title: K562 cells, no doxycycline induction, IgG antibody, batch3

first: Julia

last: Maxson

institute: Oregon Health and Science University

description: Library name: K562_noDOX_IgG_3

data_processing:
Paired-end reads were trimmed using Fastp with adapter detection and poly-G trimming.
Trimmed reads from K562 samples were aligned to the human genome (GRCh38/hg38) and trimmed reads from mouse Lin- samples were aligned to the mouse genome (GRCm38/mm10) using Bowtie2 with parameters “--local --very-sensitive-local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700”.
Duplicates were marked using the Sambamba “markdup” subcommand.
Peaks were called with GoPeaks using IgG samples as negative controls.
K562 CUT&RUN samples were sequenced across multiple batches. Samples from Batch 3 use an in-batch IgG control (K562_noDOX_IgG_3) for peak calling. K562 CUT&RUN samples from Batch 2 use the IgG control (K562_noDOX_IgG_1) from Batch 1 for peak calling. The IgG from Batch 2 yielded insufficient read depth to reliably model background signal and was therefore not used. The Batch 1 IgG sample used for peak calling was prepared using the same cell line and protocols as the experimental samples, allowing for consistent background estimation and peak calling when in-batch controls were not usable.
Mouse lineage-negative CUT&RUN samples were sequenced across multiple batches. Batch 1 samples (KAT7 and KMT2A samples) used the Batch 1 IgG control (mouse_batch1_LUC_IgG) for peak calling, which had similarly high read depth as the experimental samples in that batch, making it suitable for background modeling. Batches 2-3 used the Batch 4 IgG control (mouse_batch4_LUC_IgG) for peak calling. Batch 2 (sequenced on NovaSeq 6000) did not originally include an IgG control, and Batch 3 included an IgG control that had insufficient read depth and could not be used to reliably model background signal. The Batch 4 IgG control had read depth similar to samples in Batches 2-3, allowing for consistent background estimation and peak calling when in-batch controls were not usable.
Spike-in DNA from E. coli K-12 MG1655 was provided in the Epicypher CUT&RUN kit and used according to the manufacturer's protocols. To determine normalization factors for signal visualization, reads were aligned to host and E. coli reference genomes, and uniquely aligned reads were retained. Spike-in percentages were calculated as the proportion of reads mapping to E. coli relative to reads mapping to the host genome. Normalization factors were then calculated based on the relative spike-in percentages, scaled to the sample with the lowest spike-in recovery within each antibody target, consistent with Epicypher guidelines. Spike-in normalization was applied to the histone mark samples (H3K14ac, H3K23ac), as these samples had consistent spike-in recovery across replicates within each condition. BigWig coverage tracks for these samples were generated using spike-in derived scale factors with deepTools bamCoverage (--binSize 10 --smoothLength 50 --scaleFactor <sample-specific value>). Other antibody targets were not spike-in normalized due to variable spike-in recovery across replicates. For these samples, counts-per-million (CPM) normalized bigWig tracks were generated using deepTools bamCoverage with parameters “--binSize 10 --smoothLength 50 --normalizeUsing CPM”.
Assembly: GRCh38 (hg38) for samples from K562 cells, GRCm38 (mm10) for samples from mouse lineage-negative bone marrow cells
Supplementary files format and content: BigWig files include either spike-in normalized (for histone marks) or CPM-normalized (for other antibody targets) read signal for each sample.
Library strategy: CUT&Run

extract_protocol:
Cells were harvested following the EpiCypher CUT&RUN kit protocol (EpiCypher CUTANA 14-1048).
CUT&RUN libraries were prepared using the New England Biolabs NEBNext Ultra II DNA Library Prep Kit for Illumina.

value: myeloid leukemia cell line

value: No doxycycline induction

tag: cutrun antibody

value: IgG (EpiCypher, 13-0042k)

value: GRCh38 (hg38)


title: WT_2

status: Public on Apr 01 2026

type: SRA

city: Houston

first: Nick

last: Newkirk

state: TX

department: Genetics and Epigenetics

country: USA

institute: MD Anderson UTHealth Graduate School of Biomedical Sciences

description: Library name: N2_2

library_source: transcriptomic

data_processing:
tinyRNA (https://github.com/MontgomeryLab/tinyRNA) run using default runconfig.yml trimming of raw read files, bowtie alignment and deseq parameters
HQAlign (github.com/FireLabSoftware/HQAlign) used on raw sequencing data trimmed using cutadapt (https://cutadapt.readthedocs.io/en/) default settings and aligned to WS290: WGS, ncRNA, piRNA, mRNA, miRNA. Alignments analyzed in RStudio using the following significance cutoffs for identifying differentially expressed mappings: log2 fold change >(±)2 and >10 total RPM between the two samples being compared. Filtering for 26G (mRNA and ncRNA) and 21U (piRNA) mapped reads was performed using HQAlignSubset.py (github.com/ArurLab) and differentially expressed mappings were identified using the same significance cutoffs
Assembly: tinyRNA:  WS279; HQAlign: WS290
Supplementary files format and content: tinyRNA - .csv; feature GeneID, small RNA type and deseq data for each WS279 feature
Supplementary files format and content: HQAlign - .xlsx; aligned reads to each feature (see tab for feature), number of sense and anit-sense reads aligned and size and base histogram data provided for each replicate

molecule: total RNA

organism: Caenorhabditis elegans

source_name: whole worm

growth_protocol: hermaphrodite animals were shifted to 25C at the mid-L4 stage (after spermatogensis had completed) for 24 hours for oocytes to development

extract_protocol:
TRIzol with chloroform extraction and ethanol precipitation
TruSeq Small RNA Library Preparation Kit (Illumina)

treatment_protocol: M9 wash to collect and frozen in Trizol at -80C

tag: tissue

value: whole worm

tag: cell line

value: Bristol N2

tag: genotype

value: dcr-1 [WT]

tag: batch


title: WT_1

description: Library name: N2_1


title: K562 cells, no doxycycline induction, SETBP1 antibody, rep2

city: Portland

state: Oregon

address: 3181 SW Sam Jackson Park Rd

description: Library name: K562_noDOX_SETBP1_2

library_source: genomic

molecule: genomic DNA

organism: Homo sapiens

growth_protocol: K562 cells were grown in IMDM medium with 10% FBS, 2mM glutamine, 1x penicillin/streptomycin. Mouse lineage-negative (Lin-) bone marrow primary cells were grown in IMDM medium with 15% FBS, 2mM glutamine, 1x penicillin/streptomycin in the presence of SCF, IL6, and IL3.

treatment_protocol: K562 cells were either induced with 250ng/mL of doxycycline or not induced with any doxycycline for 24 hours. For mouse lineage-negative (Lin-) CUT&RUN, lentivirus were generated by co-transfecting psPAX2 (Addgene: 12260), pMD2.G (Addgene: 12259), and pSMAL_Luciferase (control) or pSMAL_SETBP1D868N into LentiX cells (Takara) with Fugene 6 (Promega E2691), according to the manufacturer's instructions. Mouse lineage-negative bone marrow cells were lentivirally transduced with either a control (luciferase) or SETBP1D868N mutant and then GFP-sorted.

tag: cell type

tag: treatment

value: SETBP1 (Proteintech, 16841-1-AP)

tag: genome assembly

tag: sequencing batch


title: SLAMseq_KMS11_dTAGparental_dTAGv1_0h_Rep1

city: Miami

first: sanxiong

last: liu

state: FL

address: 1501 NW 10th Avenue

department: Human Genetics

institute: University of Miami Miller School of Medicine

description: Library name: Sample 1

data_processing:
Raw sequencing reads (FASTQ files) were processed using the nf-core/slamseq pipeline (v1.0.0). Initial read quality was assessed with FastQC (v0.11.9). Adapters and low-quality sequences were trimmed using TrimGalore! (v0.6.4_dev). The core processing, including conversion-aware alignment, filtering, SNP masking, and quantification, was performed using Slamdunk (v0.4.3) with default parameters. Reads were aligned to the human genome reference (GRCh38), using gene and 3' UTR annotations derived from the UCSC Table Browser for GRCh38. Slamdunk was used to quantify reads at the gene level, specifically generating counts for total reads and counts for reads containing T>C conversions (indicative of newly synthesized RNA, defined in the analysis as reads with >2 T>C conversions). The submitted processed data files consist of these gene-level count matrices generated by Slamdunk.
Assembly: hg38
Supplementary files format and content: Each processed data file corresponds to a single experimental sample and is provided in comma-separated value (.csv) format. Within each file, rows represent individual transcripts identified by their RefSeq transcript ID. The columns contain transcript-level quantification and metric data generated by Slamdunk, including:
* readCount: Raw count of total reads mapping to the transcript.
* tcReadCount: Raw count of reads identified as containing T>C conversions (representing newly synthesized RNA, based on the pipeline's criteria, e.g., >2 T>C conversions per read).
* length: The length of the transcript.
* readsCPM: Reads Per Million mapped reads (calculated for total reads).
* conversionRate: The calculated T>C conversion rate for reads mapping to this transcript.
* Tcontent: The proportion of thymine bases within the transcript sequence.
* coverageOnTs: Coverage depth specifically over thymine bases.
* conversionsOnTs: Number of T>C conversions observed at thymine positions.
* multimapCount: Count related to multi-mapping reads assigned to the transcript.

growth_protocol: KMS-11 cells are cultured in RPMI-1640 supplemented with 10% FBS and 1% Penicillin-Streptomycin.

extract_protocol:
Cells were metabolically labeled with 100 µM 4-thiouridine (4sU) for 1 hour. Total RNA was extracted using TRIzol reagent followed by purification with the RNA Clean & Concentrator-5 kit (Zymo Research). 10 µg of RNA was then alkylated with iodoacetamide, followed by another cleanup using the RNA Clean & Concentrator-5 kit. 1 µg of the resulting RNA was used as input for library construction.
Sequencing libraries were prepared from 1 µg of alkylated total RNA using the QuantSeq 3’mRNA-Seq Library Prep FWD Kit for Illumina (Lexogen #015), following the manufacturer's protocol.

treatment_protocol: NSD2dTAG KMS-11 cells were treated with dTAGV-1 (100 nM) to induce NSD2 degradation. For specific experiments, NSD2-depleted cells were subsequently treated either with doxycycline (100 ng/ml) for reconstitution studies, or with the PRC2 inhibitors EPZ-6438 (200 nM) or MAK-658 (50 nM), or a DMSO vehicle control. The specific duration of each treatment varied according to the experimental condition and is indicated in the metadata associated with each sample.

value: WT

value: No


title: K562 cells, no doxycycline induction, KAT7 antibody, rep3

description: Library name: K562_noDOX_KAT7_3

value: KAT7 (Cell Signaling Technology, 58418)


title: HMVEC-D cells, transfected with miR-369-5p, 48 h, bio rep 3

city: Chicago

first: CRI

last: Bioinformatics

state: IL

address: 5801 S. Ellis Ave.

institute: University of Chicago

description:
Library name: GLC-xG-RS23-R-18_S56
Column name in gene_counts.csv: R-18

data_processing:
Fastq files were processed using the nf-core/rnaseq (v3.14.0) pipeline (parameters: --gencode).
Duplicated rows in terms of gene names were removed, which reduced the number of genes from 60708 to 59428.
Assembly: GRCh38
Supplementary files format and content: comma-delimited csv file includes gene counts for each sample.

source_name: HMVEC-D

growth_protocol: Human dermal microvascular endothelial cells (CC-2543) purchased from Lonza Walkersville, Inc.; Allendale, NJ), (HMVEC-D) were maintained according to manufacturer’s instructions in endothelial growth medium (EGM-2MV Bullet Kit; Lonza) supplemented with 5% fetal bovine serum (FBS; Lonza) and incubated at 37◦C, 5% CO2 in cell culture incubator. At passage 10 cells were split into 24 well plates for the following day experiment.

extract_protocol:
24 h or 48 h after transfections cells were harvested for RNA purification with miRNA easy mini kit (Qiagen). 1.3 ug of total RNA was used for the construction of sequencing libraries.
Illumina TruSEQ library

treatment_protocol:
When ~80% confluent cells were transfected with miRIDIAN microRNA
human hsa-miR-369-5p mimic or miRIDIAN microRNA mimic
negative control #1, based on cel-miR-67 (Dharmacon , Inc, Lafayette, CO ) at concentration 50 nM using 1.5 uL Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific Inc.)

value: HMVEC-D

value: endothelial cells

value: experiment

tag: time


title: K562 cells, no doxycycline induction, SETBP1 antibody, rep1

description: Library name: K562_noDOX_SETBP1_1


title: K562 cells, no doxycycline induction, KAT7 antibody, rep1

description: Library name: K562_noDOX_KAT7_1


title: K562 cells, doxycycline-induced, SETBP1 antibody, rep3

description: Library name: K562_DOX250ng_SETBP1_3

value: Doxycycline 250ng/mL induction for 24 hours


title: K562 cells, no doxycycline induction, SETBP1 antibody, rep3

description: Library name: K562_noDOX_SETBP1_3


title: K562 cells, no doxycycline induction, KAT7 antibody, rep2

description: Library name: K562_noDOX_KAT7_2


title: HMVEC-D  cells, tranfected with negative control miR, 48 h, bio rep 3

description:
Library name: GLC-xG-RS23-R-17_S55
Column name in gene_counts.csv: R-17

value: negative control


title: RNA from MDA-MB-231BR cells stably expressing ACSS2 shRNA3

city: Philadelphia

first: Mauricio J.

