the spatial barcode portion of the sequencing library. For PHO spatial barcode sequences, read 1 was used to determine cell barcode/UMI. Read 2 was used to distinguish ‘inner’ and ‘outer’ sequence reads by identifying unique ‘inner’ and ‘outer’ primer sequences as well as the downstream spatial barcode/UMI sequence. MPO barcode counts were determined similarly, but an additional multiplex identifier sequence from read 2 was used to distinguish multiplex from spatial barcodes. Unique counts for each molecule were defined as reads containing a unique combination of the UMI on read 1 and on read 2.  Plate-based scSTAMP-seq data processing. For plate-based sequencing, read 2 of the paired-end read was aligned in the sense direction using BWA (version 0.7.15-r1140) to the RefSeq gene model based on the human genome release hg19, incorporating a collection of 92 ERCC spike-in molecules. In cases where a read aligned to multiple locations, it was evenly distributed across those locations. Gene isoforms were consolidated into a single gene count, and the unique molecular identifiers (UMIs) were used to remove duplicate reads and generate transcript counts at the single-molecule level for each gene within individual cells. Genes that were not detected in at least one cell were excluded from subsequent analyses.  Droplet-based scSTAMP-seq data processing. For droplet-based sequencing, read 2 was trimmed using the default settings of TrimGalore. After trimming, STARsolo (STAR aligner version 2.7.8a) was used to map the reads to hg19 using the gene annotation file from Ensembl. Transcripts with the same UMI were deduplicated and genes that were not detected in at least one cell were removed from any downstream analysis.  scSTAMP-seq gene expression analysis. The standard analysis pipeline in Seurat (version 4.3.0) was used for single-cell RNA expression normalization and analysis36. For plate-based sequencing experiments, cells containing more than 1000 genes and more than 1000 unique transcripts, as well as less than 20% ERCC spike-ins, were used for downstream analysis. For the droplet-based sequencing experiment, cells containing more than 200 genes and more than 200 unique transcripts, as well as less than 5% mitochondrial counts, were used for downstream analysis. The default NormalizeData function was used to log normalize the data. Principal components were obtained from the 2,000 most variable genes and the elbow method was used to determine the optimal number of principal components used in clustering. UMAP based clustering was performed by running the following functions, FindNeighbors, FindClusters, and RunUMAP. After clustering, cell types were assigned to groups using known expression markers, spatial barcode counts, and multiplex barcode counts, where stated.
scSTAMP-MAT-seq genome-wide DNA accessibility and 5mC analysis. The scSTAMP-MAT-seq analysis pipeline for DNA accessibility and 5mC was performed as described in the scMAT-seq methods by Chialastri et al.31. DNase I hypersensitivity sites for Hela cells were downloaded from the UCSC table browser and sites were grouped based on detection scores35. To compare across datasets, the data was normalized to counts per million and a 75 base pair moving average was plotted for region of interest. When comparing differing genomic regions within a sample, each region was further normalized by methylated cytosines that were detected when MspJI-seq was performed on bulk Hela gDNA that had been GpC methylated after stripping off chromatin. Bulk Hela gDNA isolation, chromatin stripping, and processing was performed as described previously by Chialastri et al.31. For clustering, DNA accessibility and 5mC were quantified within 5 kb bins and then converted to binary scores. Pseudobulk profiles were generated using the assigned cell type from the transcriptome. For comparison between Hela and U2OS cells, the top 2% most variable bins between groups were retained. After removal of bins with low variance, principal component analysis was used to assign clusters. Cluster identification was performed through comparison to transcriptome derived cell types, where high similarity was observed between cluster calling for all 3 measurements. A threshold minimum of 15,000 was applied to the data shown in Supplementary Figure 14a,b.
Assembly: hg19
Library strategy: scSTAMP-seq

value: Hela cells were labeled with PHO1. Hela cell samples then received 0, 1, or 10 minutes of UV flood light illumination. Hela cell samples were dissociated, resuspended, and then processed for single cells by a 10X Genomics Chromium Controller following the workflow of the Next GEM Single Cell 3' LT kit v3.1.


title: scSTAMP-seq_CombinatorialBarcoding_Plate1_Lib3_Spatial

status: Public on Dec 04 2025

type: SRA

city: Santa Barbara

first: Robert

middle: Anthony

last: Piscopio

state: CA

address: 2002 BioEngineering Bldg, CNTR for Bioengineering

country: USA

institute: University of California, Santa Barbara

description: Method is detailed in Piscopio et al.

library_source: other

data_processing:
Supplementary files format and content: text file for PHO barcodes where each row contains counts for inner and outer spatial barcodes.
scSTAMP-seq data processing and gene expression analysis. Sequencing FASTQ data files were processed using custom Perl scripts on a Linux high performance computing (HPC) cluster at the University of California, Santa Barbara. Sequencing data was post-processed and analyzed using Seurat (version 4.3.0) in R (version 4.2.2) and RStudio (version 2022.12.0+353). Representative figures were generated in RStudio and compiled in Adobe Illustrator (version 26.5).  scSTAMP-seq barcode processing and analysis. A custom Perl script was written to evaluate the spatial barcode portion of the sequencing library. For PHO spatial barcode sequences, read 1 was used to determine cell barcode/UMI. Read 2 was used to distinguish ‘inner’ and ‘outer’ sequence reads by identifying unique ‘inner’ and ‘outer’ primer sequences as well as the downstream spatial barcode/UMI sequence. MPO barcode counts were determined similarly, but an additional multiplex identifier sequence from read 2 was used to distinguish multiplex from spatial barcodes. Unique counts for each molecule were defined as reads containing a unique combination of the UMI on read 1 and on read 2.  Plate-based scSTAMP-seq data processing. For plate-based sequencing, read 2 of the paired-end read was aligned in the sense direction using BWA (version 0.7.15-r1140) to the RefSeq gene model based on the human genome release hg19, incorporating a collection of 92 ERCC spike-in molecules. In cases where a read aligned to multiple locations, it was evenly distributed across those locations. Gene isoforms were consolidated into a single gene count, and the unique molecular identifiers (UMIs) were used to remove duplicate reads and generate transcript counts at the single-molecule level for each gene within individual cells. Genes that were not detected in at least one cell were excluded from subsequent analyses.  Droplet-based scSTAMP-seq data processing. For droplet-based sequencing, read 2 was trimmed using the default settings of TrimGalore. After trimming, STARsolo (STAR aligner version 2.7.8a) was used to map the reads to hg19 using the gene annotation file from Ensembl. Transcripts with the same UMI were deduplicated and genes that were not detected in at least one cell were removed from any downstream analysis.  scSTAMP-seq gene expression analysis. The standard analysis pipeline in Seurat (version 4.3.0) was used for single-cell RNA expression normalization and analysis36. For plate-based sequencing experiments, cells containing more than 1000 genes and more than 1000 unique transcripts, as well as less than 20% ERCC spike-ins, were used for downstream analysis. For the droplet-based sequencing experiment, cells containing more than 200 genes and more than 200 unique transcripts, as well as less than 5% mitochondrial counts, were used for downstream analysis. The default NormalizeData function was used to log normalize the data. Principal components were obtained from the 2,000 most variable genes and the elbow method was used to determine the optimal number of principal components used in clustering. UMAP based clustering was performed by running the following functions, FindNeighbors, FindClusters, and RunUMAP. After clustering, cell types were assigned to groups using known expression markers, spatial barcode counts, and multiplex barcode counts, where stated.
scSTAMP-MAT-seq genome-wide DNA accessibility and 5mC analysis. The scSTAMP-MAT-seq analysis pipeline for DNA accessibility and 5mC was performed as described in the scMAT-seq methods by Chialastri et al.31. DNase I hypersensitivity sites for Hela cells were downloaded from the UCSC table browser and sites were grouped based on detection scores35. To compare across datasets, the data was normalized to counts per million and a 75 base pair moving average was plotted for region of interest. When comparing differing genomic regions within a sample, each region was further normalized by methylated cytosines that were detected when MspJI-seq was performed on bulk Hela gDNA that had been GpC methylated after stripping off chromatin. Bulk Hela gDNA isolation, chromatin stripping, and processing was performed as described previously by Chialastri et al.31. For clustering, DNA accessibility and 5mC were quantified within 5 kb bins and then converted to binary scores. Pseudobulk profiles were generated using the assigned cell type from the transcriptome. For comparison between Hela and U2OS cells, the top 2% most variable bins between groups were retained. After removal of bins with low variance, principal component analysis was used to assign clusters. Cluster identification was performed through comparison to transcriptome derived cell types, where high similarity was observed between cluster calling for all 3 measurements. A threshold minimum of 15,000 was applied to the data shown in Supplementary Figure 14a,b.
Assembly: hg19
Library strategy: scSTAMP-seq

molecule: other

organism: Homo sapiens

source_name: Hela

growth_protocol: Cell culture. Hela and U2OS cells were cultured and maintained on tissue cultured treated plastic flasks (Thermo Scientific, 156367) or 6-well plates (Corning, 3516) in DMEM with high glucose, GlutaMAX and sodium pyruvate (Gibco, 10569044), supplemented with 10% fetal bovine serum (FBS) (Gibco, 10569044) and 1% Pen-Strep (Gibco, 15140122). All mammalian cell cultures were maintained in incubators at 37°C and 5% CO2. Cells were routinely passaged 1:6 after 70-80% confluence was reached using 0.25% trypsin-EDTA (Gibco, 25200056). For microscopy imaging, cells were seeded onto glass 96 well glass bottom plates (Cellvis, P96-1.5H-N) or 35 mm dishes with glass coverslips (MATTEK, P35G-1.5-20-C) coated with fibronectin (Advanced Biomatrix, 5050). For the combinatorial barcoding experiment, cells were seeded on a custom protein patterned 96-well plate (CYTOO, 20-950-00) treated with fibronectin.