last: Reginato

state: Pennsylvania

address: 245 N. 15th Street.

institute: Drexel University College of Medicine

description:
Library name: Sample 6
column name in core_table_gene.RNAseq.ACSS2..csv: 231BrshACSS23

data_processing:
BGI Genomics analyzes RNA-seq data by utilizing their proprietary bioinformatics pipeline, typically SOAPnuke for data filtering and RSEM or Bowtie2 for mapping and quantification. The process generates both raw data (FASTQ) and processed data (gene expression counts/normalized data)
Assembly: We used HISAT to align the clean reads to the reference genome.
Supplementary files format and content: Tab-delimited text file includes raw counts for each sample. Csv files include FPKM values for each Sample.

source_name: Breast

growth_protocol: Breast cancer brian tropic MDA-MB-231BR cells were cultured  in Dulbecco's Modified Eagle Medium (DMEM; Thermo-Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo-Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin (Corning, Glendale, AZ, USA) on 100 mm TPP Tissue Culture dishes (Midwest Scientific, Fenton, MO, USA). Cells were quarterly tested for mycoplasma (abmGood PCR Mycoplasma Detection Kit). Following thawing, cells were only used for experiments for up to ten passages.

extract_protocol:
MDA-MB-231BR control shRNA or ACSS2 shRNA whole cell lysate was collected from the monocultures for RNA extraction. RNA was extracted using Trizol (Invitrogen) as per manufacturer’s instruction from MDA-MB-231BR cells stably expressing shSCR or shACSS2 (3 biological replicates per condition). RNA-Seq and analysis was performed by BGI Tech.
In this project, we sequence 6 samples used BGISEQ platform, averagely generating about 4.58G Gb bases per sample. The average mapping ratio with reference genome is 95.65%, the average mapping ratio with gene is 70.65%; 17721 genes were identified. BGI constructs RNA libraries using their proprietary DNBseq™ technology, typically involving mRNA enrichment via oligo(dT) beads, fragmentation, double-stranded cDNA synthesis, end-repair, A-tailing, and adapter ligation followed by PCR amplification. The final library is circularized for DNBSEQ platform sequencing, producing high-quality FASTQ files

treatment_protocol: HEK-293T cells were grown to ~90% confluency and transfected. Prior to transfection, 10 μg of shRNA of shControl of shRNA targeting ACSS2, 10 μg VSVG, 5 μg RSV, and 5 μg RRE were gently mixed and incubated in 1.5 mL of Optimem for 5 minutes. Concurrently, 90 μL of PEI was added dropwise to 1.5 mL of Optimem and incubated for 5 minutes. Following the 5 minute incubation, the PEI solution was added dropwise to the DNA solution and incubated for at least 30 minutes. The PEI-DNA solution was then added dropwise to the HEK-293T cells already plated with 5 mL of Optimem, and the cells were incubated overnight in the transfection media. Approximately 16-18 hours later, the transfection media was replaced with normal growth media. Viral supernatants were collected at 24 and 48 hours following the media change. These supernatants were passed through a 0.45 μm filter and portioned into 1 mL aliquots to be stored at -80°C if not for immediate use. 1 mL aliquots from each collection time point where mixed with 2mL of growth media and 1:500 8mg/mL polybrene and added to 75% confluent cell line of interest for 6 hours. Media was replaced with 10mL of fresh growth media followed by antibiotic selection for three days.

value: Breast

value: Breast cancer

value: ACSS2 knockdown

value: ACSS2 shRNA


title: RNA from MDA-MB-231BR cells stably expressing ACSS2 shRNA2

description:
Library name: Sample 5
column name in core_table_gene.RNAseq.ACSS2..csv: 231BrshACSS22


title: HMVEC-D cells, transfected with miR-369-5p, 48 h, bio rep 2

description:
Library name: GLC-xG-RS23-R-15_S53
Column name in gene_counts.csv: R-15


title: HMVEC-D cells, untreated, 48 h, bio rep 3

description:
Library name: GLC-xG-RS23-R-16_S54
Column name in gene_counts.csv: R-16

value: untreated


title: K562 cells, doxycycline-induced, SETBP1 antibody, rep1

description: Library name: K562_DOX250ng_SETBP1_1

value: myeloid leukemia cell line

value: GRCh38 (hg38)


title: K562 cells, doxycycline-induced, KAT7 antibody, rep3

description: Library name: K562_DOX250ng_KAT7_3


title: K562 cells, doxycycline-induced, SETBP1 antibody, rep2

description: Library name: K562_DOX250ng_SETBP1_2


title: RNA from MDA-MB-231BR cells stably expressing Control shRNA3

description:
Library name: Sample 3
column name in core_table_gene.RNAseq.ACSS2..csv: 231Brcont3

value: Control

value: Control shRNA


title: HMVEC-D  cells, tranfected with negative control miR, 48 h, bio rep 2

description:
Library name: GLC-xG-RS23-R-14_S52
Column name in gene_counts.csv: R-14

value: WT


title: RNA from MDA-MB-231BR cells stably expressing ACSS2 shRNA1

description:
Library name: Sample 4
column name in core_table_gene.RNAseq.ACSS2..csv: 231BrshACSS21


title: K562 cells, doxycycline-induced, KAT7 antibody, rep2

description: Library name: K562_DOX250ng_KAT7_2


title: HMVEC-D cells, untreated, 48 h, bio rep 2

description:
Library name: GLC-xG-RS23-R-13_S51
Column name in gene_counts.csv: R-13


title: RNA from MDA-MB-231BR cells stably expressing Control shRNA2

description:
Library name: Sample 2
column name in core_table_gene.RNAseq.ACSS2..csv: 231Brcont2


title: HMVEC-D cells, transfected with miR-369-5p, 48 h, bio rep 1

description:
Library name: GLC-xG-RS23-R-12_S50
Column name in gene_counts.csv: R-12


title: HMVEC-D  cells, tranfected with negative control miR, 48 h, bio rep 1

description:
Library name: GLC-xG-RS23-R-11_S49
Column name in gene_counts.csv: R-11


title: K562 cells, doxycycline-induced, KAT7 antibody, rep1

description: Library name: K562_DOX250ng_KAT7_1


title: RNA from MDA-MB-231BR cells stably expressing Control shRNA1

description:
Library name: Sample 1
column name in core_table_gene.RNAseq.ACSS2..csv: 231Brcont1


title: CD14+ monocytes donor #5 120 hours post-MTX treatment

city: Madrid

first: Amaya

middle: Rocío

last: Puig Kröger

state: Madrid

address: Dr. Esquerdo 46

department: Immunology

country: Spain

institute: Hospital General Universitario Gregorio Marañón

description: Library name: 39

data_processing:
BGISEQ platform
Low quality reads (More than 20% of the bases qualities are lower than 10), reads with adaptors and reads with unknown bases (N bases more than 5%) were filtered to get the clean reads. Sequences were mapped to GRCh38 genome using  Bowtie2 with the following parameters: -q --phred64 --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -I 1 -X 1000 --no-mixed --no-discordant  -p 1 -k 200
Gene expression levels were determined with software RSEM
Assembly: GRCh38
Supplementary files format and content: tab-delimited text files include Entrez Gene ID and FPKM values for each Sample

source_name: peripheral blood

growth_protocol: Blood was obtained in Li-heparine tubes. Human Peripheral Blood Mononuclear Cells (PBMC) were isolated over a Lymphoprep gradient according to standard procedures. Monocytes were purified from PBMC by magnetic cell sorting using anti-CD14 microbeads (>95% CD14+ cells) and total RNA was isolated.

extract_protocol:
Total RNA was extracted using the RNeasy Micro kit (QIAgen). RNA integrity was validated by LabChip RNA Analysis (Perkin Elmer).
RNA libraries were prepared for sequencing using standard BGI protocols

treatment_protocol: Six healthy volunteers (100% men; aged from 21 to 34) were enrolled and followed-up between April to June 2018 in the open phase I clincal trial METOMAC (EUDRA-CT: 2017-002902-11).  The study was conducted in accordance with the Declaration of Helsinki, was approved by the Ethical Committee of Hospital Universitario La Princesa (8th February 2018) and the Spanish Agency of Medicines and Healthcare (AEMPS, 14th March 2018). Heparinized blood was drawn before administration of MTX and 3 h, 24 h and 120 h after single oral dose (20 mg) of MTX (Wyeth Farma).  Samples from donor number 6 were excluded due to bad quality for RNA sequencing.

value: peripheral blood

value: monocytes

value: Peripheral blood monocytes

value: Male

value: 5 days-MTX treatment


title: Myometrial spheroid, compression, pt 5

city: Cincinnati

first: Stacey

middle: C

last: Schutte

state: OH

address: 2901 Woodside Dr.

department: Biomedical Engineering

institute: University of Cincinnati

description:
Library name: MW_5
column name in raw_counts_compression_samples.csv and in TPM_compression_samples.csv: MW_5

data_processing:
Bioinformatic analysis was performed via BaseSpace SEQUECNCE HUB app RNA-Seq Alignment v2.0.2 followed by RNA-Seq Differential Expression version 1.0.1. The analysis used STAR for alignment and Salmon for quantification (Transcripts Per Million, TPM), followed by DESeq2 to identify differentially expressed genes. RNA-seq Alignment setting is Mark Duplicates unchecked.
Assembly: UCSC hg19
Supplementary files format and content: csv file includes raw counts for each sample
Supplementary files format and content: csv file includes transcripts per million for each sample

source_name: uterine myometrium

extract_protocol:
Total RNA was isolated using mirVana miRNA Isolation Kit
RNA libraries for RNA-seq were prepared using NEBNext Ultra II Directional RNA Library Prep kit

value: uterine myometrium

value: smooth muscle cell

value: compressive force


title: Fibroid spheroid, compression, pt 4

description:
Library name: FW_4
column name in raw_counts_compression_samples.csv and in TPM_compression_samples.csv: FW_4

source_name: uterine fibroid

value: uterine fibroid


title: Fibroid spheroid, compression, pt 5

description:
Library name: FW_5
column name in raw_counts_compression_samples.csv and in TPM_compression_samples.csv: FW_5


title: CD14+ monocytes donor #5 24 hours post-MTX treatment

description: Library name: 37

value: Pre-treatment


title: Fibroid spheroid, compression, pt 2

description:
Library name: FW_2
column name in raw_counts_compression_samples.csv and in TPM_compression_samples.csv: FW_2


title: Fibroid spheroid, compression, pt 1

description:
Library name: FW_1
column name in raw_counts_compression_samples.csv and in TPM_compression_samples.csv: FW_1


title: Myometrial spheroid, compression, pt 4

description:
Library name: MW_4
column name in raw_counts_compression_samples.csv and in TPM_compression_samples.csv: MW_4


title: HMVEC-D cells, untreated, 48 h, bio rep 1

description:
Library name: GLC-xG-RS23-R-10_S48
Column name in gene_counts.csv: R-10

value: HMVEC-D

value: endothelial cells


title: Fibroid spheroid, compression, pt 3

description:
Library name: FW_3
column name in raw_counts_compression_samples.csv and in TPM_compression_samples.csv: FW_3


title: Myometrial spheroid, compression, pt 3

description:
Library name: MW_3
column name in raw_counts_compression_samples.csv and in TPM_compression_samples.csv: MW_3


title: CD14+ monocytes donor #5 3 hours post-MTX treatment

description: Library name: 35

value: 30 days-MTX treatment


title: Myometrial spheroid, compression, pt 2

description:
Library name: MW_2
column name in raw_counts_compression_samples.csv and in TPM_compression_samples.csv: MW_2


title: Myometrial spheroid, compression, pt 1

description:
Library name: MW_1
column name in raw_counts_compression_samples.csv and in TPM_compression_samples.csv: MW_1


title: HMVEC-D cells, transfected with miR-369-5p, 24 h, bio rep 3

description:
Library name: GLC-xG-RS23-R-09_S47
Column name in gene_counts.csv: R-09

value: experiment


title: CD14+ monocytes donor #5 pre-MTX

description: Library name: 33


title: HMVEC-D  cells, tranfected with negative control miR, 24 h, bio rep 3

description:
Library name: GLC-xG-RS23-R-08_S46
Column name in gene_counts.csv: R-08

value: negative control


title: B_veh replicate 4

city: Brno

first: Core Facility

address: Kamenice 5

country: Czech Republic

institute: CEITEC - Masaryk University

description: Library name: B4R

data_processing:
Sequence reads were trimmed for low-quality sequence using Cutadapt (with parameters: -j 10 --quality-base=33 -q 0,20 -m 35 -M 250 -u 12)
Trimmed sequence reads were mapped to Homo sapiens genome (GRCh38-p10) using STAR v2.7.3a
Read count extraction was done using featureCount and normalization were performed using the R package DESeq2
Assembly: GRCh38-p10
Supplementary files format and content: TAB-delimited file with the read counts from featureCount.

molecule: polyA RNA

source_name: breast cancer

growth_protocol: 2.5 × 10^5 MDA-MB-231 parent and 1.5 × 10^6 MDA-MB-231 paclitaxel-resistant cells were bilaterally orthotopically injected into SCID-beige mice in 50 µl high-glucose DMEM diluted in ECM (1:1).

extract_protocol:
Snap-frozen breast tumor tissue was manually homogenized in liquid nitrogen and RNA was isolated using RNeasy® Mini Kit.
RNA libraries for RNA-seq were prepared using Lexogen QuantSeq FWD kit, 75bp Directional RNA Library Prep Kit for Illumina and polyA selection. Sequencing was made at the CEITEC Core Facility Genomics (Brno, Czech Republic)

treatment_protocol: Mice harboring tumors from either the parental or paclitaxel-resistant cell lines were treated for a total of 26 days with vehicle, doxorubicin, decitabine, or a combination of doxorubicin and decitabine. Treatment started at days 6 (MDA-MB-231 parent) and 16 (MDA-MB-231 paclitaxel-resistant) following the injection of cancer cells. Treatment consisted of two 12-day cycles, separated by 2-day rest period. In each cycle, 4 mice were treated with 0.4 mg/kg decitabine at days 1-4 and 10-11, 4 mice were treated with 1 mg/kg doxorubicin at days 5 and 12, 4 mice were treated with combination of the two agents (0.4 mg/kg decitabine at days 1-4, 10-11 and 1 mg/kg doxorubicin at days 5 and 12) and 4 mice were treated with vehicle solution at stated timepoints.

value: breast cancer

value: paclitaxel-resistant


title: HMVEC-D cells, transfected with miR-369-5p, 24 h, bio rep 2

description:
Library name: GLC-xG-RS23-R-06_S44
Column name in gene_counts.csv: R-06

value: WT


title: B_DOX replicate 3

description: Library name: B11R


title: B_veh replicate 3

description: Library name: B3R


title: B_DOX replicate 2

description: Library name: B10R


title: B_DAC_DOX replicate 3

description: Library name: B15R


title: HMVEC-D cells, untreated, 24 h, bio rep 3

description:
Library name: GLC-xG-RS23-R-07_S45
Column name in gene_counts.csv: R-07

value: untreated


title: CD14+ monocytes donor #4 120 hours post-MTX treatment

description: Library name: 31

value: peripheral blood

value: monocytes

value: Peripheral blood monocytes


title: B_DAC_DOX replicate 2

description: Library name: B14R


title: HMVEC-D cells, untreated, 24 h, bio rep 2

description:
Library name: GLC-xG-RS23-R-04_S42
Column name in gene_counts.csv: R-04


title: CD14+ monocytes donor #4 24 hours post-MTX treatment

description: Library name: 29

value: Male


title: HMVEC-D  cells, tranfected with negative control miR, 24 h, bio rep 2

description:
Library name: GLC-xG-RS23-R-05_S43
Column name in gene_counts.csv: R-05


title: B_veh replicate 1

description: Library name: B1R


title: CD14+ monocytes donor #3 120 hours post-MTX treatment

description: Library name: 23


title: CD14+ monocytes donor #4 pre-MTX

description: Library name: 25


title: B_DAC_DOX replicate 1

description: Library name: B13R


title: B_DOX replicate 4

description: Library name: B12R


title: CD14+ monocytes donor #4 3 hours post-MTX treatment

description: Library name: 27

value: 5 days-MTX treatment


title: B_veh replicate 2

description: Library name: B2R


title: B_DOX replicate 1

description: Library name: B9R


title: CD14+ monocytes donor #3 24 hours post-MTX treatment

description: Library name: 21


title: HMVEC-D cells, transfected with miR-369-5p, 24 h, bio rep 1

description:
Library name: GLC-xG-RS23-R-03_S41
Column name in gene_counts.csv: R-03


title: B_DAC replicate 1

description: Library name: B6R


title: A_veh replicate 4

description: Library name: A4R

value: parent


title: HMVEC-D  cells, tranfected with negative control miR, 24 h, bio rep 1

description:
Library name: GLC-xG-RS23-R-02_S40
Column name in gene_counts.csv: R-02


title: CD14+ monocytes donor #3 3 hours post-MTX treatment

description: Library name: 19


title: A_veh replicate 3

description: Library name: A3R


title: CD14+ monocytes donor #3 pre-MTX

description: Library name: 17


title: A_DAC_DOX replicate 4

description: Library name: A16R


title: PBMC Cad+LPS 4h, rep5

city: Saint-Malo

first: Phuong

middle: Hang

last: PHAM

address: 7 allée Métis

department: Data science

country: France

institute: Parean Biotechnologies

description: Library name: 71PBMCCadLPS4h5

data_processing:
FastQC (version 0.12) and MultiQC (version 1.12) were used for quality control of raw paired-end fastq files.
Illumina's adaptors as well as low quality reads were trimmed out using TrimGalore (version 0.6.7)
Transcript quantification was performed using Salmon (version 1.10.1) on trimmed fastqs against GENCODE V45 transcript reference database (assembly GRCh38)
Quantified transcripts were imported in R (version 4.4.0) using the package tximport (version 1.24.0)
Count data handling and differential analysis were performed using the package DESeq2 (version 1.36.0)
Genes were pre-filtered out if they expressed (count > 0) in less than 60% of samples of 1 group.
Assembly: GENCODE 45/GRCh38
Supplementary files format and content: tab delimited transcript quantification (Salmon output)

source_name: PBMC

extract_protocol:
RNA was extracted using Qiagen RNeasay kit following manufacturer instruction
mRNA library preparation were realized following manufacturer’s recommendations (Stranded Total RNA Prep Ligation with Ribo-Zero Plus from ILLUMINA). Final samples pooled library prep were sequenced on Novaseq 6000 ILLUMINA with S1-200 cycles cartridge (2x1600Millions of 100 bases reads) corresponding to 2x33Millions of reads per sample after demultiplexing.

treatment_protocol: F. prausnitzii EXL01 strain was grown at 37°C in a Exeliom Bisociences’ proprietary medium according to an in-house production process based on the know-how of Exeliom Biosciences. The test material was prepared to be ready-to-use in PBS/glycerol, characterized by enumerating the number of morphologically intact bacteria i.e. the Total Cell Count (TCC). Total human PBMCs (5. 105) were treated with control medium, LPS (100 ng/ml) or EXL01 strain at 3 doses (MOI 1, 10 and 100) for 6 or 16 h

value: PBMC

value: CD14+ monocytes

value: Cad+LPS


title: A_veh replicate 2

description: Library name: A2R


title: PBMC Cad+LPS 4h, rep6

description: Library name: 72PBMCCadLPS4h6


title: CD14+ monocytes donor #2 24 hours post-MTX treatment

description: Library name: 13

value: Pre-treatment


title: HMVEC-D cells, untreated, 24 h, bio rep 1

description:
Library name: GLC-xG-RS23-R-01_S39
Column name in gene_counts.csv: R-01

value: HMVEC-D

value: endothelial cells


title: A_DOX replicate 2

description: Library name: A10R


title: CD14+ monocytes donor #2 120 hours post-MTX treatment

description: Library name: 15


title: A_veh replicate 1

description: Library name: A1R


title: A_DOX replicate 1

description: Library name: A9R


title: B_DAC replicate 2

description: Library name: B7R


title: CD14+ monocytes donor #2 pre-MTX

description: Library name: 9


title: PBMC Cad+LPS 4h, rep3

description: Library name: 69PBMCCadLPS4h3


title: A_DAC_DOX replicate 2

description: Library name: A14R


title: A_DOX replicate 3

description: Library name: A12R


title: CD14+ monocytes donor #2 3 hours post-MTX treatment

description: Library name: 11

value: 30 days-MTX treatment


title: B_DAC replicate 3

description: Library name: B8R

value: breast cancer


title: A_DAC_DOX replicate 3

description: Library name: A15R


title: PBMC Cad+LPS 4h, rep4

description: Library name: 70PBMCCadLPS4h4


title: PBMC Cad+LPS 4h, rep2

description: Library name: 68PBMCCadLPS4h2


title: PBMC Cad+LPS 4h, rep1

description: Library name: 67PBMCCadLPS4h1


title: CD14+ monocytes donor #1 120 hours post-MTX treatment

description: Library name: 7

value: 25 hours-MTX treatment


title: A_DAC replicate 3

description: Library name: A7R


title: A_DAC_DOX replicate 1

description: Library name: A13R


title: CD14+ monocytes donor #1 24 hours post-MTX treatment

description: Library name: 5

value: 24 hours-MTX treatment


title: CD14+ monocytes donor #1 3 hours post-MTX treatment

description: Library name: 3

value: 3 hours-MTX treatment


title: A_DAC replicate 4

description: Library name: A8R


title: Fibroid cell spheroid, pt 4

description:
Library name: FC_4
column name in raw_counts_all_samples.csv and in TPM_all_samples.csv: FC_4

value: uterine fibroid

value: smooth muscle cell


title: Fibroid cell spheroid, pt 5

description:
Library name: FC_5
column name in raw_counts_all_samples.csv and in TPM_all_samples.csv: FC_5


title: PBMC Cad+LPS 2h, rep5

description: Library name: 35PBMCCadLPS2h5


title: A_DAC replicate 2

description: Library name: A6R


title: PBMC Cad+LPS 2h, rep6

description: Library name: 36PBMCCadLPS2h6


title: PBMC Cad+LPS 2h, rep4

description: Library name: 34PBMCCadLPS2h4


title: Fibroid cell spheroid, pt 1

description:
Library name: FC_1
column name in raw_counts_all_samples.csv and in TPM_all_samples.csv: FC_1


title: Myometrial cell spheroid, pt 5

description:
Library name: MC_5
column name in raw_counts_all_samples.csv and in TPM_all_samples.csv: MC_5

value: uterine myometrium


title: CD14+ monocytes patient #31 30 days post-MTX treatment

treatment_protocol: 30 patients (72% female) with inflammatory arthritis (75% RA and 25% PsA) starting MTX therapy were enrolled from November 2019 until June 2024 in Hospital Universitario La Princesa and Hospital General Universitatrio Gregorio Marañón, Madrid, Spain. The study was conducted in accordance with the Declaration of Helsinki, was approved by the Spanish Agency of Medicines and Healthcare (AEMPS, 05th August 2019) and the Ethical Committee of Hospital Universitario La Princesa and Hospital General Universitario Gregorio Marañón (protocol code: METOMAC-PAC, IGA-MET-2019-01). Starting MTX dose was mean (SD) 12.9 mg/week (2.01) and folic acid was prescribed at 5 mg/week (24 hours after MTX). 30 ml of peripheral blood was extracted in lythium heparinized tubes before the start of MTX treatment (pre-MTX), 5 days after the first MTX intake (1×MTX) and 30 days later after four MTX shots (4×MTX). Patients were seen at months 1, and 3 after initiation of MTX. 6 patients were excluded for RNA sequencing.