extract_protocol:
For FACS sorting, a single-cell suspension was made by dissociating cells using 0.25% trypsin-EDTA and inactivated using DMEM containing serum. Afterwards, cells were washed and resuspended in 1 mL of cold 1x DPBS-/- (without calcium or magnesium) (Gibco, 14040133) before being passed through a 35 µm nylon mesh cell strainer into a 5 mL round bottom polystyrene tube (Corning, 352235) that was then kept on ice. For plate-based single-cell capture, cell suspensions were FACS sorted for single cells into 384-well plates (Bio-Rad, HSP3801). For droplet-based single-cell capture, cell suspensions were processed for single cells by a 10X Genomics Chromium Controller following the workflow of the 10X Genomics Chromium Next GEM Single Cell 3’ LT Kit v3.1 (10X Genomics, 1000325).
scSTAMP-seq plate-based library preparation. 4 µL of Vapor-Lock (QIAGEN, 981611) was dispensed into each well of a 384-well plate using a 12-channel pipette. All downstream dispensing into 384-well plates was performed using a Nanodrop II liquid handling robot (BioNex Solutions). To each well, 100 nL of uniquely barcoded 7.5 ng/µL reverse transcription primers containing 6-nucleotide UMI was added. The reverse transcription primers used here have been described by Grün et al.33. Next, 100 nL of lysis buffer (0.175% IGEPAL CA-630, 1.75 mM dNTPs, 1:1,250,000 ERCC RNA spike-in mix (Ambion, 4456740), 0.19 U RNase inhibitor (Clontech, 2313A)) was added to each well. Single cells were sorted into individual wells of a 384-well plate using FACS. After sorting, plates were heated to 65°C for 3 minutes and returned to ice. Reverse transcription was conducted next by adding 150 nL of reverse transcription mix (0.7 U RNase OUT (Invitrogen, 10777-019), 2.34x first strand buffer (Invitrogen, 10777-019), 23.34 mM DTT (Invitrogen, 10777-019), 3.5 U Superscript II (Invitrogen, 18064-071)) to each well and the plates were incubated at 42°C for 1 hour and 15 minutes, 4°C for 5 minutes, 70°C for 10 minutes, and then held at 4°C. Next, second strand synthesis was carried out by adding 1.75 µL of second strand synthesis mix (1.74x second strand buffer (Invitrogen, 10812-014), 0.35 mM dNTP (Invitrogen, 10812-014), 0.14 U E. coli DNA Ligase (Invitrogen, 18052-019), 0.56 U E. coli DNA Polymerase I (Invitrogen, 18010-025), 0.03 U RNase H (Invitrogen 18021-071)) to each well and the plates were incubated at 16°C for 2 hours and then held at 4°C. 1 µL of nuclease-free water was added into each well to minimize losses during downstream pooling. The PHO, MPO, and endogenous mRNA molecules in a well now all contain the same RNA capture barcode, indicating a unique cell, which allows for pooling. Reaction wells receiving different barcodes were pooled using a multichannel pipette, and the oil phase was discarded. The small molecule spatial barcode portion was separated from endogenous transcript cDNA using a 3.2x SPRI bead cleanup with 1.8x 100% isopropanol in 2 steps. First, 0.7x AMPure XP beads (Beckman Coulter, A63881) were added and incubated at room temp for 15 minutes. At this step, it is critical to not dispose of the supernatant, as the spatial barcode library portion is found in the supernatant and the endogenous cDNA is bound to SPRI beads. The supernatant containing the spatial barcode fragments was then transferred to a fresh Eppendorf tube. The endogenous cDNA portion was prepared as described previously by Chialastri et al.31. Second, to the spatial barcode sample, an additional 2.5x beads and 1.8x 100% isopropanol (of the original pre-cleanup sample volume) were added and incubated at room temp for 15 minutes. The samples were placed on a magnetic stand for 5 minutes. The supernatant was removed and beads were washed twice with 80% ethanol. The sample was then eluted in 30 µL of nuclease-free water. The sample was then concentrated to 15 µL using an Eppendorf Vacufuge plus. Next, samples were prepared for PCR amplification by mixing 15 µL of the concentrated elute from the previous step, 25 µL of 2x PCR master mix (NEB, M0541S), 2 µL Illumina RPI primer (10 µM), 2 µL each of an ‘inner’ and ‘outer’ PCR primer (10 µM), and nuclease-free water up to 50 µL. This first PCR was performed at 94°C for 10 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, for 10 cycles. A subsequent ExoSAP-IT treatment was then applied to cleanup the PCR product as follows. 2 µL of ExoSAP-IT (Applied Biosystems, 78200.200.UL) was added to 5 µL of sample from the previous PCR (for a total volume of 7 µL) and incubated at 37°C for 15 minutes. Another PCR was performed by mixing 7 µL of the cleaned-up PCR with 22.5 µL 2x PCR master mix, 2 µL Illumina RPI primer, 2 µL Illumina sample index primer, and nuclease-free water up to 45 µL. This second PCR was performed at 94°C for 10 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, for 6-12 cycles.  Another ExoSAP-IT treatment was then applied to the 45 µL reaction by adding 5 µL of ExoSAP-IT to the previous PCR (for 50 µL total) and incubated at 37°C for 15 minutes. Finally, a 1.2x double-sided selection (0.90x left/0.30x right) was used to purify the spatial library, and a final ethanol wash was performed twice. The product was then eluted in 20 µL of nuclease-free water. Library fragment size distribution was determined using a 2100 Bioanalyzer. A schematic depicting each step of this process can be found in Supplementary Figure 1.  scSTAMP-seq droplet-based library preparation. Sequencing libraries were prepared using a custom protocol based on the 10X Genomics Chromium Next GEM Single Cell 3’ LT Kit v3.1 (10X Genomics, 1000325) adapted from McGinnis et al.29. The 10X workflow was followed until cDNA amplification. We designed custom ‘inner’ and ‘outer’ PHO handle primers that allow scSTAMP-seq to be adapted for the 10X workflow (Supplementary Table 1). The scSTAMP-seq 10X ‘inner’ and ‘outer’ PHO primers were mixed to a working concentration of 2.5 µM. Next, the following cDNA amplification master-mix was made: 50 µL 10X amplification master mix, 15 µL 10X cDNA primer mix, 1 µL scSTAMP-seq 10X primers. Then, cDNA amplification was performed and an SPRI size selection was performed by incubating samples for 30 minutes with 0.6x AMPure XP beads to separate spatial and endogenous cDNA fractions. Next, the small molecule spatial barcode fraction was separated from endogenous transcript cDNA using the same strategy as described in the plate-based library preparation method above. The endogenous cDNA portion was then processed to generate Illumina libraries according to the 10X workflow and the spatial portion was processed to generate Illumina libraries as described in the plate-based library preparation workflow. A schematic depicting each step of this process can be found in Supplementary Figure 10.  scSTAMP-MAT-seq multiomic library preparation. The DNA accessibility and 5mC sequencing pipeline was performed as described previously in the scMAT-seq methods by Chialastri et al.31 up to the first SPRI bead clean up. At this step, the small molecule spatial barcode portion was separated from endogenous molecules using the same strategy as described in the plate-based library preparation workflow. The transcriptomic and epigenomic libraries were processed to generate Illumina libraries as described in scMAT-seq by Chialastri et al.31 and the spatial portion was processed to generate Illumina libraries as described in the plate-based library preparation workflow above. A schematic depicting each step of this process can be found in Supplementary Fig. 12.  Library sequencing was performed by Novogene on an Illumina HiSeq 4000 platform or at the Biological Nanostructures Laboratory (BNL) in the California NanoSystems Institute (CNSI) at the University of California Santa Barbara (UCSB) on an Illumina NextSeq 500 platform.

treatment_protocol: PHO labeling of cell membranes. First, cell culture media was removed, and cells were washed three times with cold DPBS+/+ (with calcium and magnesium) (Gibco, 14040133). Hybridized PHO/CMO-anchor at 500 nM was then incubated with live-cell samples over ice for 15 minutes. Next, cells were washed again three times with cold DPBS+/+. After washing, CMO co-anchors were incubated with live-cell samples over ice for 15 minutes. Cells were then washed again three times with cold DPBS+/+. This subsequent two-step anchor/co-anchor incubation is optimal for oligo embedding rate and retention time, as described previously by Weber et al.28. Cells were then imaged and illuminated with UV light. After illumination, cells were either processed for single cell sorting, or the labeling procedure was repeated for experiments that utilized iterative rounds of unique spatial barcode labeling. For experiments that incorporated MPOs, both PHOs and MPOs were incubated simultaneously.  Microscopy and photomask illumination. All microscopy was performed using a Nikon Ti-2 inverted confocal microscope equipped with a Yokogawa CSU-W1 SoRa spinning disk and an Andor iXon Life 888 EMCCD camera. Images were captured with a Nikon CFI S Plan Fluor ELWD 20x objective lens and a Nikon CFI Plan Apo Lambda 10x objective lens. For fluorescence confocal imaging, 488 nm, 561 nm, and 640 nm lasers at 100% power were delivered through liquid light guides for FAM, Cy3, and Cy5 fluorophores, respectively. Digital light processing (DLP) patterned light projection was done using a Mightex Polygon400-G digital micromirror device (DMD) coupled with an Excelitas X-Cite 360-380 nm UVA liquid light guide-coupled LED, or a 365 nm 50W LED flood light (Everbeam, B08635F9CX) with an acrylic light diffusion film and custom 3D printed plate stand for consistent and uniform illumination.

tag: cell line

value: Hela

tag: treatment

value: Hela cell samples were labeled with PHO1, PHO2, and PHO3. Hela cell samples then received 60 seconds of UV DLP light illumination. After PHO1 labeling, Hela cell samples received 60 seconds of a three quarters area UV DLP light illumination photomask. After PHO2 labeling, Hela cell samples received 60 seconds of a one half area UV DLP light illumination photomask. After PHO3 labeling, Hela cell samples received 60 seconds of a one quarter area UV DLP light illumination photomask.


title: 2_V278_ST

city: Vienna

first: Andreas

last: Lackner

address: Schwarzspanierstrasse 17

department: Center for Anatomy and Cell Biology

country: Austria

institute: Medical University of Vienna

library_source: transcriptomic

data_processing:
adapter trimming; bbduk
UMI extraction, UMItools
alignment; STAR (2.7.3c)
UMI collapsing; UMItools
Read count; htseq (version 0.11.2)
Assembly: GENCODE V38
Supplementary files format and content: count table of each submitted sample

molecule: polyA RNA

source_name: placenta

extract_protocol:
RNA extraction was carried out using RNeasy mini/micro and DNAse I kit (all from QIAGEN) according to the manufacturer’s instruction. RNA quantification was carried out by Nanodrop (ThermoFisher) or RNA broad range QUBIT assay kit (ThermoFisher, Q10211).
Libraries were prepared using QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina - Dual Indexing, 96 UDI (Workflow B), Set B1 (Lexogen) following the manufacturer’s instructions.

tag: tissue

value: placenta

tag: cell type

value: ST

tag: genotype

value: WT


title: 3_V188_SR

value: hTSC

value: shNeg


title: Ctrl_rep_2

city: shenzhen

first: Xiaona

last: Chen

state: China

address: No. 113 Baohe Road, Longgang District, Shenzhen

country: China

institute: Southern University of Science and Technology

description: Library name: Ctrl_2

data_processing:
RNA-seq: Reads were aligned to the mouse Ensemble genome GRCm39 using Hisat2 aligner (v2.2.1). The reads mapped the genome were calculated using StringTie (v2.1.6). Differential gene expression analysis was performed using the DESeq2 R-package.
Assembly: GRCm39
Supplementary files format and content: read counts table and FPKM table

molecule: total RNA

organism: Mus musculus

source_name: cell line

extract_protocol:
Total RNA was extracted using TRIzol™ Reagent (Invitrogen, 15596018).
dUTP method was used for library preparation.

value: cell line

value: Macrophage; Abelson murine leukemia virus transformed

value: overexpressed control

value: without LPS treatment


title: Ctrl_rep_3

description: Library name: Ctrl_3


title: Ctrl_rep_1

description: Library name: Ctrl_1


title: 2_V188_ST


title: scSTAMP-seq_CombinatorialBarcoding_Plate1_Lib1_Spatial

description: Method is detailed in Piscopio et al.


title: Cerebellum, Het-DMSO, 411

city: Albany

first: Cara

middle: T

last: Pager

state: NY

address: 1400 Washington Avenue

institute: University at Albany, State University of New York

data_processing:
After sequencing the quality of the fastq files were checked using the program FastQC (version 0.11.9).
Both paired-end files for samples 409, 422, 425, and 426 were downsampled using Seqtk (version 1.4) to either ~65 or 70 million reads per file to more closely match the read depth of the other samples in the dataset.
STAR (version 2.7.10a) was used to align the fastq files to the GRcm39/mm39 genome with the --quantModeGeneCounts option added to count the reads per gene.
Assembly: GRCm39/mm39
Supplementary files format and content: Tab-delimited text files contain the raw read count abundances for each sample.

source_name: Cerebellum

extract_protocol:
RNA forwild-type and SCA1 KI mice was extracted using TRIzol (Ambion, Life Technologies),following the manufacturers protocol and aDNA digestion was performed usingthe TURBO DNA-free Kit (Invitrogen). The Qubit RNA high sensitivity assay wasused to obtain RNA concentrations(Thermo Fisher Scientific). RNA quality waschecked via capillary electrophoresis on a 5300 fragment analyzer (AgilentBioanalyzer) using the RNA AnalysisDNF-471 RNA kit (Agilent).
The NEBNext Ultra IIDirectional RNA Library Prep Kit (Illumina) with NEBNext rRNA Depletion Kit(New EnglandBiolabs) was used to prepare RNA-seq libraries, with a total of500 ng input RNA from each sample. The manufacturer’sprotocols were followed,with the following exceptions: 40× adaptor dilutions were used, and 13 cyclesof libraryamplification were performed. The resulting libraries were pooled inequimolar amounts, quantified using the NEBNextLibrary Quant Kit for Illumina(New England Biolabs), quality checked via capillary electrophoresis on the5300fragment analyzer (Agilent Bioanalzyer) using the DNF-474 HS NGS kit(Agilent). Libraries with sufficient quantitywere loaded into P2 flow cell1000/2000 by Illumina and were sequenced using paired-end, 100 base pairsequencing onthe Illumina NextSeq 2000 sequencer at the University at AlbanyRNA Institute. All datasets had total read depths greaterthan 40 million paired-end reads.

treatment_protocol: Starting at 18 weeks ofage, WT and Atxn1154Q/2Q mice were treated with 25mg/ml Hit 2 dissolved in DMSO or DMSO alone (DMSO<4ml/kg) via intraperitoneal (IP) injection daily for 5 days, followed by tworest days and an additional 5 days of IPinjection. All injections were performedbetween 8 and 9am in the morning and on the final day of injections (20 weeksofage), after 3pm, mice were anesthetised using urethane in saline (1.2-1.5 g/kg)followed by a double thoracotomy andperfusion through the ascending aorta with20 mLs 1x PBS. Cerebellum was removed and stored at -70°C.

value: Cerebellum

value: Het

value: DMSO


title: 2_V281_SR


title: scSTAMP-seq_CombinatorialBarcoding_Plate1_Lib2_Spatial

description: Method is detailed in Piscopio et al.


title: Cerebellum, Het-Hit2, 410


title: Cerebellum, Het-DMSO, 409


title: scSTAMP-seq_ErrorCorrection_Plate6_Lib4_Spatial

description: Method is detailed in Piscopio et al.

value: Hela cell samples were labeled with PHO1, PHO2, PHO4, PHO5, PHO7, and PHO8. Hela cell samples then received 0, 1.5, 4.5, 13.5, 18, or 25 seconds of UV DLP light illumination.


title: 2_V278_SR


title: Caerulein pancreatitis - Elp3WT - Mmu 71

city: Liège

first: Arnaud

last: Lavergne

state: Liège

address: Avenue de l'hôpital 11

department: GIGA

country: Belgium

institute: University of Liège

description: Library name: d6-wt-71

data_processing:
Trimming (Trimgalore)
Mapping (STAR)
Quantification (Salmon)
QCs
DESeq2 downstream analysis
Assembly: GRCm39.107
Supplementary files format and content: salmon.merged.gene_counts.tsv : raw genes counts from salmon quantification
Supplementary files format and content: Counts_norm.tsv : DESeq2 normalized counts table

source_name: pancreas

extract_protocol:
TriPure isolation method (11667157001, Roche)
Illumina totalRNA stranded ligation protocol

treatment_protocol: RNAlater™ Stabilization Solution (AM7021, Invitrogen), overnight, 4°C

value: pancreas

value: caerulein (day 6)


title: scSTAMP-seq_DropletBasedSequencing_Hela_Spatial

value: Hela

value: Hela cells were labeled with PHO1. Hela cell samples then received 0, 1, or 10 minutes of UV flood light illumination. Hela cell samples were dissociated, resuspended, and then processed for single cells by a 10X Genomics Chromium Controller following the workflow of the Next GEM Single Cell 3' LT kit v3.1.


title: Caerulein pancreatitis - Elp3WT - Mmu 74

description: Library name: d6-wt-74


title: T-bet ZsGreen Low rep2 (ATAC-seq)

city: Berlin

first: Max

last: Löhning

address: Chariteplatz 1

country: Germany

institute: DRFZ

description:
step3.2_normalized_per_10M_3L_S4
full_count_ATAC.txt

data_processing:
QC and analysis on ATAC-seq libraries was performed using AIAP pipeline. The reads were mapped to mouse mm10 genome.
The generated peaks files for each library were annotated using Homer package.
Genome_build: mm10
Supplementary files format and content: Tab-delimited text file including raw counts for peaks

source_name: Spleen & lymph node

extract_protocol:
For each sort, spleens and lymph nodes of 4-5 mice were pooled. Thy1.1+ CD4 T cells were magnetically enriched with CD90.1 beads. Live, single CD90.1+ lymphocytes were FACS sorted according to their T-bet ZsGreen reporter brightness into high and low expressors. For RNA-Seq: Cells were lysed using RA1 buffer and stored at -80°C until further processing. Total RNA was purified with Nucleospin RNA XS Micro Kit without carrier RNA. For ATAC-Seq: The cell sample preparation manual from the ATAC-Seq Kit (activemotif) was followed for DNA tagmentation and purification. The samples were stored at -20°C until further processing.
For RNA-Seq: cDNA library was prepared using Smart-Seq v4 mRNA Ultra Low Input RNA Kit. For ATAC-Seq: PCR Amplification of tagmented DNA was performed using the ATAC-Sequencing Kit (activemotif). With an Illumina NextSeq2000 device, paired-end sequencing (2x75bp) of the libraries was performed.

tag: infection

value: LCMV Arm

tag: time

value: d10 p.i. LCMV Arm

value: Spleen & lymph node

value: Smarta+ activated CD4 T cells

tag: t-bet zsgreen brightness

value: Low


title: 2_V188_SR

value: placenta

value: hTSC

value: WT

value: shNeg


title: scSTAMP-seq_ErrorCorrection_Plate6_Lib3_Spatial

description: Method is detailed in Piscopio et al.


title: T-bet ZsGreen Low rep1 (ATAC-seq)

description:
step3.2_normalized_per_10M_2L_S2
full_count_ATAC.txt


title: 1_V278_ST

value: ST


title: scSTAMP-seq_ErrorCorrection_Plate6_Lib2_Spatial

description: Method is detailed in Piscopio et al.


title: T-bet ZsGreen High rep2 (ATAC-seq)

description:
step3.2_normalized_per_10M_3H_S3
full_count_ATAC.txt

value: High


title: Caerulein pancreatitis - Elp3WT - Mmu 68

description: Library name: d6-wt-68


title: 1_V281_SR


title: 1_V188_ST


title: Caerulein pancreatitis - Elp3KO - Mmu 79

description: Library name: d6-ko-79

value: Elp3(fl/fl); Pdx-Cre


title: scSTAMP-seq_ErrorCorrection_Plate6_Lib1_Spatial

description: Method is detailed in Piscopio et al.

value: Hela cell samples were labeled with PHO1, PHO2, PHO4, PHO5, PHO7, and PHO8. Hela cell samples then received 0, 1.5, 4.5, 13.5, 18, or 25 seconds of UV DLP light illumination.


title: scSTAMP-seq_ErrorCorrection_Plate5_Lib4_Spatial

description: Method is detailed in Piscopio et al.


title: T-bet ZsGreen Low rep3 (RNA-seq)

description:
3L_S8
featureCounts_RNA-seq.txt

data_processing:
Sequence reads from RNA-seq were mapped to the mm39 genome (annotation release: M27_GRCm39) using Hisat2 with default settings and quality was controlled with Trimmomatic package
Gene expression was quantified by featureCounts.
Genome_build: mm39
Supplementary files format and content: Tab-delimited text file including raw counts for genes


title: scSTAMP-seq_ErrorCorrection_Plate5_Lib3_Spatial

description: Method is detailed in Piscopio et al.


title: 1_V278_SR


title: Caerulein pancreatitis - Elp3KO - Mmu 77

description: Library name: d6-ko-77

value: pancreas


title: T-bet ZsGreen Low rep2 (RNA-seq)

description:
2L_S5
featureCounts_RNA-seq.txt


title: T-bet ZsGreen Low rep1 (RNA-seq)

description:
1L_S2
featureCounts_RNA-seq.txt


title: Caerulein pancreatitis - Elp3KO - Mmu 69

description: Library name: d6-ko-69

value: caerulein (day 6)


title: scSTAMP-seq_ErrorCorrection_Plate5_Lib1_Spatial

description: Method is detailed in Piscopio et al.

value: Hela


title: 1_V188_SR


title: Control pancreas - Elp3WT - Mmu 88

description: Library name: ctrl-wt-88


title: T-bet ZsGreen High rep2 (RNA-seq)

description:
2H_S4
featureCounts_RNA-seq.txt


title: scSTAMP-seq_ErrorCorrection_Plate4_Lib4_Spatial

description: Method is detailed in Piscopio et al.