value: peripheral blood

value: monocytes

tag: disease state

value: RA

tag: Sex

value: Female


title: Fibroid cell spheroid, pt 2

description:
Library name: FC_2
column name in raw_counts_all_samples.csv and in TPM_all_samples.csv: FC_2


title: CD14+ monocytes donor #1 pre-MTX

description: Library name: 1

value: Peripheral blood monocytes

value: Male


title: Fibroid cell spheroid, pt 3

description:
Library name: FC_3
column name in raw_counts_all_samples.csv and in TPM_all_samples.csv: FC_3


title: Myometrial cell spheroid, pt 2

description:
Library name: MC_2
column name in raw_counts_all_samples.csv and in TPM_all_samples.csv: MC_2


title: A_DAC replicate 1

description: Library name: A5R

value: parent


title: PBMC Cad+LPS 2h, rep2

description: Library name: 32PBMCCadLPS2h2

value: PBMC

value: CD14+ monocytes

value: Cad+LPS


title: CD14+ monocytes patient #31 5 days post-MTX treatment

value: 5 days-MTX treatment


title: Myometrial cell spheroid, pt 3

description:
Library name: MC_3
column name in raw_counts_all_samples.csv and in TPM_all_samples.csv: MC_3


title: PBMC Cad+LPS 2h, rep3

description: Library name: 33PBMCCadLPS2h3


title: Myometrial cell spheroid, pt 4

description:
Library name: MC_4
column name in raw_counts_all_samples.csv and in TPM_all_samples.csv: MC_4


title: PC9,scrDSeq

city: Jerusalem

first: Xue

last: Sun

address: Hebrew University, Givat Ram campus

department: Biochemistry department

country: Israel

institute: The Hebrew University of Jerusalem

description: Raw reads from all lanes are merged

library_source: transcriptomic single cell

data_processing:
The raw sequencing data were paired-end fastq files. In data QC, Read1 from all lanes was iterated through and filtered for reads possessing the correct barcode structure, and single-cell barcodes and UMIs were extracted. Barcodes that could be uniquely assigned to an accepted barcode with a Hamming distance of 2 nt or less were merged. UMI deduplication was performed by removing reads with duplicated UMIs under the same single-cell barcode. Unique UMI reads per barcode were counted and plotted in a histogram. Thresholds were determined based on the separation between the first and second peaks. This threshold was then applied in the filtering step, where a similar process was performed as in QC, but outputting reads with UMI counts greater than the threshold into barcode-specific fastq files. Single cell fastqs were then trimmed for Illumina adaptors, barcode spacers and repeated elements (20x A/T/G/C). Finally reads aligned to ribosomal RNA were removed. Raw data were then aligned in multiple rounds to exclusively map to total RNA. Reads were first mapped to the whole genome, then counted with different GTF files in each round. Only reads that failed to align proceeded to the next round. The alignment order was short RNA, long RNA exons, long RNA introns, and pseudogenes. Single cell matrix obtained from each round were combined in downstream analysis.
Assembly: GRCh38
Supplementary files format and content: Tab-separated values files and matrix files
Library strategy: sc-rDSeq; scRNAseq

growth_protocol: PC9 cells were grown in DMEM supplemented with 10% fetal bovine serum, 50 μg/ml penicillin-streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate. HCC827 cells were grown in RPMI 1640 (+Glu) supplemented with 10% fetal bovine serum, 50 μg/ml penicillin-streptomycin and 10mM HEPES. All cells were maintained in 5% CO2 in a humidified incubator at 37 °C.

extract_protocol:
Cells were detached from culture dish with treatment of 2mL trypsin for 10cm2 dish. After counting and checking viability, single cell suspension were prepared at concentration of 300,000 cells/mL in PBS containing 10% Optiprep.
The total RNA library was prepared in two steps. First, total RNA was captured in nanodroplets, and single-cell barcodes were assigned to reversely transcribed cDNA. Subsequently, the cDNA was pooled for in vitro amplification and integration of Illumina adapters. Briefly, using a microfluidics device, single cells were encapsulated in water-in-oil droplets containing a lysis buffer with reverse transcription (RT) enzymes and hydrogel beads carrying primers. These primers included single-cell barcodes and rDS/polyT sequences, as well as the Illumina adapter PE1 and a T7 promoter for in vitro transcription (IVT). Following reverse transcription, the cDNA from all droplets was pooled, unused primers were removed, and the cDNA was cleaned. The second strand was then synthesized prior to IVT amplification. The amplified RNA was slightly fragmented, and the Illumina adapter PE2 was integrated via a random RT step. Finally, the library was amplified by PCR, during which Illumina indexes were incorporated.

treatment_protocol: For drug treatment experiments, stock solutions of Osimertinib and Gefitinib were first diluted in DMSO and then in culture medium, maintaining a final DMSO concentration of 1% across all dilutions. Untreated cells were given 1% DMSO as a control. As start, 1 x 106 PC9 or HCC827 cells were seeded to a 10 cm2 plate. 24h after seeding, 1μM Osimertinib and Gefitinib were given to cells. Every 3 days, medium was changed with fresh drugs. After 9 days, remained cells were collected for sc-rDSeq. Control groups were treated with DMSO for 3 days. Cell survival relative to the control was about 1% for all groups.


title: CD14+ monocytes patient #31 pre-MTX

value: Pre-treatment


title: PBMC Cad+LPS 2h, rep1

description: Library name: 31PBMCCadLPS2h1


title: CD14+ monocytes patient #30 30 days post-MTX treatment

value: 30 days-MTX treatment


title: N2_wildtype_embryo_rep3

city: Bloomington

first: Leanne

last: Kelley

state: IN

address: 1001 E Third St

institute: Indiana University

data_processing:
Reads mapped to the WS272 C. elegans genome using HISAT2 with the –rna-strandness R parameter because the library preparation method used retains the reads from the opposite strand that need to be identified as the reverse strand. SAM files were converted to BAM format and sorted with SAMtools. Reads were identified by CDS regions using featureCounts with the WS272 canonical gene set GTF obtained from ftp.wormbase.org. The resulting counts tables were used for data analysis in R.
Assembly: C. elegans WS272
Supplementary files format and content: Total counts in a matrix

source_name: in utero embryos at 2- to ~30-cell stages

growth_protocol: 1 day old adult hermaphrodites or early embryos extracted from 1 day old adult hermaphrodites; raised at 22°C

extract_protocol:
RNA was isolated using the TRIzol method
Ribosomal-depleted RNA was used as the input for the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760S)


title: PBMC Cad 4h, rep3

description: Library name: 63PBMCCad4h3

value: Cad


title: N2_wildtype_embryo_rep1

value: in utero embryos at 2- to ~30-cell stages


title: N2_wildtype_embryo_rep2


title: PBMC Cad 4h, rep6

description: Library name: 66PBMCCad4h6


title: PBMC Cad 4h, rep5

description: Library name: 65PBMCCad4h5


title: PC9,inDrops

description: Raw reads from all lanes are merged


title: Myometrial cell spheroid, pt 1

description:
Library name: MC_1
column name in raw_counts_all_samples.csv and in TPM_all_samples.csv: MC_1

value: smooth muscle cell


title: CD14+ monocytes patient #27 30 days post-MTX treatment


title: pup1_pup2_embryo_adult_rep3


title: CD14+ monocytes patient #27 5 days post-MTX treatment


title: pup1_pup2_embryo_adult_rep2


title: Healhy Subject

city: Manchester

first: Angela

last: Simeone

address: 200 Hathersage Rd

country: United Kingdom

institute: Genomic Services QIAGEN

description: exosomes

library_source: other

data_processing:
All primary analysis was conducted with CLC Genomics Server 23.0.5
The CLC workflow “QIAseq miRNA quantification” with standard parameters to map sequencing reads to miRBase version 22 (prioritizing the Homo Sapiens specie).
The reads are processed by (1) trimming of the common sequence (AACTGTAGGCACCATCAAT, 19nt), UMI and adapters, and (2) filtering of reads with length < 15 nt or length > 55 nt. They are then deduplicated using their UMI. Reads are grouped into UMI groups when they (1) start at the same position based on the end of the read to which the UMI is ligated (i.e., Read2 for paired data), (2) are from the same strand, and (3) have identical UMIs. Groups that contain only one read (singletons) are merged into non-singleton groups if the singleton’s UMI can be converted to a UMI of a non-singleton group by introducing an SNP (the biggest group is chosen).
Read counts extraction and normalisation was perfomed using CLC Genomic Workbench
Assembly: miRBAse V22 - homo sapiens
Supplementary files format and content: xls file includes the feature ID (miRNA), miRNA identifier, expression (counts), miRNA name counts, match type counts, miRBase accession counts, exact mature counts, mature counts, unique exact mature counts and unique mature counts.

molecule: other

source_name: Endometrial biopsy

growth_protocol: Endometrial Tissue Collection: Twenty-two fresh endometrial biopsies were retrieved from CUSL patients by means of a Novak curette.Biopsies retrieved from the operating room were immediately placed in cold sterile phosphate-buffered saline (PBS) pH 7.4 (Thermo Fisher Scientific, MA, USA) until use. They were processed according to an established protocol (Defrère et al., 2005). Briefly, the tissue was mechanically minced with surgical scissors and enzymatically digested in 1 mg/ml collagenase (Sigma, MO, USA) for one hour. The suspension was strained through a 300-µm sieve to exclude undigested fragments, followed by a 100-µm filter to separate epithelial glands and ESCs. ESCs passing through the sieve were centrifuged for 5 minutes at 1000 rpm and resuspended in Dulbecco’s modified Eagle medium F-12 nutrient mixture (DMEM/F-12) (Thermo Fisher Scientific, MA, USA) enriched with 10% heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, MA, USA) and 1% antibiotic-antimycotic (Thermo Fisher Scientific, MA, USA). After 20 minutes, the medium was replaced with a fresh mixture to exclude blood cells and any residual epithelial cells. The ESCs were cultured at 37°C with 5% CO2 for expansion, with medium replacement every 2 days. The ESC phenotype was tested before adding exosome-depleted FBS to the culture medium (10%) by immunocytochemistry on five representative samples. After fixation in 4% paraformaldehyde, 300 µL resuspended cells (5x104 – 5x105 cells/ml) were aggregated on slides using the Shandon Cytospin 2 (Thermo Fisher Scientific, MA, USA) for 10 minutes at 700 rpm, then stained with mouse anti-human CD10 antibody (ready to use, Sanbio, Uden, the Netherlands). Human endometrium served as positive controls. Culture purity was 92% (91.8 ± 1.79). Exosome isolation from ESCs by ultracentrifugation: ESCs were cultured in exosome-depleted FBS for 48 hours based on three considerations: i) ESC medium is routinely changed every 2 days; ii) after culturing ESCs in EV-depleted FBS for both 48 and 72 hours higher exosome concentrations were observed at 48 hours; and iii) exosome secretion tends to decrease as cells approach confluency, so the 48-hour culture period was chosen for optimal exosome collection. FBS used for culture was subjected to ultracentrifugation at 36600 rpm for 16 hours at 4°C (Ti70 21U8118 rotor, LE-80K, Beckman Coulter, CA, USA) to eliminate exosomes. To confirm particle depletion, flow cytometry analysis was conducted. PE anti-human CD9 antibody and APC anti-human CD81 TAPA-1 (BioLegend, CA, USA), two widely known exosome biomarkers, were used. The supernatant was then filtered (0.22 µm) and stored at -20°C prior to use (Supplementary Figure S2). ESC supernatant was collected from each sample at the end of culture. Differential ultracentrifugation was performed to purify and isolate exosomes according to an established protocol (Zipponi et al., 2024b). Briefly, culture medium was centrifuged twice (Sigma 2-16PK, Merck, NJ, USA) at 2000 rpm for 10 minutes at room temperature to remove the bulk of the cell debris. The supernatant was then ultracentrifuged (LE-80K, Beckman Coulter, CA, USA) at 17300 rpm for 20 minutes at 4°C (Ti50 E4280 rotor, Beckman Coulter, CA, USA) and 4000 rpm for 120 minutes at 4°C (Ti80 1377 rotor, Beckman Coulter, CA, USA). After discarding the supernatant, the pellets were resuspended in 1 ml 0.22-µm-filtered PBS. Finally, the samples were subjected to ultracentrifugation at 4000 rpm for 120 minutes at 4°C (Ti80 1377 rotor, Beckman Coulter, CA, USA) to obtain pellet particles. Half of the particles were resuspended in 200 µL PBS for exosome characterization by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and flow cytometry. The rest were resuspended in 800 µL QIAzol lysis reagent (Qiagen, Hilden, Germany) for RNA extraction. Total RNA was extracted using the miRNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Two microliters of the remaining RNAse-free water were kept for quantification by Quant-IT RiboGreen RNA assays kit (Thermo Fisher Scientific, MA, USA). The RNA was stored at -80°C prior to sequencing. Maximum storage time of RNA samples was 3 months.

extract_protocol:
Culture medium was centrifuged twice (Sigma 2-16PK, Merck, NJ, USA) at 2000 rpm for 10 minutes at room temperature to remove the bulk of the cell debris. The supernatant was then ultracentrifuged (LE-80K, Beckman Coulter, CA, USA) at 17300 rpm for 20 minutes at 4°C (Ti50 E4280 rotor, Beckman Coulter, CA, USA) and 4000 rpm for 120 minutes at 4°C (Ti80 1377 rotor, Beckman Coulter, CA, USA). After discarding the supernatant, the pellets were resuspended in 1 ml 0.22-µm-filtered PBS. Finally, the samples were subjected to ultracentrifugation at 4000 rpm for 120 minutes at 4°C (Ti80 1377 rotor, Beckman Coulter, CA, USA) to obtain pellet particles. Half of the particles were resuspended in 200 µL PBS for exosome characterization by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and flow cytometry. The rest were resuspended in 800 µL QIAzol lysis reagent (Qiagen, Hilden, Germany) for RNA extraction. Total RNA was extracted using the miRNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Two microliters of the remaining RNAse-free water were kept for quantification by Quant-IT RiboGreen RNA assays kit (Thermo Fisher Scientific, MA, USA). The RNA was stored at -80°C prior to sequencing. Maximum storage time of RNA samples was 3 months.
The library preparation was done using the QIAseq miRNA Library Kit (QIAGEN). A total of 10ng total RNA was converted into miRNA NGS libraries. After adapter ligation, UMIs were introduced in the reverse transcription step. The cDNA was amplified using PCR (19 cycles) and during the PCR indices were added. After PCR the samples were purified. Library preparation was quality controlled using capillary electrophoresis (Tape D1000). Based on quality of the inserts and the concentration measurements the libraries were pooled in equimolar ratios. The library pool(s) were quantified using qPCR. The library pool(s) were then sequenced on a NextSeq (Illumina Inc.) sequencing instrument according to the manufacturer instructions (1x75, 2x10). Raw data was de-multiplexed and FASTQ files for each sample were generated using the bcl2fastq2 software (Illumina inc.).