title: Control pancreas - Elp3WT - Mmu 85

description: Library name: ctrl-wt-85


title: Caerulein pancreatitis - Elp3KO - Mmu 75

description: Library name: d6-ko-75


title: 3_ST_72h_BRM014

value: Forskolin + BRM014


title: T-bet ZsGreen High rep3 (RNA-seq)

description:
3H_S7
featureCounts_RNA-seq.txt

value: LCMV Arm


title: Caerulein pancreatitis - Elp3WT - Mmu 70

description: Library name: d6-wt-70


title: 3_ST_72h

value: Forskolin


title: scSTAMP-seq_ErrorCorrection_Plate4_Lib3_Spatial

description: Method is detailed in Piscopio et al.


title: Control pancreas - Elp3WT - Mmu 80

description: Library name: ctrl-wt-80


title: T-bet ZsGreen High rep1 (RNA-seq)

description:
1H_S1
featureCounts_RNA-seq.txt

value: d10 p.i. LCMV Arm

value: Spleen & lymph node


title: 3_SR_72h_BRM014


title: scSTAMP-seq_ErrorCorrection_Plate3_Lib4_Spatial

description: Method is detailed in Piscopio et al.


title: T-bet ZsGreen High rep1 (ATAC-seq)

description:
step3.2_normalized_per_10M_2H_S1
full_count_ATAC.txt

value: Smarta+ activated CD4 T cells


title: Control pancreas - Elp3WT - Mmu 76

description: Library name: ctrl-wt-76


title: scSTAMP-seq_ErrorCorrection_Plate5_Lib2_Spatial

description: Method is detailed in Piscopio et al.


title: 3_SR_72h

value: placenta


title: Apple peel “Red Love” sample 3

city: Kunming

first: Yun

last: Zheng

state: Yunnan

address: 452 Fengyuan Road

institute: Yunnan Agricultural University

description: Library name: Rpe3

data_processing:
The raw sequening profiles in FASTQ format were aligned to reference genome with the Burrows-Wheeler algorithm.
The obtained alignments in the SAM format were sorted with SAMTools (v1.12)
The transcripts were assembled with Cufflinks (v2.2.1).
The transcripts in different profiles were merged with cuffmerge in the Cufflinks package.
The merged transcripts were then compared the original gene annotation of apple (cultivar: Golden Delicious) with cuffcompare in the Cufflinks package.
The expression levels of transcripts were obtained using cuffquant in the Cufflinks package.
The normalized expression levels of transcripts were obtained by cuffnorm in the Cufflinks package.
Assembly: Golden Delicious (NCBI Accession No. GCA_002114115.1)
Supplementary files format and content: Excel file includes transcript IDs, annotations, and expression levels of transcripts (in count).

organism: Malus domestica

source_name: Apple peel

growth_protocol: The apple trees of three selected cultivars were grown in Xiaolong Village, Malong County, Qujing, Yunnan, China, which had an altitude of 2,030 meters, an average annual temperature of 13.8 degree Celsius, and an annual rainfall of 1020 mm.

extract_protocol:
The total RNA of each sample was extracted using TRIZOL reagent.
Ribosomal RNA is removed from the total RNA, resulting in mRNA. Subsequently, fragmentation buffer was added to break the RNA into short fragments, and the short RNA fragments were used as a template with a six-base random primer(random hexamers) to synthesize first-strand cDNA, followed by the addition of buffers, dNTPs (dUTP, dATP, dGTP, and dCTP), and DNA polymeraseI synthesized two-stranded cDNA, followed by purification of double-stranded cDNA using AMPure XPbeads. Purified double-stranded cDNA is re-entered Row end repair, A-tail addition, and sequencing adapter ligation, followed by fragment size selection with AMPure XPbeads, and finally PCR Enrichment yields the final cDNA library. Input cDNAs were sequenced using the Illumina Novaseq 6000 platform.

treatment_protocol: No special treatments were applied.

value: Apple peel

tag: cultivar

value: Red Love (Rpe)


title: scSTAMP-seq_ErrorCorrection_Plate3_Lib3_Spatial

description: Method is detailed in Piscopio et al.


title: Control pancreas - Elp3KO - Mmu 86

description: Library name: ctrl-ko-86


title: scSTAMP-seq_ErrorCorrection_Plate3_Lib2_Spatial

description: Method is detailed in Piscopio et al.


title: Apple peel “Red Love” sample 2

description: Library name: Rpe2


title: 2_ST_72h_BRM014

value: WT


title: Control pancreas - Elp3KO - Mmu 84

description: Library name: ctrl-ko-84


title: Apple peel “Venus Gold” sample 1

description: Library name: Ype1

value: Venus Gold (Ype)


title: Apple peel “Venus Gold” sample 2

description: Library name: Ype2


title: Apple peel “Venus Gold” sample 3

description: Library name: Ype3


title: 1_ST_72h_BRM014

value: ST


title: scSTAMP-seq_ErrorCorrection_Plate3_Lib1_Spatial

description: Method is detailed in Piscopio et al.


title: 2_SR_72h

value: hTSC


title: Control pancreas - Elp3KO - Mmu 83

description: Library name: ctrl-ko-83

value: Elp3(fl/fl); Pdx-Cre


title: scSTAMP-seq_MultiplexOligoExp_Plate4_Lib2_Spatial

description: Method is detailed in Piscopio et al.

data_processing:
Supplementary files format and content: text file for PHO barcodes where each row contains counts for inner and outer spatial barcodes. text file for MPO barcodes where each row contains counts for multiplex barcodes.
scSTAMP-seq data processing and gene expression analysis. Sequencing FASTQ data files were processed using custom Perl scripts on a Linux high performance computing (HPC) cluster at the University of California, Santa Barbara. Sequencing data was post-processed and analyzed using Seurat (version 4.3.0) in R (version 4.2.2) and RStudio (version 2022.12.0+353). Representative figures were generated in RStudio and compiled in Adobe Illustrator (version 26.5).  scSTAMP-seq barcode processing and analysis. A custom Perl script was written to evaluate the spatial barcode portion of the sequencing library. For PHO spatial barcode sequences, read 1 was used to determine cell barcode/UMI. Read 2 was used to distinguish ‘inner’ and ‘outer’ sequence reads by identifying unique ‘inner’ and ‘outer’ primer sequences as well as the downstream spatial barcode/UMI sequence. MPO barcode counts were determined similarly, but an additional multiplex identifier sequence from read 2 was used to distinguish multiplex from spatial barcodes. Unique counts for each molecule were defined as reads containing a unique combination of the UMI on read 1 and on read 2.  Plate-based scSTAMP-seq data processing. For plate-based sequencing, read 2 of the paired-end read was aligned in the sense direction using BWA (version 0.7.15-r1140) to the RefSeq gene model based on the human genome release hg19, incorporating a collection of 92 ERCC spike-in molecules. In cases where a read aligned to multiple locations, it was evenly distributed across those locations. Gene isoforms were consolidated into a single gene count, and the unique molecular identifiers (UMIs) were used to remove duplicate reads and generate transcript counts at the single-molecule level for each gene within individual cells. Genes that were not detected in at least one cell were excluded from subsequent analyses.  Droplet-based scSTAMP-seq data processing. For droplet-based sequencing, read 2 was trimmed using the default settings of TrimGalore. After trimming, STARsolo (STAR aligner version 2.7.8a) was used to map the reads to hg19 using the gene annotation file from Ensembl. Transcripts with the same UMI were deduplicated and genes that were not detected in at least one cell were removed from any downstream analysis.  scSTAMP-seq gene expression analysis. The standard analysis pipeline in Seurat (version 4.3.0) was used for single-cell RNA expression normalization and analysis36. For plate-based sequencing experiments, cells containing more than 1000 genes and more than 1000 unique transcripts, as well as less than 20% ERCC spike-ins, were used for downstream analysis. For the droplet-based sequencing experiment, cells containing more than 200 genes and more than 200 unique transcripts, as well as less than 5% mitochondrial counts, were used for downstream analysis. The default NormalizeData function was used to log normalize the data. Principal components were obtained from the 2,000 most variable genes and the elbow method was used to determine the optimal number of principal components used in clustering. UMAP based clustering was performed by running the following functions, FindNeighbors, FindClusters, and RunUMAP. After clustering, cell types were assigned to groups using known expression markers, spatial barcode counts, and multiplex barcode counts, where stated.
scSTAMP-MAT-seq genome-wide DNA accessibility and 5mC analysis. The scSTAMP-MAT-seq analysis pipeline for DNA accessibility and 5mC was performed as described in the scMAT-seq methods by Chialastri et al.31. DNase I hypersensitivity sites for Hela cells were downloaded from the UCSC table browser and sites were grouped based on detection scores35. To compare across datasets, the data was normalized to counts per million and a 75 base pair moving average was plotted for region of interest. When comparing differing genomic regions within a sample, each region was further normalized by methylated cytosines that were detected when MspJI-seq was performed on bulk Hela gDNA that had been GpC methylated after stripping off chromatin. Bulk Hela gDNA isolation, chromatin stripping, and processing was performed as described previously by Chialastri et al.31. For clustering, DNA accessibility and 5mC were quantified within 5 kb bins and then converted to binary scores. Pseudobulk profiles were generated using the assigned cell type from the transcriptome. For comparison between Hela and U2OS cells, the top 2% most variable bins between groups were retained. After removal of bins with low variance, principal component analysis was used to assign clusters. Cluster identification was performed through comparison to transcriptome derived cell types, where high similarity was observed between cluster calling for all 3 measurements. A threshold minimum of 15,000 was applied to the data shown in Supplementary Figure 14a,b.
Assembly: hg19
Library strategy: scSTAMP-seq

source_name: Hela, U2OS

value: Hela, U2OS

value: Hela and U2OS cell samples were labeled with PHO1. U2OS cell samples labeled with MPO2 received 0 minutes of UV flood light illumination. Hela cell samples labeled with MPO3 received 0 minutes of UV flood light illumination. U2OS cell samples labeled with MPO5 received 1 minute of UV flood light illumination. Hela cell samples labeled with MPO6 received 1 minute of UV flood light illumination.


title: 2_ST_72h


title: Control pancreas - Elp3KO - Mmu 67

description: Library name: ctrl-ko-67

value: pancreas


title: Apple peel “Red Love” sample 1

description: Library name: Rpe1


title: 2_SR_72h_BRM014


title: scSTAMP-seq_MultiplexOligoExp_Plate4_Lib1_Spatial

description: Method is detailed in Piscopio et al.


title: 1_SR_72h_BRM014


title: scSTAMP-seq_MultiplexOligoExp_Plate4_Lib1_Transcriptome

description: Method is detailed in Piscopio et al.