value: Endometrial biopsy

value: ESCs exosomes; ESCs from endometrial tissue (biopsy); ESCs were separatedfrom epithelial glands.

tag: conditions

value: Healthy


title: PBMC Cad 4h, rep4

description: Library name: 64PBMCCad4h4


title: CD14+ monocytes patient #30 5 days post-MTX treatment

value: peripheral blood

value: monocytes

value: RA


title: pup1_pup2_embryo_adult_rep1


title: Adenomyosis subject

description: exosomes

value: Adenomyosis


title: CD14+ monocytes patient #30 pre-MTX

value: Female


title: CD14+ monocytes patient #27 pre-MTX


title: pup2_tm4344_embryo_rep2


title: PBMC Cad 4h, rep2

description: Library name: 62PBMCCad4h2

value: PBMC


title: pup2_tm4344_embryo_rep3


title: PBMC Cad 4h, rep1

description: Library name: 61PBMCCad4h1

value: CD14+ monocytes


title: CD14+ monocytes patient #26 30 days post-MTX treatment


title: PBMC Cad 2h, rep5

description: Library name: 29PBMCCad2h5


title: PBMC Cad 2h, rep4

description: Library name: 28PBMCCad2h4


title: CD14+ monocytes patient #26 5 days post-MTX treatment


title: PBMC Cad 2h, rep6

description: Library name: 30PBMCCad2h6


title: pup1_tm1021_embryo_rep2


title: N2_wildtype_adult_rep2

source_name: whole animal

value: whole animal


title: pup1_tm1021_embryo_rep1


title: pup1_pup2_om129_adult_rep2


title: pup1_tm1021_adult_rep2


title: pup2_tm4344_adult_rep2


title: pup2_tm4344_adult_rep1


title: CD14+ monocytes patient #25 30 days post-MTX treatment


title: PBMC Cad 2h, rep3

description: Library name: 27PBMCCad2h3


title: CD14+ monocytes patient #25 5 days post-MTX treatment

value: 5 days-MTX treatment


title: pup1_tm1021_embryo_rep3


title: pup1_pup2_om129_adult_rep1


title: CD14+ monocytes patient #25 pre-MTX

value: Pre-treatment


title: PBMC Cad 2h, rep2

description: Library name: 26PBMCCad2h2


title: pup2_tm4344_embryo_rep1


title: N2_wildtype_adult_rep1


title: pup1_tm1021_adult_rep1


title: 193-MDCK-wt-B

city: Tampere

first: Elina

last: Mantyla

address: Arvo Ylpön katu 34

department: Faculty of Medicine and Health Technology

country: Finland

institute: Tampere University Foundation

description: Library name: 2 h grown MDCK II B

data_processing:
Read alignment was done using Bowtie2 to CanFam3.1 reference genome. Macs2 was used for peak calling.
Assembly: CanFam3.1
Supplementary files format and content: Raw count matrix (.tsv)

organism: Canis lupus familiaris

source_name: In vitro epithelial monolayer

growth_protocol: MEM with 10 % FBS, 1 % P/S

extract_protocol:
50,000 cells was collected and centrifuged at 500 x g for 5 min, 4°C. Cells were washed once with 50 µL of cold 1xPBS buffer and centrifuged again. Supernatant was removed, and cells resuspended in 50 µL of cold lysis buffer followed by centrifugation with the same settings. Supernatant was removed.
Libraries were produced as described in Buenrostro, J., Giresi, P., Zaba, L. et al. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods 10, 1213–1218 (2013). https://doi.org/10.1038/nmeth.2688. Briefly, a transposition mix (25 μL 2× TD buffer, 2.5 μL transposase (Tn5, 100 nM final), 22.5 μL water) was added to the nuclear pellet. Reaction was incubated at 37 °C for 45 minutes and amplified using PCR. Samples were purified using Qiagen MinElute PCR Purification Kit and again using Agencourt AMPure XP magnetic beads.

value: In vitro epithelial monolayer

value: Madine Darby canine kidney type II

value: Epithelial

value: Grown for 7d, detached and reseeded for 2 h


title: 193-MDCK-wt-A

description: Library name: 2 h grown MDCK II A


title: CD14+ monocytes patient #24 30 days post-MTX treatment

value: 30 days-MTX treatment


title: 133-MDCK-wt-B

description: Library name: 7 d grown MDCKII B

value: Grown for 7 d until full maturity


title: PBMC Cad 2h, rep1

description: Library name: 25PBMCCad2h1

value: Cad


title: CD14+ monocytes patient #24 5 days post-MTX treatment

value: peripheral blood


title: 133-MDCK-wt-A

description: Library name: 7 d grown MDCKII A


title: P29236

first: Chad

last: Creighton

address: One Baylor Plaza, Mail Stop: BCM305

department: Biostatistics, Ducan Cancer Center

institute: Baylor College of Medicine

description:
Library name: P29236
genotype Nf1KO.Tp53KO, virus AAV-NP, vehicle-treated

data_processing:
RNA-seq reads were aligned to the rat reference genome (Rnor_6.0, 620 NCBI, along with refGene.gtf file for gene annotations) using Bowtie2, and gene-level expression quantification was performed using RSEM. RSEM estimated transcript abundance and generated gene-level expected counts and RPM values.
Assembly: Rnor_6.0
Supplementary files format and content: The resulting expression data were log transformed and quantile normalized 54 to correct for differences in sequencing depth and RNA composition across samples by organism type. All tools were run with default parameters unless otherwise specified.
Supplementary files format and content: tab-delimited text file include RSEM values for each sample

organism: Rattus norvegicus

source_name: tumor

growth_protocol: LE-ROSA26tm1(LSL-Cas9)Ottc and Wistar-Tg(CAG-Ncre)81Jmsk rats were purchased from Rat Resource & Research Center (RRRC#: 00833 and 301, respectively). Rats constitutively expressing Cas9 were generated by breeding LE-ROSA26tm1(LSL-Cas9)Ottc and Wistar-Tg(CAG-Ncre)81Jmsk rats and genotyped according to the protocol provided by RRRC.

extract_protocol:
Total RNA was extracted using the Direct-zol RNA Miniprep Plus Kit (Zymo Research, R2072) according to the manufacturer’s instructions. The purified RNA samples were then submitted to Novogene Corporation Inc.  (Sacramento, CA, USA) for RNA-Seq. Quality control was performed using the Agilent 5400 Fragment Analyzer System to evaluate RNA quantity, purity, and integrity. RNA libraries were prepared using poly(A) enrichment, and sequencing was carried out on the Illumina NovaSeq X Plus platform with a 150 bp paired-end strategy.
RNA libraries were prepared using poly(A) enrichment, and sequencing was carried out on the Illumina NovaSeq X Plus platform with a 150 bp paired-end strategy.

treatment_protocol: Tumors used for RNA-Seq were snap-frozen in liquid nitrogen and subsequently stored at −80 °C before RNA extraction.

value: tumor

tag: virus used

value: AAV-NP

value: Vehicle


title: CD14+ monocytes patient #23 30 days post-MTX treatment

value: monocytes

value: PsA

value: Female


title: P29250

description:
Library name: P29250
genotype Nf1KO.Tp53KO, virus AAV-NP, vehicle-treated


title: P29253

description:
Library name: P29253
genotype Nf1KO.Tp53KO, virus AAV-NP, vehicle-treated


title: PBMC LPS 4h, rep4

description: Library name: 46PBMCLPS4h4

value: PBMC

value: CD14+ monocytes

value: LPS


title: CD14+ monocytes patient #24 pre-MTX

value: RA

value: Pre-treatment


title: PBMC LPS 4h, rep5

description: Library name: 47PBMCLPS4h5


title: CD14+ monocytes patient #23 5 days post-MTX treatment

value: 5 days-MTX treatment


title: PBMC LPS 4h, rep3

description: Library name: 45PBMCLPS4h3


title: P29227

description:
Library name: P29227
genotype Nf1KO.Tp53KO, virus AAV-NP, fulvestrant-treated

value: Fulvestrant


title: PBMC LPS 4h, rep6

description: Library name: 48PBMCLPS4h6


title: P29233

description:
Library name: P29233
genotype Nf1KO.Tp53KO, virus AAV-NP, fulvestrant-treated


title: PBMC LPS 4h, rep2

description: Library name: 44PBMCLPS4h2


title: P29604

description:
Library name: P29604
genotype Pik3caH1047R.Tp53KO, virus AAV-rPP

value: AAV-rPP

value: Non


title: P29607

description:
Library name: P29607
genotype Pik3caH1047R.Tp53KO, virus AAV-rPP


title: CD14+ monocytes patient #23 pre-MTX


title: P29572

description:
Library name: P29572
genotype Pik3caH1047R.Tp53KO, virus AAV-rPP


title: P29548

description:
Library name: P29548
genotype Pik3caH1047R.Tp53KO, virus AAV-rPP


title: P29230

description:
Library name: P29230
genotype Nf1KO.Tp53KO, virus AAV-NP, fulvestrant-treated


title: PBMC LPS 2h, rep6

description: Library name: 9PBMCLPS2h3


title: CD14+ monocytes patient #22 5 days post-MTX treatment


title: CD14+ monocytes patient #22 30 days post-MTX treatment

value: 30 days-MTX treatment


title: P29449

description:
Library name: P29449
genotype Pik3caH1047R, virus AAV-rPik3ca


title: PBMC LPS 4h, rep1

description: Library name: 43PBMCLPS4h1


title: PBMC LPS 2h, rep4

description: Library name: 7PBMCLPS2h1


title: PBMC LPS 2h, rep5

description: Library name: 8PBMCLPS2h2


title: CD14+ monocytes patient #22 pre-MTX

value: peripheral blood


title: P29446

description:
Library name: P29446
genotype Pik3caH1047R, virus AAV-rPik3ca

value: tumor


title: CD14+ monocytes patient #21 30 days post-MTX treatment


title: P29439

description:
Library name: P29439
genotype Nf1KO.Tp53KO.Pik3caH104R, virus AAV-rPNP

value: AAV-rPNP


title: P29185

description:
Library name: P29185
genotype Pik3caH1047R, virus AAV-rPik3ca


title: P29443

description:
Library name: P29443
genotype Pik3caH1047R, virus AAV-rPik3ca


title: PBMC LPS 2h, rep3

description: Library name: 12PBMCLPS2h6


title: CD14+ monocytes patient #21 5 days post-MTX treatment


title: P29272

description:
Library name: P29272
genotype Pik3caH1047R, virus AAV-rPik3ca


title: P29247

description:
Library name: P29247
genotype Nf1KO.Tp53KO.Pik3caH104R, virus AAV-rPNP


title: PBMC LPS 2h, rep2

description: Library name: 11PBMCLPS2h5


title: P29244

description:
Library name: P29244
genotype Nf1KO.Tp53KO.Pik3caH104R, virus AAV-rPNP


title: P29269

description:
Library name: P29269
genotype Pik3caH1047R, virus AAV-rPik3ca


title: P29241

description:
Library name: P29241
genotype Nf1KO.Tp53KO.Pik3caH104R, virus AAV-rPNP


title: P29171

description:
Library name: P29171
genotype Nf1KO, virus AAV-Nf1, fulvestrant-treated


title: P29173

description:
Library name: P29173
genotype Nf1KO, virus AAV-Nf1, fulvestrant-treated


title: PBMC LPS 2h, rep1

description: Library name: 10PBMCLPS2h4


title: CD14+ monocytes patient #20 5 days post-MTX treatment

value: Male


title: P29176

description:
Library name: P29176
genotype Nf1KO, virus AAV-Nf1, vehicle-treated

value: Vehicle


title: P29174

description:
Library name: P29174
genotype Nf1KO, virus AAV-Nf1, vehicle-treated


title: P29175

description:
Library name: P29175
genotype Nf1KO, virus AAV-Nf1, vehicle-treated


title: PBMC Fp100 4h, rep5

description: Library name: 59PBMCFp1004h5


title: CD14+ monocytes patient #20 30 days post-MTX treatment

value: monocytes


title: PBMC Fp100 4h, rep4

description: Library name: 58PBMCFp1004h4


title: CD14+ monocytes patient #19 30 days post-MTX treatment

value: PsA


title: CD14+ monocytes patient #21 pre-MTX

value: Female


title: PBMC Fp100 4h, rep6

description: Library name: 60PBMCFp1004h6

value: PBMC

value: CD14+ monocytes


title: P28993

description:
Library name: Nf1_5
genotype Nf1KO, virus AAV-Nf1


title: P26488

description:
Library name: Nf1_4
genotype Nf1KO, virus AAV-Nf1


title: P29172

description:
Library name: P29172
genotype Nf1KO, virus AAV-Nf1, fulvestrant-treated