data_processing:
Supplementary files format and content: text file where column 1 contains gene names and columns 2-97 contain gene counts for 96 single cells.
scSTAMP-seq data processing and gene expression analysis. Sequencing FASTQ data files were processed using custom Perl scripts on a Linux high performance computing (HPC) cluster at the University of California, Santa Barbara. Sequencing data was post-processed and analyzed using Seurat (version 4.3.0) in R (version 4.2.2) and RStudio (version 2022.12.0+353). Representative figures were generated in RStudio and compiled in Adobe Illustrator (version 26.5).  scSTAMP-seq barcode processing and analysis. A custom Perl script was written to evaluate the spatial barcode portion of the sequencing library. For PHO spatial barcode sequences, read 1 was used to determine cell barcode/UMI. Read 2 was used to distinguish ‘inner’ and ‘outer’ sequence reads by identifying unique ‘inner’ and ‘outer’ primer sequences as well as the downstream spatial barcode/UMI sequence. MPO barcode counts were determined similarly, but an additional multiplex identifier sequence from read 2 was used to distinguish multiplex from spatial barcodes. Unique counts for each molecule were defined as reads containing a unique combination of the UMI on read 1 and on read 2.  Plate-based scSTAMP-seq data processing. For plate-based sequencing, read 2 of the paired-end read was aligned in the sense direction using BWA (version 0.7.15-r1140) to the RefSeq gene model based on the human genome release hg19, incorporating a collection of 92 ERCC spike-in molecules. In cases where a read aligned to multiple locations, it was evenly distributed across those locations. Gene isoforms were consolidated into a single gene count, and the unique molecular identifiers (UMIs) were used to remove duplicate reads and generate transcript counts at the single-molecule level for each gene within individual cells. Genes that were not detected in at least one cell were excluded from subsequent analyses.  Droplet-based scSTAMP-seq data processing. For droplet-based sequencing, read 2 was trimmed using the default settings of TrimGalore. After trimming, STARsolo (STAR aligner version 2.7.8a) was used to map the reads to hg19 using the gene annotation file from Ensembl. Transcripts with the same UMI were deduplicated and genes that were not detected in at least one cell were removed from any downstream analysis.  scSTAMP-seq gene expression analysis. The standard analysis pipeline in Seurat (version 4.3.0) was used for single-cell RNA expression normalization and analysis36. For plate-based sequencing experiments, cells containing more than 1000 genes and more than 1000 unique transcripts, as well as less than 20% ERCC spike-ins, were used for downstream analysis. For the droplet-based sequencing experiment, cells containing more than 200 genes and more than 200 unique transcripts, as well as less than 5% mitochondrial counts, were used for downstream analysis. The default NormalizeData function was used to log normalize the data. Principal components were obtained from the 2,000 most variable genes and the elbow method was used to determine the optimal number of principal components used in clustering. UMAP based clustering was performed by running the following functions, FindNeighbors, FindClusters, and RunUMAP. After clustering, cell types were assigned to groups using known expression markers, spatial barcode counts, and multiplex barcode counts, where stated.
scSTAMP-MAT-seq genome-wide DNA accessibility and 5mC analysis. The scSTAMP-MAT-seq analysis pipeline for DNA accessibility and 5mC was performed as described in the scMAT-seq methods by Chialastri et al.31. DNase I hypersensitivity sites for Hela cells were downloaded from the UCSC table browser and sites were grouped based on detection scores35. To compare across datasets, the data was normalized to counts per million and a 75 base pair moving average was plotted for region of interest. When comparing differing genomic regions within a sample, each region was further normalized by methylated cytosines that were detected when MspJI-seq was performed on bulk Hela gDNA that had been GpC methylated after stripping off chromatin. Bulk Hela gDNA isolation, chromatin stripping, and processing was performed as described previously by Chialastri et al.31. For clustering, DNA accessibility and 5mC were quantified within 5 kb bins and then converted to binary scores. Pseudobulk profiles were generated using the assigned cell type from the transcriptome. For comparison between Hela and U2OS cells, the top 2% most variable bins between groups were retained. After removal of bins with low variance, principal component analysis was used to assign clusters. Cluster identification was performed through comparison to transcriptome derived cell types, where high similarity was observed between cluster calling for all 3 measurements. A threshold minimum of 15,000 was applied to the data shown in Supplementary Figure 14a,b.
Assembly: hg19
Library strategy: scSTAMP-seq


title: 1_ST_72h


title: Apple peel “YanFu8” sample 2

description: Library name: Mpe2

value: Yanfu8 (Mpe)


title: WT5, L-fucose

city: Sacramento

first: Jacopo

last: Di Lucente

institute: UC Davis

description: Library name: L-fucose 5

data_processing:
Glaxy version 24.0.3
Sequence reads were trimmed Trimmomatic (version 0.39)
Trimmed reads were allign to mm10 using HISAT2 (version 2.2.1)
Gene expression was measured using featureCounts (version 2.0.3)
Differentially expressed features were determined using DESeq2 (version 2.11.40.8)
Assembly: mm10
Supplementary files format and content: Normalized counts

source_name: Brain

extract_protocol:
RNA was harvested using Rneasy plus universal mini kit (quiagen)
Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. Following is the workflow of library construction. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.

value: Brain

value: aCSF+L-fucose 5 hours


title: Apple peel “YanFu8” sample 1

description: Library name: Mpe1


title: WT4, L-fucose

description: Library name: L-fucose 4


title: scSTAMP-seq_MultiplexOligoExp_Plate3_Lib2_Hela_Spatial

description: Method is detailed in Piscopio et al.


title: r3_ST_BRM014_24h

value: Forskolin + BRM014


title: scSTAMP-seq_MultiplexOligoExp_Plate3_Lib2_Transcriptome

description: Method is detailed in Piscopio et al.


title: WT3, L-fucose

description: Library name: L-fucose 3


title: HFD+LPS tumor

type: RNA

city: Sendai

first: Kaname

last: Uno

address: 1-1 Seiryo Aoba-ku

department: Gastroenterology

country: Japan

institute: Tohoku University

description: gene expression data from HFD+LPS-stimulated Gan mice

hyb_protocol: not provided

scan_protocol: not provided

label: biotin

source_name: HFD+LPS

label_protocol: not provided

extract_protocol: not provided

value: HFD+LPS-stimulated tumor tissue


title: WT6, L-fucose

description: Library name: L-fucose 6


title: scSTAMP-seq_MultiplexOligoExp_Plate4_Lib2_Transcriptome

description: Method is detailed in Piscopio et al.


title: Apple peel “YanFu8” sample 3

description: Library name: Mpe3


title: Control tumor

description: gene expression data from control Gan mice

source_name: Control Diet

value: Control Tumor tissue


title: scSTAMP-seq_MultiplexOligoExp_Plate3_Lib1_Spatial

description: Method is detailed in Piscopio et al.


title: iPSC-derived spinal motor neurons, Control, IgG IP, rep4

city: Chicago

first: Evangelos

last: Kiskinis

state: IL

address: 303 E. Superior Street

department: Neurology

institute: Northwestern University Feinberg School of Medicine

description: Library name: rIgG_MN_4

data_processing:
Adapter removal and quality filtering was performed using cutadapt. Illumina adapters were removed (-a AGATCGGAAGAGC) along with N calls (--trim-n) and bases with Phred score < 20 (-q 20). A trimmed length of at least 25bp (-m 25) was required to pass filtering. Trimmed reads were aligned to GencodeV38 (GRCh38.p13) with the following flags: --twopassMode Basic; --outSAMtype BAM SortedByCoordinate; --outSAMstrandField intronMotif.
Assembly: hg38 (GRCh38.p13, Gencode V38 gene annotations)
Supplementary files format and content: tab-delimited text file includes gene-level counts for each sample

source_name: iPSC-derived spinal motor neurons

growth_protocol: iPSCs were differentiated into spinal motor neurons (MNs) following a standard 2D differentiation protocol (Ortega et al., 2020). iPSCs were dissociated into single cells with Accutase and plated in mTESR1 supplemented with 10μM ROCK inhibitor (Y-27632, DNSK International)at a density of 1.2million/well of a 6-well plate. After two days, cells were washed with PBS, then fed daily with N2B27 media (50% DMEM:F12 / 50% Neurobasal media, supplemented with Non-Essential Amino Acids, Glutamax, N2, B27, all from Thermo Fischer Scientific) supplemented with 10μM SB431542 (DNSK International), 100nM LDN-193189 (DNSK International), 1μM Retinoic Acid (RA, Sigma-Aldrich), 1μM of Smoothened-Agonist (SAG, DNSK International) to induce spinal neural progenitors. After 6 days, the cells were fed daily with N2B27 medium supplemented with 1μM RA, 1μM SAG, 5μM DAPT (DNSK International) and 4μM SU5402 (DNSK International)to generate postmitotic spinal MNs. After an additional 7 days (day 14), the cells were dissociated using TryplE (Thermo Fischer Scientific) with added DNaseI (Worthington) and plated onto plates previously coated with Matrigel (BD Bioscences) at a density of 1 million/well of a 6-well plate. Cells were then fed every other day with Neurobasal media supplemented with Non-Essential Amino Acids, Glutamax, N2, B27 and the neurotrophic factors BDNF, CNTF, GDNF (10ng/mL,R&D systems) and Ascorbic acid (0.2μg/ml). In the first four days after plating, the medium was supplemented with EdU (10μM) to inhibit the growth of remaining proliferating progenitor cells. All cell cultures were maintained at 37°C, 5% CO2 without antibiotics and tested regularly for mycoplasma.

extract_protocol:
iPSC-derived MNs (~1-2*107 cells) were cultured for 2 hours in okadaic acid (50 nM) before harvesting. The cells were lysed with hypotonic gentle lysis buffer (10 mM Tris (pH 7.4), 10 mM NaCl, 10 mM EDTA, 0.5% w/w Triton X-100) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). The lysates underwent immunoprecipitation using Dynabeads Protein A (Thermo Fisher Scientific) with either anti-p- UPF1 S1116 (MilliporeSigma; 07-1016) or rabbit IgG (Sigma Aldrich; I5006) as a control. Cellular RNAs captured by bead-bound antibody complexes were digested to <100 nt by incubating for 30 min at 4°C with RNase I (1 U/μl; Thermo Fisher Scientific Ambion; AM2294). After extensive washing, bound complexes were eluted using IP Elution Buffer (125mM Tris-HCl [pH 6.8], 4% v/v SDS, 20% v/v glycerol, 10% v/v 2-mercaptoethanol, bromophenol blue), and RNA fragments were purified using TRIzol Reagent (Thermo Fisher Scientific). Electrophoresis in 6M urea–15% polyacrylamide parallel to a DynaMarker Prestain Marker for Small RNA (BioDynamics Laboratory; DM253S) was performed. Fragments in the ~25−50-nt size range were excised, agitated overnight at 25°C in RNA Extraction Buffer (20 mM Tris, 300mM sodium acetate, 2 mM EDTA, 0.2% v/v SDS), and purified using a Coaster Spin-X Centrifuge Tube Filter (Corning; 8162). Further extraction with TRIzol Reagent and ethanol precipitation concentrated the RNA. Control IPs using rabbit IgG were performed, and size- matched Input control samples before IP were prepared by incubating cell lysate for 30 min at 4°C with RNase I in parallel.
The cDNA library is prepared as described(Kurosaki et al., 2021a; Kurosaki et al., 2022). Briefly, to generate libraries for p-UPF1 RIP-seq, purified ~25−50-nt RNA fragments underwent treatment with recombinant shrimp alkaline phosphatase (New England Biolabs; M0371) to remove 5'- and 3'-phosphates. The fragments were re- phosphorylated at their 5'-hydroxyl groups using T4 polynucleotide kinase (New England Biolabs; M0201S) and purified using the miRNeasy Mini Kit (Qiagen; 217004). A 5'-adenylated DNA adapter (5'- rAppAGATCGGAAGAGCACACGTCT-NH2-3') was added to 3'-ends using truncated T4 RNA ligase 2 (New England Biolabs; M0242S). After ligation of the 5'-RNA adapter (5'-GUUCAGAGUUCUACAGUCCGACGAUC- 3') using T4 RNA ligase 1 (New England Biolabs; M0204S), the RT primer was annealed to the 3'-adapter. Reverse transcription of the adapter-ligated RNAs using SuperScript III reverse transcriptase (Thermo Fisher Scientific) was followed by PCR amplification using Phusion high-fidelity DNA polymerase (New England Biolabs; M0530S) for 15 cycles. PCR products were purified in 8% polyacrylamide, and cDNA quality and quantity were assessed using the 2100 Agilent Bioanalyzer and qPCR. The cDNAs were then sequenced using the Illumina NextSeq550 System.

value: iPSC-derived spinal motor neurons


title: scSTAMP-seq_MultiplexOligoExp_Plate3_Lib1_Transcriptome

description: Method is detailed in Piscopio et al.


title: r2_SR_BRM014_24h

value: placenta


title: WT1, L-fucose

description: Library name: L-fucose 1


title: WT2, L-fucose

description: Library name: L-fucose 2


title: r1_ST_BRM014_12h


title: scSTAMP-seq_MultiplexOligoExp_Plate2_Lib2_Spatial

description: Method is detailed in Piscopio et al.


title: WT6, aCSF

description: Library name: Control 6

value: aCSF 5 hours


title: 1_SR_72h

value: WT


title: WT5, aCSF

description: Library name: Control 5


title: iPSC-derived spinal motor neurons, Control, IgG IP, rep3

description: Library name: rIgG_MN_3

treatment_protocol: iPSC-derived MNs (~1-2*107 cells) were cultured for 2 hours in okadaic acid (50 nM) before harvesting.


title: r1_SR_BRM014_24h


title: scSTAMP-seq_MultiplexOligoExp_Plate2_Lib2_Transcriptome

description: Method is detailed in Piscopio et al.


title: scSTAMP-seq_MultiplexOligoExp_Plate2_Lib1_Spatial

description: Method is detailed in Piscopio et al.


title: iPSC-derived spinal motor neurons, Control, IgG IP, rep1

description: Library name: rIgG_MN_1


title: r1_SR_BRM014_12h


title: WT3, aCSF

description: Library name: Control 3


title: r3_ST_BRM014_12h


title: iPSC-derived spinal motor neurons, Control, IgG IP, rep2

description: Library name: rIgG_MN_2


title: WT4, aCSF

description: Library name: Control 4


title: scSTAMP-seq_MultiplexOligoExp_Plate2_Lib1_Transcriptome

description: Method is detailed in Piscopio et al.


title: r3_ST_24h

value: Forskolin


title: WT2, aCSF

description: Library name: Control 2


title: scSTAMP-seq_MultiplexOligoExp_Plate1_Lib2_Spatial

description: Method is detailed in Piscopio et al.


title: iPSC-derived spinal motor neurons, Control, pUPF1 IP, rep3

description: Library name: p_UPF1_MN_3


title: WT1, aCSF

description: Library name: Control 1


title: r3_ST_12h

value: ST


title: scSTAMP-seq_MultiplexOligoExp_Plate1_Lib2_Transcriptome

description: Method is detailed in Piscopio et al.


title: iPSC-derived spinal motor neurons, Control, pUPF1 IP, rep2

description: Library name: p_UPF1_MN_2


title: r3_SR_BRM014_24h

value: hTSC


title: r3_SR_BRM014_12h


title: scSTAMP-seq_MultiplexOligoExp_Plate1_Lib1_Spatial

description: Method is detailed in Piscopio et al.


title: scSTAMP-seq_MultiplexOligoExp_Plate1_Lib1_Transcriptome

description: Method is detailed in Piscopio et al.


title: RNA-Seq of mouse kidney (WT_6h_3)

city: Xiamen

first: Jianxin

address: Jinhu Road No.668

department: Department of Urology

institute: Zhongshan Hospital (Xiamen), Fudan University

description: Library name: WT_6h_3

data_processing:
Raw paired-end RNA-seq reads were first subjected to quality control using FastQC. Adapter sequences and low-quality bases were trimmed using Trimmomatic. Clean reads were then aligned to the Mus musculus reference genome (GRCm39) using HISAT2. Read counts for each gene were quantified using featureCounts based on GENCODE or Ensembl gene annotation. Transcript expression levels were calculated using the transcripts per kilobase per million mapped reads (TPM) method. Downstream differential expression analysis was conducted using DESeq2 in R. Genes with an adjusted p-value < 0.05 and absolute log2 fold change > 1 were considered significantly differentially expressed.
Assembly: GRCm39
Supplementary files format and content: The processed data files include: A gene-level TPM expression matrix (tab-delimited text format), where rows represent genes (Ensembl IDs) and columns represent samples. A raw read count matrix (tab-delimited), suitable for downstream differential expression analysis using tools like DESeq2. A differential expression results file including log2 fold changes, p-values, and adjusted p-values.

source_name: kidney

extract_protocol:
Total RNA was extracted from mouse kidney tissues using TRIzol reagent according to the manufacturer’s instructions. RNA quantity and purity were assessed using a NanoDrop 2000 spectrophotometer. RNA integrity was evaluated by agarose gel electrophoresis and Agilent 2100 Bioanalyzer. Samples with RNA Integrity Number (RIN) ≥ 7.0, concentration ≥ 35 ng/μL, and total RNA ≥ 1 µg were used for library preparation.
mRNA was enriched from total RNA using oligo(dT) magnetic beads. The purified mRNA was fragmented using fragmentation buffer, and first-strand cDNA was synthesized using random hexamer primers and reverse transcriptase. Second-strand cDNA synthesis was then performed to generate double-stranded cDNA. The cDNA ends were repaired and adenylated at the 3' ends, followed by ligation of Illumina paired-end sequencing adapters. The ligated products were enriched by PCR amplification (15 cycles). Libraries were quantified, size-selected, and sequenced on the Illumina NovaSeq 6000 platform with paired-end 150 bp reads using the Illumina TruSeq RNA sample prep Kit.

value: kidney

value: acetaminophen


title: RNA-Seq of mouse kidney (WT_6h_4)

description: Library name: WT_6h_4


title: r3_SR_24h


title: r3_SR_12h


title: iPSC-derived spinal motor neurons, Control, Input, rep4

description: Library name: IP_MN_4


title: scSTAMP-seq_ProofOfConcept_Plate2_Lib4_Spatial

description: Method is detailed in Piscopio et al.

value: Hela

value: Hela cells were labeled with PHO1. Hela cell samples then received 0, 1, or 10 minutes of UV flood light illumination.


title: iPSC-derived spinal motor neurons, Control, pUPF1 IP, rep4

description: Library name: p_UPF1_MN_4


title: RNA-Seq of mouse kidney (WT_6h_1)

description: Library name: WT_6h_1


title: Rned500_rep4

city: Taipei

first: Sue

last: Lin-Chao

address: 128 Academia Road, Section 2, Nankang,

department: Institute of Molecular Biology

country: Taiwan

institute: Academia Sinica

description: Library name: R419-R169

data_processing:
The single-end, 76-bp raw sequencing reads for each sample were aligned to the reference genome and transcriptome of E. coli. (GCF_000005845.2; ASM584v2) using STAR (v 2.7.10)
The read counts of 4,626 genes, including 92 spike-in ERCC RNAs, were estimated by RSEM (v1.3.3)
The spike-in ERCC RNAs as the internal control showed the highly correlation (> 0.9) with the input concentration. Subsequently, the read-count-per-gene measurements were normalized across all samples using ERCC RNAs according to the method in Potts AH et. al.
The normalization factors, k values, were calculated for the 24 most abundant RNAs in the ERCC spike-in (top 6 abundances from each A, B, C and D group) from a WT sample, R419-R-177_S1, and the geometric average of these was then used as the library normalization factor.
Assembly: ASM584v2
Supplementary files format and content: tab-delimited text files includes raw read counts and ERCC normalized counts for each sample

organism: Escherichia coli str. K-12 substr. MG1655

source_name: bacterial cells

growth_protocol: Overnight E. coli liquid culture were stab into 0.35 % soft agar plate and incubate at 30 ℃ for 24 h. Plasmid ectopic expressed proteins are under the transcription control of rne native promoter.

extract_protocol:
To extract RNA from plate culture, we collected ~ 3 to 4 ml of soft-agar into 50 ml falcon tube. Bacterial enzymatic activity was stop by incubating in 8 ml of stop solution for 3 min. Soft-agar that contained bacterial cells were pelleted by centrifugation at 12,000 rpm, 5 min, 4 ℃. After that, agar pellets were resuspended in 4 ml of KJ medium and 4 ml of lysis buffer. To lyse the bacteria in soft-agar, the mixture was boiled in hot water for 3 min. Total RNE were extracted by adding 4 ml of acid phenol (pH 4.5) and mixed thoroughly by slowly inverting the tube ~ 30 times. Total RNA were extracted in aqueous phase by centrifugation at 4000 g, 1 h, 4 ℃. Total RNA were precipitated in 1 volume of isopropanol and 1/10 volume of 3 M sodium acetate (pH 7.8) at – 20 ℃ for at least 2 h as described above. All RNA samples were maintained in isopropanol at – 20 ℃ before further experiments.
five ug total RNA with ERCC control RNA were prepared for libraries using  Ribominus bacteria 2.0  Kit (ThermoFisher A47335) and Illumina TruSeq total  RNA standard Sample Prep protocol.RNAseq on Illumina NextSeq 500 platform as single read 75 bp.