title: P27161

description:
Library name: Nf1_3
genotype Nf1KO, virus AAV-Nf1


title: CD14+ monocytes patient #19 5 days post-MTX treatment


title: P27700

description:
Library name: Nf1_2
genotype Nf1KO, virus AAV-Nf1


title: P26471

description:
Library name: Nf1_1
genotype Nf1KO, virus AAV-Nf1


title: CD14+ monocytes patient #19 pre-MTX


title: PBMC Fp100 4h, rep2

description: Library name: 56PBMCFp1004h2


title: PBMC Fp100 4h, rep3

description: Library name: 57PBMCFp1004h3


title: CD14+ monocytes patient #18 5 days post-MTX treatment

value: RA


title: P27751

description:
Library name: P53_2
genotype Tp53KO, virus AAV-rP53


title: PBMC FP100 2h, rep6

description: Library name: 24PBMCFp1002h6


title: CD14+ monocytes patient #18 30 days post-MTX treatment


title: P27698

description:
Library name: Nf1_6
genotype Nf1KO, virus AAV-Nf1


title: PBMC Fp100 4h, rep1

description: Library name: 55PBMCFp1004h1


title: P27754

description:
Library name: P53_4
genotype Tp53KO, virus AAV-rP53


title: P27685

description:
Library name: P53_1
genotype Tp53KO, virus AAV-rP53


title: P27748

description:
Library name: P53_5
genotype Tp53KO, virus AAV-rP53


title: CD14+ monocytes patient #18 pre-MTX

value: Pre-treatment


title: PBMC FP100 2h, rep5

description: Library name: 23PBMCFp1002h5


title: P27948

description:
Library name: P53_3
genotype Tp53KO, virus AAV-rP53


title: F_27304

description:
Library name: F_27304
genotype Nf1KO.Tp53KO, virus AAV-NP, fulvestrant-treated

value: AAV-NP


title: F_27309

description:
Library name: F_27309
genotype Nf1KO.Tp53KO, virus AAV-NP, fulvestrant-treated

value: Fulvestrant


title: PBMC FP100 2h, rep4

description: Library name: 22PBMCFp1002h4


title: F_27308

description:
Library name: F_27308
genotype Nf1KO.Tp53KO, virus AAV-NP, fulvestrant-treated


title: F_27310

description:
Library name: F_27310
genotype Nf1KO.Tp53KO, virus AAV-NP, fulvestrant-treated


title: CD14+ monocytes patient #26 pre-MTX


title: CD14+ monocytes patient #17 5 days post-MTX treatment

value: 5 days-MTX treatment


title: PBMC FP100 2h, rep3

description: Library name: 21PBMCFp1002h3


title: PBMC FP100 2h, rep2

description: Library name: 20PBMCFp1002h2


title: N_26430

description:
Library name: N_26430
genotype Nf1KO.Tp53KO, virus AAV-NP

value: Non


title: N_26329

description:
Library name: N_26329
genotype Nf1KO.Tp53KO, virus AAV-NP


title: CD14+ monocytes patient #17 pre-MTX


title: Sample 50

type: genomic

city: Albuquerque

first: Gurdeep

last: Singh

state: New Mexico

address: 915 Camino de Salud NE, Fitz Hall, Rooms 155 (office) & 154 (lab)

institute: UNM School of Medicine

description: Giga Mouse Universal Genotyping Array (GigaMUGA)

hyb_protocol: standard protocol

scan_protocol: standard protocol

data_processing:
GenomeStudio v2.0
Normalized signal intensity score, GC score and Genotype values were processed

label: Cy3, Cy5

organism: Mus musculus

source_name: SNP analysis based on GigaMUGA mouse genotyping array data for Modifiers of Placental Function That Mediate Survival of Sfmbt2 Deficient Embryos

label_protocol: Infinium kit from Illumina, Steemers FJ, Chang W, Lee G, Barker DL, Shen R, Gunderson KL. Whole-genome genotyping with the single-base extension assay. Nat Methods. 2006 Jan;3(1):31-3. doi: 10.1038/nmeth842. PMID: 16369550.

growth_protocol: Mice from various genetic backgrounds were bred in the Biological Sciences Facility (BSF) at the University of Toronto according to guidelines set out by the Canadian Council on Animal Care. Pups were collected on the day of their birth and brain tissue was processed for DNA extraction.

extract_protocol: Extraction of genomic DNA from brain tissue was performed using proteinase K followed by purification using QIAamp™ DNA micro kit

value: Brain

tag: breeding strategy

value: (CD1 X C57BL/6J) X C57BL/6Jgt


title: CD14+ monocytes patient #17 30 days post-MTX treatment

value: peripheral blood

value: 30 days-MTX treatment


title: PBMC Fp100 2h, rep1

description: Library name: 19PBMCFp1002h1


title: CD14+ monocytes patient #16 pre-MTX


title: N_26429

description:
Library name: N_26429
genotype Nf1KO.Tp53KO, virus AAV-NP

value: tumor


title: Sample 47


title: Sample 51


title: CD14+ monocytes patient #15 30 days post-MTX treatment

value: Male


title: Sample 48


title: PBMC Fp10 4h, rep6

description: Library name: 54PBMCFp104h6


title: CD14+ monocytes patient #16 30 days post-MTX treatment

value: monocytes


title: PBMC Fp10 4h, rep5

description: Library name: 53PBMCFp104h5


title: Sample 52


title: CD14+ monocytes patient #16 5 days post-MTX treatment


title: Sample 49


title: PBMC Fp10 4h, rep4

description: Library name: 52PBMCFp104h4


title: Sample 45


title: N_26469

description:
Library name: N_26469
genotype Nf1KO.Tp53KO, virus AAV-NP


title: PBMC Fp10 4h, rep3

description: Library name: 51PBMCFp104h3


title: Sample 44


title: CD14+ monocytes patient #15 5 days post-MTX treatment

value: PsA


title: Sample 43


title: Sample 37


title: Sample 42


title: PBMC Fp10 4h, rep1

description: Library name: 49PBMCFp104h1


title: Sample 40


title: PBMC Fp10 2h, rep5

description: Library name: 17PBMCFp102h5


title: Sample 39


title: CD14+ monocytes patient #15 pre-MTX


title: Sample 38


title: Sample 36


title: CD14+ monocytes patient #14 pre-MTX


title: CD14+ monocytes patient #14 5 days post-MTX treatment


title: CD14+ monocytes patient #14 30 days post-MTX treatment


title: PBMC Fp10 2h, rep4

description: Library name: 16PBMCFp102h4


title: Sample 33


title: Sample 35


title: PBMC Fp10 2h, rep6

description: Library name: 18PBMCFp102h6


title: Sample 34


title: Sample 32


title: CD14+ monocytes patient #13 30 days post-MTX treatment


title: PBMC Fp10 2h, rep3

description: Library name: 15PBMCFp102h3


title: PBMC Fp10 2h, rep2

description: Library name: 14PBMCFp102h2


title: Sample 27


title: Sample 24


title: CD14+ monocytes patient #13 pre-MTX


title: Sample 21


title: CD14+ monocytes patient #12 30 days post-MTX treatment

value: Female


title: PBMC Fp10 2h, rep1

description: Library name: 13PBMCFp102h1

value: PBMC


title: CD14+ monocytes patient #12 5 days post-MTX treatment


title: Sample 29


title: Sample 31


title: Sample 20


title: CD14+ monocytes patient #12 pre-MTX


title: PBMC Control 4h, rep6

description: Library name: 42PBMCCo4h6

value: CD14+ monocytes

value: Control


title: PBMC Control 4h, rep4

description: Library name: 40PBMCCo4h4


title: Sample 19


title: Sample 25


title: CD14+ monocytes patient #13 5 days post-MTX treatment


title: Sample 23


title: PBMC Fp10 4h, rep2

description: Library name: 50PBMCFp104h2


title: PBMC Control 4h, rep5

description: Library name: 41PBMCCo4h5


title: Sample 26


title: CD14+ monocytes patient #11 30 days post-MTX treatment


title: Sample 22


title: Sample 18


title: PBMC Control 4h, rep3

description: Library name: 39PBMCCo4h3


title: Sample 17


title: Sample 15


title: Sample 14


title: Sample 5


title: Sample 6


title: Sample 16


title: PBMC Control 4h, rep1

description: Library name: 37PBMCCo4h1


title: Sample 13


title: PBMC Control 2h, rep5

description: Library name: 5PBMCCo2h5


title: Sample 11


title: Sample 4


title: Sample 12


title: CD14+ monocytes patient #11 5 days post-MTX treatment


title: PBMC Control 4h, rep2

description: Library name: 38PBMCCo4h2


title: CD14+ monocytes patient #10 pre-MTX

value: RA

value: Pre-treatment


title: PBMC Control 2h, rep6

description: Library name: 6PBMCCo2h6


title: Sample 3


title: CD14+ monocytes patient #10 30 days post-MTX treatment


title: Sample 2


title: CD14+ monocytes patient #9 30 days post-MTX treatment


title: CD14+ monocytes patient #11 pre-MTX


title: Sample 1


title: PBMC Control 2h, rep4

description: Library name: 4PBMCCo2h4


title: CD14+ monocytes patient #10 5 days post-MTX treatment


title: CD14+ monocytes patient #9 5 days post-MTX treatment


title: PBMC Control 2h, rep3

description: Library name: 3PBMCCo2h3


title: CD14+ monocytes patient #9 pre-MTX


title: day 43, 2D NMP, RA_BMP, rep 2

city: Los Angeles

first: Samantha

last: Butler

state: California

address: 610 Charles E Young Drive East

department: Neurobiology

institute: University of California, Los Angleles

description: Library name: Sample 9

data_processing:
Single-cell libraries were processed with the 10x Genomics Cell Ranger Count pipeline (v9.0.1)
Assembly: GRCh38
Supplementary files format and content: Tab-seperated values files and matrix files

growth_protocol: H9 hESCs are thawed and mantained for two passages post-thaw in mTeSR+ media. NMPs are induced by the addition of FGF (10ng/mL) for 1 day and FGF + CHIR-99021 (3µM) for 1 day/3 days/9 days to generate 2/4/10-day NMPs respectively. GDF11 was added on the 6th day for 4 days as a separate condition to generate 10-day NMPs + GDF11.

extract_protocol:
Single cell suspension from EBs is generated via papain dissociation method using the Worthington papain dissociation kit. Briefly, EBs are first collected in 15mL centrifuge tubes and washed twice with DPBS. The papain solution is prepared by mixing papain with DNAse, and the solution is allowed to activate for 10 minutes at 37ºC before adding to the EBs followed by triturating 10 times with a p1000 pipette. EBs are then preferably placed on an orbital shaker at 37ºC and 5% CO2 for 1 hour and 15 minutes and triturated 10 times every 5 minutes or until no visible EB clumps remain. Single-cell suspension of EBs are then centrifuged at 300 x g for 7 minutes. The cell pellet is resuspended in 2mLs of Ovomucoid protease inhibitor solution to stop the papain digestion and passed through a a 40µm cell strainer to remove undigested organoid debris. Single cells are then centrifuged once more at 300 x g for 7 minutes and the cell pellet is then resuspended in 1mL of 0.04% BSA in DPBS for GEM preparation using 10X genomics.
~ 10,000 live cells/condition were used to generate single-cell cDNA libraries using protocol described by the 10X Genomics 3’GEX library preparation kit. Cells were first partitioned into GEMs (gel-beads-in-emulsion) containing using the 10x Genomics Chromium controller. Further, 3’ mRNA-seq gene libraries were prepared using the Chromium Single Cell 3′ Library & Gel Bead Kit v2 (10x Genomics), according to the manufacturer’s instructions. Library quality control was assessed using Agilent and Qubit assays. If quality control is within acceptable conditions (e.g., library concentration of 20-40ng/ul), libraries were sequenced on the Illumina NovaSeq X Plus 10B. For 3’GEX libraries targeting 10,000 cells 1,800 million reads/sample were obtained.

treatment_protocol: From NMPs, EBs are generated and treated with RA for 14 days/RA for 2 days and then RA+BMP4 for 12 days/RA for 2 days and then RA+GDF11 for 12 days. EBs are then cultured in maturation media (DMEM:F12, N2, B27+vitA, NEAA, cyclicAMP, ascorbic acid) until day 43.

value: RA, BMP4


city: Atlanta

first: Gregory

middle: K

last: Tharp

state: GA

address: 954 Gatewood Dr

department: Developmental and Cognitive Neuroscience

institute: Yerkes National Primate Research Center

description: Library name: p23259_Gerardo_30RNASeq-s015_Z15408-RNA-Seq-08-22-23

data_processing:
Sequencing data were aligned to Macaca nemestrina Ensembl release 95 by using STAR 2.7.9a. Transcript abundance estimates were calculated by using the STAR “quantMode GeneCounts” option.
Assembly: Mnem_1.0, Macaca_nemestrina.Mnem_1.0.95.gtf, GCA_000956065.1
Supplementary files format and content: Comma delimited text with counts for each sample in columns and transcript in rows.