treatment_protocol: Stop solution (95 % EtOH, 5 % phenol saturated with buffer, pH 6.6-7.9) was added to plate culture to stop all enzymatic activity before sujected for RNA isolation.

value: bacterial cells

value: low catalytic activity N-terminal RNase E (Rne1-499)


title: RNA-Seq of mouse kidney (WT_0h_4)

description: Library name: WT_0h_4

value: PBS


title: r2_ST_BRM014_12h

value: Forskolin + BRM014


title: r2_ST_BRM014_24h


title: iPSC-derived spinal motor neurons, Control, Input, rep2

description: Library name: IP_MN_2


title: iPSC-derived spinal motor neurons, Control, Input, rep1

description: Library name: IP_MN_1


title: RNA-Seq of mouse kidney (WT_0h_3)

description: Library name: WT_0h_3


title: r2_ST_24h


title: RNA-Seq of mouse kidney (WT_0h_2)

description: Library name: WT_0h_2


title: Rned500_rep2

description: Library name: R419-R167


title: iPSC-derived spinal motor neurons, Control, pUPF1 IP, rep1

description: Library name: p_UPF1_MN_1


title: RNA-Seq of mouse kidney (WT_0h_1)

description: Library name: WT_0h_1


title: Macrophages, IL4 stimulation + Ti64_CaP_Zn75, donor 4

city: Mannheim

first: Guanhao

last: Li

state: Baden-Württemberg

address: Ludolf-Krehl Strasse 13-17

department: Institute of Transfusion Medicine and Immunology

institute: Medical Faculty Mannheim, Heidelberg University

data_processing:
FastQC and Cutadapt were used to assess the quality of raw sequencing data (fastq data).
HISAT2 was used to align the cleaned reads to the reference genome (hg38).
FeatureCounts was used to count the number of reads mapping to each gene.
Assembly: GRCh38
Supplementary files format and content: A text file including featureCounts was generated to count the number of reads for each sample.

source_name: Macrophages

growth_protocol: Monocytes were cultivated for 6 days on the sample disks at a concentration of 2×10^6cells/mL in macrophage serum-free medium X-Vivo 10 (Lonza) supplemented with 5 ng/mL M-CSF (Peprotech, Hamburg, Germany) and cytokines as follows: For M0, no cytokines were added; for M1, 100 ng/mL of IFNγ (Peprotech, Hamburg, Germany) was added; and for M2, 10 ng/mL of IL-4 was added. The cells were cultured in a 37°C incubator with an atmosphere of 7.5% CO2.

extract_protocol:
Total RNA was isolated using the E.Z.N.A. total RNA kit I (Omega Bio-tek, Norcross, USA) according to the manufacturer’s instructions. 500ng of total RNA was used for construction of sequencing libraries.
The quality of total RNA samples was checked on BioAnalyzer2100 (G2939BA, Agilent, USA) using Eukaryotic Total RNA Nano Kit (5067-1512, Agilent, USA) according to manufacturer’s protocol. Samples with RNA integrity number (RIN) value >8 were accepted for the library preparation process.
RNA-Seq libraries were prepared from Illumina Stranded mRNA Prep and Ligation Kit (20040534, Illumina, USA) according to the manufacturer’s protocol. Briefly, Oligo(dT) magnetic beads purify and capture the mRNA molecules containing polyA tails. Then the purified mRNA was fragmented and copied into first strand complimentary DNA (cDNA) using reverse transcriptase and random primers. In the second strand cDNA synthesis step, dUTP replaces dTTP to achieve strand specificity. The final steps add adenine (A) and thymine (T) bases to fragment ends and ligate adapters. Sequencing runs are performed on an Illumina NextSeq 550 instrument using single-end 75-cycle sequencing by the NextSeq 500/550 High Output Kit v2.5 (75 Cycles) (20024906, Illumina, USA).

value: Macrophages


title: Macrophages, IL4 stimulation + Ti64_CaP_Zn50, donor 4


title: Rned500_rep1

description: Library name: R419-R165


title: r2_ST_12h


title: RNA-Seq of mouse kidney (WT_6h_2)

description: Library name: WT_6h_2


title: MG1655_rep4

description: Library name: R419-R172

value: full-length RNase E


title: r2_SR_BRM014_12h


title: scSTAMP-seq_ProofOfConcept_Plate1_Lib4_Spatial

description: Method is detailed in Piscopio et al.


title: r2_SR_24h


title: r2_SR_12h


title: MG1655_rep3

description: Library name: R419-R171


title: ICH-Fabp4CKO-3

city: Shaanxi Xi'an

first: Jia qing

last: He

state: -- None selected --

address: Tangdu Hospital, Xi'an City, Shaanxi Province

institute: Air Force Medical University Tangdu Hospital Department of Neurosurgery

description:
Library name: ICH-Fabp4CKO-3
RNA Extraction, library construction and sequencing Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit(Qiagen, Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).

data_processing:
Reads obtained from the sequencing machines includes raw reads containing adapters or low quality bases which will affect the following assembly and analysis
Short reads alignment tool Bowtie2 (version 2.2.8) was used for mapping reads to ribosome RNA (rRNA) database. The rRNA mapped reads then will be removed. The remaining clean reads were further used in assembly and gene abundance calculation.
An index of the reference genome was built, and paired-end clean reads were mapped to the reference genome using HISAT2.2.4 and other parameters set as a default
The mapped reads of each sample were assembled by using StringTie v1.3.1 in a reference-based approach
RNAs differential expression analysis was performed by DESeq2 software between two different groups
Assembly: Ensembl_release111
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample

source_name: brain tissue

extract_protocol:
Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol
RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads.Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reversly transcribed into cDNA by using NEBNext Ultra RNA Library Prep Kit for Illumina(NEB #7530,New England Biolabs, Ipswich, MA, USA).The purified double-stranded cDNA fragments were end repaired, A base added, and ligated to Illumina sequencing adapters.The ligation reaction was purified with the AMPure XP Beads(1.0X).Ligated fragments were subjected to size selection by agarose gel electrophoresis and polymerase chain reaction (PCR) amplified.The resulting cDNA library was sequenced using Illumina Novaseq6000.

value: brain tissue

value: intracerebral hemorrhage


title: Macrophages, IL4 stimulation + Ti64_CaP_Zn100, donor 4


title: iPSC-derived spinal motor neurons, Control, Input, rep3

description: Library name: IP_MN_3

value: iPSC-derived spinal motor neurons


title: MG1655_rep2

description: Library name: R419-R164


title: Rned500_rep3

description: Library name: R419-R168


title: ICH-Fabp4fl_fl-3-1

description:
Library name: ICH-Fabp4fl_fl-3-1
RNA Extraction, library construction and sequencing Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit(Qiagen, Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).


title: MG1655_rep1

description: Library name: R419-R163


title: r1_ST_BRM014_24h

value: placenta

value: WT


title: r1_ST_24h

value: ST

value: Forskolin


title: ICH-Fabp4fl_fl-2-1

description:
Library name: ICH-Fabp4fl_fl-2-1
RNA Extraction, library construction and sequencing Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit(Qiagen, Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).


title: Macrophages, IL4 stimulation + Ti64, donor 4


title: r1_ST_12h


title: Macrophages, IL4 stimulation + plastic, donor 4


title: GCMD3

city: guangzhou

first: Weiyong Wang

last: Wang

address: 382 Outer Ring East Road, Guangzhou Higher Education Mega Center

institute: south china university of technology

description: Library name: Sample 6

data_processing:
Filter out unqualified sequences from the raw data using Cutadapt to obtain Clean data.
Alignment to the reference genome was performed using Hisat2.
The mapped reads of each sample were assembled using StringTie (http://ccb.jhu.edu/software/stringtie/,version:stringtie-1.3.4d) with default parameters. Then, all transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using gffcompare software(http://ccb.jhu.edu/software/stringtie/gffcompare.shtml,version:gffcompare-0.9.8). After the final transcriptome was generated, StringTie and ballgown (http://www.bioconductor.org/packages/release/bioc/html/ballgown.html) were used to estimate the expression levels of all transcripts and perform expression abundance for mRNAs by calculating FPKM (fragment per kilobase of transcript per million mapped reads) value
3.4 Differentially expressed genes (DEGs) Analysis Genes differential expression analysis was performed by DESeq2 software between two different groups (and by edgeR between two samples). The genes with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change ≥ 2 were considered differentially expressed genes.
Assembly: GRCm39 mm10
Supplementary files format and content: txt file includes FPKM values for each sample

source_name: Testis

extract_protocol:
RNA was harvested using  Trizol reagent. 5.0 ug of total RNA was used for the construction of sequencing libraries
total RNA (5ug) using Dynabeads Oligo (dT) (Thermo Fisher, CA, USA) with two rounds of purification. Following purification, the mRNA was fragmented into short fragments using divalent cations under elevated temperature (Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 94℃ 5-7min). Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA). An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min.

value: Testis

value: Spermatogonia


title: Macrophages, IL4 stimulation + Ti64_CaP_Zn25, donor 4


title: ICH-Fabp4fl_fl-1-1

description:
Library name: ICH-Fabp4fl_fl-1-1
RNA Extraction, library construction and sequencing Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit(Qiagen, Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).


title: GCMD2

description: Library name: Sample 5


title: Macrophages, IFNgamma stimulation + Ti64_CaP_Zn75, donor 4


title: r1_SR_24h

value: hTSC


title: Macrophages, IFNgamma stimulation + Ti64_CaP_Zn50, donor 4


title: GCMD1

description: Library name: Sample 4


title: Macrophages, IL4 stimulation + Ti64_CaP, donor 4


title: r1_SR_12h


title: ICH-Fabp4CKO-1

description:
Library name: ICH-Fabp4CKO-1
RNA Extraction, library construction and sequencing Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit(Qiagen, Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).


title: Macrophages, IFNgamma stimulation + Ti64_CaP_Zn25, donor 4


title: ICH-Fabp4CKO-2

description:
Library name: ICH-Fabp4CKO-2
RNA Extraction, library construction and sequencing Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit(Qiagen, Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).


title: Sham-Fabp4CKO-2

description:
Library name: Sham-Fabp4CKO-2
RNA Extraction, library construction and sequencing Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit(Qiagen, Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).