organism: Macaca nemestrina

extract_protocol:
Two million PBMCs were lysed with 350 µL of Buffer RLT and then extracted by using the RNeasy Mini kit (Qiagen, Germantown, MD, USA) with on-column DNase digestion.
RNA quality was assessed by using a TapeStation 4200 (Agilent, Alpharetta, GA, USA) and then ten nanograms of total RNA were used as input for cDNA synthesis by using the SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio, San Jose, CA, USA) according to the manufacturer’s instructions. Amplified cDNA was fragmented and appended with dual-indexed barcodes by using the Nextera XT DNA Library Preparation kit (Illumina, San Diego, CA,USA). Libraries were validated by capillary electrophoresis on a TapeStation 4200, pooled at equimolar concentrations, and sequenced with PE100 reads on an Illumina NovaSeq 6000, yielding ~23 million reads per sample on average.

tag: subjectid

value: Islatravir

tag: timepoint


title: day 43, 10D NMP, RA_BMP4

description: Library name: Sample 5


title: day 43, 2D NMP, RA, rep 2

description: Library name: Sample 12


title: PBMC Control 2h, rep2

description: Library name: 2PBMCCo2h2


title: CD14+ monocytes patient #4 30 days post-MTX treatment


title: day 43, 2D NMP, RA_BMP, rep 1

description: Library name: Sample 1


title: CD14+ monocytes patient #7 30 days post-MTX treatment


description: Library name: p23259_Gerardo_30RNASeq-s014_Z15303-RNA-Seq-08-22-23


title: CD14+ monocytes patient #7 pre-MTX


title: day 43, 10D NMP, RA

description: Library name: Sample 6


title: day 43, 2D GDF(100) NMP, RA_BMP

description: Library name: Sample 8


title: day 43, 2D GDF(50) NMP, RA

description: Library name: Sample 10


title: day 43, 2D GDF(100) NMP, RA

description: Library name: Sample 11


title: day 43, 4D NMP, RA_BMP4

description: Library name: Sample 3


title: CD14+ monocytes patient #7 5 days post-MTX treatment

value: 5 days-MTX treatment


title: PBMC Control 2h, rep1

description: Library name: 1PBMCCo2h1


description: Library name: p23259_Gerardo_30RNASeq-s012_Z13076-RNA-Seq-08-08-23


description: Library name: p23259_Gerardo_30RNASeq-s013_Z15339-RNA-Seq-08-22-23


title: day 43, 2D GDF(50) NMP, RA_BMP

description: Library name: Sample 7


title: day 43, 4D NMP, RA

description: Library name: Sample 4


title: day 43, 2D NMP, RA, rep 1

description: Library name: Sample 2


title: day 27, 10D GDF NMP, RA

description: Library name: Sample 8

treatment_protocol: From NMPs, EBs are generated and treated with RA for 14 days/RA for 2 days and then RA+BMP4 for 12 days/RA for 2 days and then RA+GDF11 for 12 days. EBs are then cultured in maturation media (DMEM:F12, N2, B27+vitA, NEAA, cyclicAMP, ascorbic acid) for 6 days until day 20.

value: day 27, 10D GDF NMP, RA


title: CD14+ monocytes patient #4 5 days post-MTX treatment


description: Library name: p23259_Gerardo_30RNASeq-s009_Z15303-RNA-Seq-07-25-23


title: CD14+ monocytes patient #2 5 days post-MTX treatment


title: day 27, 10D NMP, RA_BMP

description: Library name: Sample 7

value: day 27, 10D NMP, RA_BMP


title: day 27, 4D NMP, RA

description: Library name: Sample 3

value: day 27, 4D NMP, RA


title: day 27, 10D NMP, RA_GDF

description: Library name: Sample 5

value: day 27, 10D NMP, RA_GDF


description: Library name: p23259_Gerardo_30RNASeq-s010_Z15408-RNA-Seq-07-25-23


description: Library name: p23259_Gerardo_30RNASeq-s008_Z15339-RNA-Seq-07-25-23


description: Library name: p23259_Gerardo_30RNASeq-s011_Z15228-RNA-Seq-08-01-23


title: day 27, 4D NMP, RA_BMP

description: Library name: Sample 4

value: day 27, 4D NMP, RA_BMP


title: CD14+ monocytes patient #20 pre-MTX

value: peripheral blood

value: Male


description: Library name: p23259_Gerardo_30RNASeq-s007_Z13076-RNA-Seq-07-11-23


title: hESC control, rep 3

description: Library name: Sample 15

data_processing:
Reads were obtained as raw FASTQ files, assessed for quality with FASTQC, and aligned to the human reference genome (GRCh38, GENCODE v29 annotation) using STAR. Gene-level counts were obtained with featureCounts (exon-level, paired-end read counting).
Assembly: GRCh38
Supplementary files format and content: Tab-delimited text file generated using featureCounts containing gene-level read counts for each sample.

growth_protocol: H9 hESCs are thawed and mantained for two passages post-thaw in mTeSR+ media.

extract_protocol:
NMP cultures subjected to bulk-RNA sequencing were first washed with 1X DPBS to remove the media. Cells were then lysed by directly adding 600μL of RLT buffer in the culture wells, and the lysate is then collected and flash frozen with liquid nitrogen to be stored at -80ºC. The total RNA was then purified using Qiagen’s RNeasy kit, and eluted in 50μL of RNAse-free water. Using a microvolume spectrophotometer, RNA concentrations were determined and diluted to 1μg/μL.
RNA samples were further subjected to quality control assessment using Agilent Technologies 2100 Bioanalyzer and only samples with a RIN score >8.0 were used for library construction using Universal plus mRNA sequencing kit (NuGEN).

treatment_protocol: NMPs are induced by the addition of FGF (10ng/mL) for 1 day and FGF + CHIR-99021 (3µM) for 1 day/3 days/9 days to generate 2/4/10-day NMPs respectively. GDF11 was added on the 6th day for 4 days as a separate condition to generate 10-day NMPs + GDF11.

value: hESC control


description: Library name: p23259_Gerardo_30RNASeq-s006_Z15228-RNA-Seq-07-05-23


title: Western Diet+auranofin 3 mg/kg/day 3

city: New York

first: Michael

last: Gildea

state: New York

address: 550 1st avenue

institute: NYU Langone Health

description:
Library name: M23
auranofin effect on atherosclerosis progression

data_processing:
Trimmed reads were mapped using STAR v2.6.1d
Alignments mapping to genes were counted using FeatureCounts from the subread package v1.6.3
Assembly: GRCm39 GENCODE M32
Supplementary files format and content: gene counts per sample

source_name: whole aorta

growth_protocol: 6-weeks old ApoE-/- mice were fed western diet (WD) -/+ auranofin or atorvastatin for 16 weeks to follow effect of the drug on atherosclerosis progression.

extract_protocol:
Isolated mouse atherosclerotic aortas were stored in RNA later (Cat.# AM7021, Invitrogen) for 24 hours, then tissue was stored at -80 C until processed for RNA extraction using the gentleMACSTM Octo Dissociator (Miltenyi Biotec) homogenization protocol for total RNA isolation, the QIAzol Lysis Reagent (Qiagen, Cat.# 79306) and RNA clean-up using the RNAeasy Mini kit (Qiagen).
Sample quality and quantity was determined with Agilent 2100 bioanalyzer, using the Agilent RNA 6000 Nano Kit (Agilent Technologies, Cat.# 5067-1511). For RNA-sequencing, 250ng of RNA were used as input for poly-A library construction.
Transcriptome Library - non-stranded mRNA sequencing on DNBseq platform

treatment_protocol:
Mice were randomized into 4 groups and fed Western diet (WD, % by kcal: protein, 17.3%, carbohydrate, 43%, fat, 40%; catalog no. D12079B, Research Diet, Inc.), alone or in combination with either 10 mg/kg/day atorvastatin, (catalog no. D16102901, Research Diet, Inc.),
3 mg/kg/day auranofin, (catalog no. D16102902, Research Diet, Inc.) or auranofin plus atorvastatin (catalog no. D16102903, Research Diet, Inc.) for 16 weeks.

value: whole aorta

value: ApoE-/-

value: Western Diet+auranofin 3 mg/kg/day

value: Week 16


title: Western Diet+auranofin 3 mg/kg/day 2

description:
Library name: M20
auranofin effect on atherosclerosis progression


title: day 27, 10D NMP, RA

description: Library name: Sample 6

value: day 27, 10D NMP, RA


title: CD14+ monocytes patient #4 pre-MTX

value: monocytes

value: RA

value: Female

value: Pre-treatment


title: hESC control, rep 2

description: Library name: Sample 14


title: day 27, 2D NMP, RA

description: Library name: Sample 1

value: day 27, 2D NMP, RA


title: CD14+ monocytes patient #2 pre-MTX

value: PsA


title: 4D NMP, day 0

description: Library name: Sample 2

extract_protocol:
Worthington's Papain dissociation system was used to generate single cells in 0.04% BSA in PBS from 3D cultured embryoid bodies (EBs). EBs are first incubated at 37˚C with 20 units/mL papain and 0.005% DNase solution and triturated every 10 minutes for about 1 hour until tissue is fully dissociated. To stop the dissociation the dissociated cells are spun down and resuspended in EBSS, albumin-ovomucoid inhibitor, and DNase. Cells are then passed through a 40μm filter and spun down to be resuspended in 0.04% BSA in PBS.
~ 10,000 live cells/condition were used to generate single-cell cDNA libraries using protocol described by the 10X Genomics 3’GEX library preparation kit. Cells were first partitioned into GEMs (gel-beads-in-emulsion) containing using the 10x Genomics Chromium controller. Further, 3’ mRNA-seq gene libraries were prepared using the Chromium Single Cell 3′ Library & Gel Bead Kit v2 (10x Genomics), according to the manufacturer’s instructions. Library quality control was assessed using Agilent and Qubit assays. If quality control is within acceptable conditions (e.g., library concentration of 20-40ng/ul), libraries were sequenced on the Illumina NovaSeq X Plus 10B. For 3’GEX libraries targeting 10,000 cells 1,800 million reads/sample were obtained.

treatment_protocol: NMPs are induced by the addition of FGF (10ng/mL) for 1 day and FGF + CHIR-99021 (3µM) for 1 day/3 days/9 days to generate 2/4/10-day NMPs respectively.

value: 4D NMP, day 0


title: day 27, 2D NMP, RA_BMP

description: Library name: Sample 2

value: day 27, 2D NMP, RA_BMP


title: CD14+ monocytes patient #2 30 days post-MTX treatment

value: 30 days-MTX treatment


description: Library name: p23259_Gerardo_30RNASeq-s005_Z15408-RNA-Seq-05-30-23

value: PBMC

value: Control

value: pre-treatment


title: 10D NMP, day 0

description: Library name: Sample 3

value: 10D NMP, day 0


title: 2D NMP, day 0

description: Library name: Sample 1

value: 2D NMP, day 0


title: hESC control, rep 1

description: Library name: Sample 13

value: hESC control


title: Western Diet+auranofin 3 mg/kg/day 1

description:
Library name: M6
auranofin effect on atherosclerosis progression

value: whole aorta

value: ApoE-/-

value: Western Diet+auranofin 3 mg/kg/day

value: Week 16


title: 10D GDF11 NMP, rep 3

description: Library name: Sample 12

value: 10D GDF11 NMP


description: Library name: p23259_Gerardo_30RNASeq-s003_Z15339-RNA-Seq-05-30-23


title: Western Diet+auranofin 3 + atoravaststin 10 mg/kg/day 2

description:
Library name: M7
auranofin effect on atherosclerosis progression


description: Library name: p23259_Gerardo_30RNASeq-s002_Z13076-RNA-Seq-05-16-23


description: Library name: p23259_Gerardo_30RNASeq-s004_Z15303-RNA-Seq-05-30-23


title: 4D NMP, rep 1

description: Library name: Sample 4

value: 4D NMP


title: Western Diet+atorvastatin 10 mg/kg/day 2

description:
Library name: M9
auranofin effect on atherosclerosis progression

value: Western Diet+atorvastatin 10 mg/kg/day


title: Western Diet+auranofin 3 + atoravaststin 10 mg/kg/day 1

description:
Library name: M3
auranofin effect on atherosclerosis progression


title: 10D GDF11 NMP, rep 2

description: Library name: Sample 11


title: 10D NMP, rep 3

description: Library name: Sample 9

value: 10D NMP


title: 10D GDF11 NMP, rep 1

description: Library name: Sample 10


title: Western Diet+atorvastatin 10 mg/kg/day 3

description:
Library name: M19
auranofin effect on atherosclerosis progression


title: 10D NMP, rep 2

description: Library name: Sample 8


title: 10D NMP, rep 1

description: Library name: Sample 7


title: 4D NMP, rep 2

description: Library name: Sample 5


description: Library name: p23259_Gerardo_30RNASeq-s001_Z15228-RNA-Seq-05-09-23


title: Western Diet+atorvastatin 10 mg/kg/day 1

description:
Library name: M2
auranofin effect on atherosclerosis progression


title: Experiment 24-022, dCas9/dCas12a system testing

city: Stockholm

first: Jens

middle: Peter

last: Magnusson

address: Alfred Nobels Allé 8

department: Laboratory Medicine

country: Sweden

institute: Karolinska Institutet

description:
Library name: Exp24022
See Exp24022_README.md

data_processing:
zUMIs (v.2.8.2 or newer) was used to process raw FASTQ files as described in Hagemann-Jensen et al., Nature Biotechnology, 2022. QC was performed using thresholds for reads per cell and percent exonic reads (see specific settings in each sample's individual README.md file.
Assembly: Homo_sapiens.GRCh38.95.chr.gtf
Supplementary files format and content: Expression matrices in .txt format with cells as columns and genes (HGNC format) as rows. Expression is given as All Reads (as opposed to only UMI-containing reads) mapping to both introns and exons.