value: Sham


title: GCNC3

description: Library name: Sample 3


title: Macrophages, IFNgamma stimulation + Ti64_CaP_Zn100, donor 4


title: RHE KO PNPLA1 E11 R2

city: Toulouse

first: Nathalie

last: JONCA

state: France

address: CHU Purpan

country: France

institute: INSERM UMR1291 - CNRS UMR 5051 - Université Toulouse III

description: E11R2: Gene counts

data_processing:
Sequencing analysis was performed in Galaxy software
Sequence reads were trimmed for adaptor and low quality sequences using trimmomatic
HISAT2 was used to align sequencing reads to a reference genome ( Hs).
Featurecounts function is used to produce counts for each gene of how many aligned reads overlap its exons.
For differential expression analysis we used the DESeq2 version 1.22.1
Assembly: hg38
Supplementary files format and content: Tabular data files for gene_counts contains raw counts for each sample
Supplementary files format and content: Tabular data file contains normalized counts for each sample in the same file

growth_protocol: N/TERT-2G keratinocytes were grown in EpiLife culture medium supplemented with Human KGF,  1%penicillinandstreptomycin at 37°C in a humidified containing 5% CO2 until subconfluence. They were seeded into polycarbonate inserts to allow keratinocyte stratification and diferentiation

extract_protocol:
RNA was harvested using RNeasy mini kit (Qiagen)
RNA-seq paired-end libraries were prepared  using the TruSeq Stranded mRNA library prep Kit, according to Illumina’s protocol with some adjustments

treatment_protocol: keratinocyte differentiation was induced by adding 50 mg/ml vitamin C and 10 ng/ml keratinocyte growth factor into the lower compartment of the insert

value: keratinocyte

value: PNPLA1 KO

value: keratinocyte differentiation


title: Macrophages, IFNgamma stimulation + Ti64_CaP, donor 4


title: GCNC2

description: Library name: Sample 2


title: RHE KO PNPLA1 E11 R3

description: E11R3: Gene counts


title: Sham-Fabp4CKO-3

description:
Library name: Sham-Fabp4CKO-3
RNA Extraction, library construction and sequencing Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit(Qiagen, Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).


title: Sham-Fabp4fl_fl-3

description:
Library name: Sham-Fabp4fl_fl-3
RNA Extraction, library construction and sequencing Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit(Qiagen, Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).


title: Macrophages, IFNgamma stimulation + Ti64, donor 4


title: RHE KO PNPLA1 E11 R1

description: E11R1: Gene counts


title: Sham-Fabp4fl_fl-2

description:
Library name: Sham-Fabp4fl_fl-2
RNA Extraction, library construction and sequencing Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit(Qiagen, Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).


title: Lung, quad_p21_4

city: Melbourne

first: Dylan

last: Sheerin

state: VIC

address: 1G Royal Parade

country: Australia

institute: WEHI

data_processing:
Forward and reverse reads were then aligned to the reference genome index using the Rsubread align function.
Gene counts were quantified using the BAM files generated from this step, featureCounts (v2.0.3).
Assembly: GRCm38 DNA primary assembly
Supplementary files format and content: .txt formatted gene count matrix

source_name: Lung

extract_protocol:
Lungs were homogenised using pellet pestles and RNA extraction was performed using the Isolate II RNA mini kit according to manufacturer’s instruction An input of 100 ng of total RNA was prepared and indexed for illumina sequencing using the TruSeq RNA sample Prep Kit with RiboGlobin depletion as per manufacturer’s instruction. The indexed libraries were pooled and diluted to 1.5 pM for paired end sequencing (2 x 81 cycles) on a NextSeq 500 instrument using the v2 150 cycle High Output kit as per manufacturer’s instructions.
1000 ng of the isolated total RNA was globin reduced and TruSeq Stranded kit was used for library preparation.

value: Lung

value: p21 virus, 3 d.p.i.


title: Sham-Fabp4fl_fl-1

description:
Library name: Sham-Fabp4fl_fl-1
RNA Extraction, library construction and sequencing Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit(Qiagen, Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).


title: RHE KO PNPLA1 C1 R3

description: C1R3: Gene counts


title: Lung, quint_mock_1

value: mock virus, 3 d.p.i.


title: CM without GF, shBeclin1, replicate 2, Day10

city: wuhan

first: yu

middle: zhang

last: yi

state: hubei

address: donghu street

department: wuhan

institute: wuhan university

description:
Library name: CM without GF shBeclin1_2
q60

data_processing:
Raw sequencing data was initially filtered by Trimmomatic (version 0.36), with the discarding of low-quality reads and the trimming of reads contaminated with adaptor sequences.
Clean reads were mapped to the reference genome of mouse (mm10) using STRA software (version 2.5.3a) with default parameters.
The counting of reads mapped to the exon regions of each gene was carried out by featureCounts (Subread-1.5.1; Bioconductor).
Resulting BAM files were sorted and indexed using SAMtools.
Assembly: mm10
Supplementary files format and content: shBeclin1_exp.xlsx includes raw counts for each sample

source_name: heart

extract_protocol:
RNA was harvested using TRIzol (Invitrogen, cat. NO15596026). 2ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using Ribo-off rRNA Depletion Kit (Human/Mouse/Rat) (Catalog NO. MRZG12324, Illumina) and KCTM Stranded mRNA Library Prep Kit for Illumina® (Catalog NO. DR08402, Wuhan Seqhealth Co., Ltd. China) following the manufacturer’s instruction. The library products corresponding to 200-500 bps were enriched, quantified and finally sequenced on Hiseq X 10 sequencer (Illumina) with PE150 model.

value: heart

value: mouse cardiomyocyte

value: CM differentiation without GF+shBeclin1

tag: batch


title: Macrophages, without stimulation + Ti64_CaP_Zn75, donor 4

value: Macrophages


title: RHE KO PNPLA1 C1 R2

description: C1R2: Gene counts


title: Macrophages, IFNgamma stimulation + plastic, donor 4


title: RHE KO PNPLA1 A5 R3

description: A5R3: Gene counts


title: Lung, quint_p21_2


title: CM without GF, shNT, replicate 1, Day10

description:
Library name: CM without GF shNT_1
q60

value: WT

value: CM differentiation without GF+shNT


title: Macrophages, without stimulation + Ti64_CaP_Zn25, donor 4


title: CM, shBeclin1, replicate 2, Day10

description:
Library name: CM shBeclin1_2
q60

value: CM differentiation+shBeclin1


title: Lung, quint_p21_1


title: CM without GF, shNT, replicate 2, Day10

description:
Library name: CM without GF shNT_2
q60


title: GCNC1

description: Library name: Sample 1

value: Testis


title: RHE KO PNPLA1 A5 R2

description: A5R2: Gene counts


title: CM without GF, shBeclin1, replicate 1, Day10

description:
Library name: CM without GF shBeclin1_1
q60


title: Lung, wt_p21_6

value: wild-type


title: RHE KO PNPLA1 A5 R1

description: A5R1: Gene counts


title: Macrophages, without stimulation + Ti64_CaP, donor 4


title: RHE WT R3

description: WTR3: Gene counts


title: CM, shBeclin1, replicate 1, Day10

description:
Library name: CM shBeclin1_1
q60


title: Lung, quad_p21_3


title: Lung, quint_p21_4


title: RHE WT R2

description: WTR2: Gene counts


title: Macrophages, IL4 stimulation + Ti64_CaP_Zn75, donor 3


title: Macrophages, without stimulation + Ti64_CaP_Zn50, donor 4


title: RHE KO PNPLA1 C1 R1

description: C1R1: Gene counts


title: Lung, quint_p21_3


title: RHE WT R1

description: WTR1: Gene counts


title: Sham-Fabp4CKO-1

description:
Library name: Sham-Fabp4CKO-1
RNA Extraction, library construction and sequencing Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit(Qiagen, Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).

value: brain tissue


title: Lung, wt_mock_4


title: CM, shNT, replicate 2, Day10

description:
Library name: CM shNT_2
q60

value: CM differentiation+shNT


title: Macrophages, IL4 stimulation + Ti64_CaP_Zn50, donor 3


title: CM, shNT, replicate 1, Day10

description:
Library name: CM shNT_1
q60


title: Macrophages, without stimulation + Ti64_CaP_Zn100, donor 4


title: PC9RESISTANT-3

city: Nanjing

first: yuxian

last: Qian

address: baiziting no.42

institute: Nanjing medical university

data_processing:
After sequencing by Illumina NovaSeq 6000 sequencer, image analysis, base identification and quality control, Raw reads (Raw Data) were produced. First use Q30 for quality control. Then, cutadapt[1] software (v1.9.3) was used to disconnect low-quality reads and obtain high-quality clean reads. STAR software was used to match clean reads from input library to mouse reference genome (MM10), and then DCC software was used to identify circRNA. The de-coupling reads were compared to the human reference genome using Hisat2 software (v2.0.4). MACS software was then used to identify methylated genes in each sample. Differential methylation gene identification was performed using diffReps software. The peaks located on the exon of mRNA, LncRNA and circRNA were screened by our own procedures and annotated accordingly. The coding genes of differential methylation, LncRNA associated genes and circRNA source genes were analyzed by GO and Pathway, respectively.
Assembly: hg19
Supplementary files format and content: Excel format of methylated sites of circRNAs

extract_protocol:
m6A RNA immunoprecipitation was performed using GenSeqTM m6A RNA IP kit (GenSeq).
Utilize NEBNext® The Ultra II Directional RNA Library Prep kit was used to detect Input RNA samples without immunoprecipitation. RNA sequencing library was constructed from IP RNA samples after immunoprecipitation. Analysis by BioAnalyzer 2100 Agilent performs library quality inspection. The Illumina NovaSeq 6000 sequencer was run in 150 bp double-ended mode High-throughput sequencing was performed.
MeRIP-seq

value: lung adenocacinoma cell

value: Osimertinib

tag: phenotype

value: RESISTANT (T)


title: Lung, wt_mock_2

value: Lung


title: Macrophages, IL4 stimulation + Ti64_CaP_Zn25, donor 3


title: EB, shBeclin1, replicate 2, Day4

description:
Library name: EB shBeclin1_2
q60

value: mouse Embryoid body

value: EB differentiation+shBeclin1


title: PC9RESISTANT-1


title: Lung, wt_mock_1

value: mock virus, 3 d.p.i.


title: PC9RESISTANT-2


title: Macrophages, IL4 stimulation + Ti64_CaP, donor 3


title: Macrophages, IL4 stimulation + Ti64_CaP_Zn100, donor 3


title: Lung, wt_mock_3


title: EB, shBeclin1, replicate 1, Day4

description:
Library name: EB shBeclin1_1
q60

value: heart


title: Macrophages, IL4 stimulation + Ti64, donor 3


title: EB, shNT, replicate 2, Day4

description:
Library name: EB shNT_2
q60

value: EB differentiation+shNT


title: EB, shNT, replicate 1, Day4

description:
Library name: EB shNT_1
q60


title: Macrophages, IL4 stimulation + plastic, donor 3

value: Macrophages


title: PC9SENSITIVE-2

value: WT

value: Vehicle

value: SENSITIVE (N)


title: Lung, wt_p21_4

value: p21 virus, 3 d.p.i.


title: PC9SENSITIVE-1


title: Lung, wt_p21_3

value: wild-type


title: Lung, wt_p21_2