growth_protocol:
HEK293T growth protocol. Cells were grown in DMEM with high glucose (4.5 g/L), GlutaMAX, and pyruvate (Gibco; 10569-010) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA; F7524-500ML) and 1% penicillin-streptomycin (Gibco; 15070063). For passaging, cells were dissociated with TrypLE (Thermo Fisher; 12604039).
iPSC growth protocol. Cells were grown in 6-well plates coated with Laminin-521 (BioLamina, Sundbyberg, Sweden; 17187501) according to BioLamina’s protocol (1/10 dilution in PBS for 2 hr). Cells were kept in mTeSR Plus (Stemcell Technologies; 17187501), which was changed daily. For passaging, cells were dissociated with Accutase (Corning Inc., Corning, NY, USA; 15323609) followed by addition of 1 volume of mTeSR Plus containing 1x RevitaCell Supplement (Gibco; A2644501). Cells were transferred to a 15 ml Falcon tube and centrifuged at 300*g for 4 minutes. Thereafter, supernatant was removed, 2 ml new mTeSR Plus containing RevitaCell Supplement was added. Cells were counted using a Countess 3 Automated Cell Counter (Thermo Fisher) by performing four counts on the same chip and using the average cell number to increase seeding precision. We seeded 150,000 or 300,000 cells per well in a 6-well plate in mTeSR Plus containing RevitaCell Supplement. For subsequent medium changes, RevitaCell Supplement was excluded. Cell cryopreservation was performed using BamBanker (Fujifilm Wako, Neuss, Germany; 306-14684).

extract_protocol:
We performed flow cytometry and sorting using BD FACSMelody (100-µm nozzle) or BD Influx (140-µm nozzle) or BD FACSAria Fusion (100-140-µm nozzle) (BD Biosciences). We generated scRNA-seq libraries using the Smart-seq3xpress method and sequenced on a DNBSeq-G400RS platform (MGI, Shenzhen, China), as described in ref.26, using either HotMPS or StandardMPS chemistry. Smart-seq3xpress involves cell sorting by FACS into 384-well plates and downstream processing to achieve full-transcript coverage.
Library construction protocol Exp23-001. At the end of the experiment (96 hr post-transfection), cells were dissociated and suspended in PBS containing DAPI (4 µg/ml) as a live/dead marker. FACS sorting was done using a BD Influx (BD Biosciences), using a 140-um nozzle. Because transfected plasmids have largely been diluted out at this time point, we did not gate for expression of dCas9-mCherry-KRAB or dCas12a-miniVPR-GFP. This meant that many sorted cells were untransfected. During library preparation, we performed 12 cycles of pre-amplification PCR and 12 cycles of index PCR and used 0.002 ul TDE1 Tn5 enzyme per cell. Sequencing was performed using a SE100 sequencing kit (MGI). After sequencing and quality-control, the dataset consisted of 2959 cells.
Library construction protocol Exp23-006. At the end of the experiment (72 hr post-transfection), cells were dissociated and suspended in PBS containing DAPI (4 µg/ml) as a live/dead marker. FACS sorting was done using a BD Influx (BD Biosciences) using a 140-um nozzle, gating for dCas9-mCherry-KRAB expression and dCas12a-miniVPR-GFP expression. During library preparation, we performed 12 cycles of pre-amplification PCR and 12 cycles of index PCR and used 0.003 ul TDE1 Tn5 enzyme per cell. Sequencing was performed using a PE100 sequencing kit (MGI). After sequencing and quality-control, the dataset consisted of 1179 cells. We further computationally subsetted the dataset by including only cells that contained ≥1 read of any transfected construct, yielding a final dataset of 717 cells.
Library construction protocol Exp24-010. At the end of the experiment (96 hr post-seeding), cells were dissociated and suspended in PBS containing DAPI (4 µg/ml) as a live/dead marker. FACS sorting was done using a BD Influx (BD Biosciences) using a 140-um nozzle. During library preparation, we performed 12 cycles of pre-amplification PCR and 12 cycles of index PCR and used 0.003 ul TDE1 Tn5 enzyme per cell. Sequencing was performed using a PE100 sequencing kit (MGI). After sequencing and quality-control, the dataset consisted of 476 cells.
Library construction protocol Exp24-019. Forty-eight hr post-transfection, cells were dissociated and suspended in PBS containing a live/dead marker (NucRed Dead 647; Thermo Fisher). FACS sorting was done using a BD FACS Melody (BD Biosciences) using a 100-um nozzle. Cells were sorted based on Lb-dCas12a-mCherry-KRAB expression and low fluorescence of the Live/Dead marker. During library preparation, we performed 12 cycles of pre-amplification PCR and 12 cycles of index PCR and used 0.003 ul TDE1 Tn5 enzyme per cell. Sequencing was performed using a PE100 sequencing kit (MGI). After sequencing and quality-control, the dataset consisted of 1334 cells. However, because our BD FACS Melody was not equipped with a UV laser, we had been unable to sort for cells expressing the As-dCas12a-VPR-BFP construct. We therefore subsetted the dataset to include only cells expressing high levels of the As-dCas12a-VPR-BFP (>100 reads per cell), yielding a final dataset of 543 cells.
Library construction protocol Exp24-022. Forty-eight hr post-transfection, cells were dissociated and suspended in PBS. FACS sorting was done using a BD FACS Melody (BD Biosciences) using a 100-um nozzle. Cells were sorted based on dCas9-mCherry-KRAB expression and dCas12a-miniVPR-GFP expression. During library preparation, we performed 12 cycles of pre-amplification PCR and 12 cycles of index PCR and used 0.004 ul TDE1 Tn5 enzyme per cell. Sequencing was performed using a PE100 sequencing kit (MGI). After sequencing and quality-control, the dataset consisted of 1003 cells.

treatment_protocol:
Transfection of HEK293T cells. Cells were forward- or reverse-transfected using Mirus TransIT-LT1 (Mirus Bio, Madison, WI; MIR2304-70) according to manufacturer’s protocol, using a 1:3 DNA:Mirus ratio. We used 500 ng total DNA per well in a 24-well plate. To activate rtTA-inducible expression of endogenous GFP (Fig. 2), we added doxycycline (1 µg/ml) 24 hr after transfection and analyzed samples 48 hr after transfection. For experiments in Fig. 2c and 2f, we omitted doxycycline and relied on the (low) leaky GFP expression to minimize putative disturbances in the cells’ transcriptomes caused by GFP overexpression.
Transfection of iPSCs. To optimize DNA delivery and balance transfection efficiency and cell survival, we tried several transfection reagents (Mirus TransIT-LT1 [MIR-2304], Mirus TransIT-2020 [MIR-5404], Mirus TransIT-X2 [MIR-6004], Lipofectamine Stem Transfection Reagent [STEM00001]). We also tried both forward and reverse transfection; we tried growing cells in Essential-8 (Thermo Fisher; A1517001) or mTeSR Plus (Stemcell Technologies; 17187501); we tried ROCK inhibitor Y-27632 (Merck; SCM075) versus RevitaCell Supplement (Gibco; A2644501, which contains a ROCK inhibitor), and we tested different cell seeding densities. All these parameters powerfully influenced transfection efficiency and cell survival. In our hands, the condition that generated the best balance between efficiency and survival was reverse transfection using Lipofectamine Stem Reagent (2 ul reagent and 500 ng DNA per well in a 24-well plate), growing cells in wells coated with Laminin-521 (BioLamina; 17187501) in mTeSR Plus. We added RevitaCell Supplement for the first 12-24 hr after reverse transfection but not in subsequent medium changes. We found the optimal cell seeding density during reverse transfection to be 150,000 cells per well in a 24-well plate. Amounts were scaled accordingly when using 48-well plates. With these parameters, co-transfection of two constructs was ~30-40% and was tolerated relatively well by the cells.
Exp24-010 treatment protocol. For mesoderm differentiation using small molecule CHIR99021, we adapted a previously published protocol57. Briefly, wells in a 24-well plate were coated with Laminin-521 (BioLamina, LN521-02; Thermo Fisher, #A29249) at 1/10 dilution in PBS for 2 hr at 37°C. iPSCs were seeded at a density of 100,000 cells per well in mTeSR Plus (#100-0274 containing supplement #100-0275, STEMCELL Technologies, BC, Canada), containing 1x RevitaCell Supplement (#A2644501, Thermo Fisher). Twenty-four hr after seeding, medium was changed to mTeSR Plus without RevitaCell Supplement. Forty-eight hr after seeding, medium was changed to RPMI supplemented with L-glutamine and HEPES (#22400089, Gibco, MT, USA) and 1x B27-minus-insulin (#A1895601, Gibco) and containing 10 µm CHIR99021 (#SML1046, Sigma-Aldrich, MO, USA). Exactly 72 hr after seeding, medium was changed to RPMI (with L-glutamine and HEPES) containing 1x B27-minus-insulin, but without CHIR99021. Ninety-six hr after seeding, cells were dissociated and FACS-sorted for scRNA-seq.
Exp23-006 treatment protocol. For mesoderm differentiation using single-gene upregulation (Fig. 4), we used the dCas9/dCas12a system (Fig. 2G; “Experiment #23-006”) to make results comparable to subsequent differentiation experiments, even though no dCas9-based gene repression was performed in this experiment. We targeted each gene with 5 dCas12a gRNAs in a single array (Table S1). We co-transfected iPSCs with the dCas12a-Activator and dCas9-Repressor constructs using reverse transfection (see above). Twenty-four hr after transfection, medium was changed to RPMI supplemented with L-glutamine and HEPES (#22400089, Gibco, MT, USA) and 1x B27-minus-insulin (#A1895601, Gibco). Forty-eight hr after transfection, medium was again changed to RPMI containing B27-minus-insulin. Seventy-two hr after transfection (a time point where pilot experiments showed MESP1 upregulation and morphological changes, both suggestive of mesoderm differentiation), cells were dissociated and resuspended in PBS containing RevitaCell Supplement and FACS-sorted into 384-well plates for scRNA-seq (see below), gating for GFP and mCherry present on the dCas12a-Activator and dCas9-Repressor constructs, respectively.
Exp23-001 treatment protocol. For mesoderm and endoderm differentiation using multi-gene up- and downregulation (Fig. 5; “Experiment #23-001”), we used the dCas9/dCas12a system (Fig. 2G) similar to the setup in the previous paragraph. But instead of changing to RPMI + B27-minus-insulin medium already 24 hr post-transfection, we instead changed to mTeSR Plus (without RevitaCell Supplement) 24 hr post-transfection. At 48 and 72 hr post-transfection, we changed medium to RPMI + B27-minus-insulin. At 96 hr post-transfection (a time point where pilot experiments showed MESP1 upregulation and morphological changes (both suggestive of mesoderm differentiation), cells were analyzed, either by dissociation and FACS-sorting for scRNA-seq, or by lysis and downstream processing for RT-qPCR. For Fig. 5A-C, we sorted cells without gating for the fluorescent proteins fused to the Cas protein, as these protein constructs had largely been diluted out by cell division at this time point.
Exp24-019, Exp24-022 treatment protocol. Cells were analyzed 48 hr post-transfection.

value: CRISPRa/i


title: 2D NMP, rep 3

description: Library name: Sample 3

value: 2D NMP


title: 2D NMP, rep 2

description: Library name: Sample 2


title: 2D NMP, rep 1

description: Library name: Sample 1


title: Western Diet 5

description:
Library name: M18
auranofin effect on atherosclerosis progression

value: Western Diet


title: Exp23-006: CRISPRa for iPSC meso diff. Exp24-010: Small-molecule meso diff. Exp24-019: Lb/As-dCas12 system test

description:
Library name: Exp23006_Exp24010_Exp24019
See Exp23006_Exp24010_Exp24019_README.md

source_name: iPSC, HEK293T

value: iPSC, HEK293T

value: iPSC:Female, african american, healthy


title: Experiment 23-001: iPSC mesoderm diff with CRISPRa+i

description:
Library name: Exp23001
See Exp23001_README.md

source_name: iPSC

value: iPSC


title: Western Diet 4

description:
Library name: M14
auranofin effect on atherosclerosis progression