title: fetal striatum; 21 PCW

first: Kevin

last: Kelley

address: 318 Campus Drive, W200

description: 10x 3' v3

data_processing:
For the multiplex organoid scRNAseq screen and SAG secondary screen, gene expression levels were quantified for each putative cell using the Parse Biosciences analysis software suite (split-pipe command, version 0.9.3p for the primary screen and version 1.0.4p for the SAG secondary screen). Specifically, reads were mapped to a human (GRCh38, Ensemble release 93) reference genome created using the mkref mode and quantified using default parameters. All subsequent analyses were performed on the filtered count matrices using the R (version 4.1.2) package Seurat (version 4.3.0)
For the 10x fetal sample, filtered count matrices were obtained using the CellRanger analysis soft-ware suite (version 6.1.2)
To ensure only high-quality cells were included for downstream anal-yses, an iterative filtering process was implemented. First, cells with less than the 5th percentile within the dataset of unique genes detected and with mitochondrial counts greater than 20% of the total counts were identified and removed. Subsequently, raw gene count matrices were normalized by regularized negative binomial regression using the sctrans-form function (vst.flavor=”v2”), which also identified the top 3,000 highly variable genes using default parameters. Dimensionality reduction using principal component analysis (PCA) on the top variable genes was performed and clusters of cells were identified in PCA space by shared nearest-neighbor graph construction and modularity detection implemented by the Find-Neighbors and FindClusters functions using a dataset dimension of 50 (dims=50 chosen based on visual inspection of elbow plot) with default parameters. We subsequently performed iterative rounds of clustering (resolution = 2) to identify and remove clusters of putative low-quality cells based on outlier low gene counts (median below the 10th percentile), outlier high fraction mitochondrial genes (median above the 95th percentile), outlier high fraction ribosomal genes (median above the 95th percentile), and/or high proportions of putative doublets identified by the DoubletFinder package (median DoubletFinder score above the 95th percentile). We iteratively removed low-quality clusters until no further clusters met the above thresh-olds. Following low quality cell removal, an additional round of filtering of putative stressed cells was performed using the Gruffi R package in addition to manual inspection and removal of clusters expressing markers associated with unfolded protein response (e.g. DDIT3, EIF2AK3).
Assembly: GRCh38, Ensemble release 93, for the organoid scRNA-seq; GRCh38, Ensemble release 98, for the human fetal scRNA-seq
Supplementary files format and content: csv or tsv files with feature (gene) and cell barcode information corresponding to row and column indices of gene count data for each sample
Supplementary files format and content: matrix exchange format for sparse matrices of gene count data for each sample (rows: genes; columns: cells)
Supplementary files format and content: csv file with annotated cell clusters of processed/filtered cells each sample
Supplementary files format and content: Seurat object (.rds) of processed and filtered scRNA-seq data

source_name: human fetal striatum

growth_protocol: Prior to neural differentiation, hiPS cells were cultured on vitronectin-coated plates (5 µg ml–1, Thermo Fisher Scientific, A14700) in Essential 8 medium with supplement (Thermo Fisher Scientific, A1517001). Cells were dissociated every 5 to 6 days with UltraPureTM 0.5 mM EDTA, pH 8.0 (Thermo Fisher Scientific, 15575) and passaged onto new plates. For the generation of 3D neural spheroids (day –1), hiPS cells were washed with PBS and incubated with Accutase (Innovative Cell Technologies, AT104) at 37C for 7 min. Cells were manually triturated with a P1000 into a single cell suspension and were passed through a 40 µM filter (FloMi). To generate spheroids, 3 x 106 hiPS cells were added to each AggreWell-800 well in Essential 8 medium supplemented with the ROCK inhibitor Y27632 (10 µM, Selleckchem, S1049), centrifuged at 100 g for 1 min, and then incubated at 37C in 5 % CO2. After 24 hours, spheroids of approximately 10,000 cells were formed. Spheroids were lifted from each microwell by pipetting medium in the well up and down with a cut P1000 pipet tip and were placed in Essential 6 medium (Thermo Fisher Scientific, A1516401) with the SMAD pathway inhibitors dorsomorphin (2.5 µM, Sigma-Aldrich, P5499) and SB-431542 (10 µM, R&D Systems, 1614). Molecules in the screen were added to the following basal media: from day 0 to day 6, spheres were cultured in Essential 6 medium and media was exchanged daily. From day 6 to day 21, spheres were cultured in neural medium consisting of Neurobasal™-A Medium (Thermo Fisher Scientific, 10888022), B-27™ Supplement, minus vitamin A (Thermo Fisher Scientific, 12587010), GlutaMAX™ Supplement (1:100, Thermo Fisher Scientific, 35050079), and Penicillin-Streptomycin (1:100, Thermo Fisher Scientific, 15070063) and media was exchanged daily. From day 21 onwards, spheres were cultured in Neurobasal™-A Medium (Thermo Fisher Scientific, 10888022), B-27™ Supplement, minus vitamin A (Thermo Fisher Scientific, 12587010), GlutaMAX™ Supplement (1:100, Thermo Fisher Scientific, 35050079), N-2 supplement (Life Technologies, 17502048) and Penicillin-Streptomycin (1:100, Thermo Fisher Scientific, 15070063) with BDNF (20 ng/ml; Peprotech), NT3 (20 ng/ml; Peprotech), (cAMP; 50 µM, Millipore Sigma, D0627), L-Ascorbic Acid 2-phosphate Trisodium Salt (AA; 200 µM, Wako, 323-44822) and media was exchanged every 2-3 days.

extract_protocol:
Three organoids from each condition were pooled at day 72–74, incubated in 30 U/mL papain enzyme solution (Worthington Biochemical, LS003126) and 0.4% DNase (12,500 U/mL, Worthington Biochemical, LS2007) at 37 °C for 45 minutes. Organoids were triturated with a P1000 pipette and were resus-pended in FBS followed by trituration with a P200 pipette tip. Cells were resuspended in PBS with 0.4% DNase and filtered through a 70 μm Flowmi Cell Strainer (Bel-Art, H13680-0070). The samples were centrifuged at 200 x g for 3 minutes and were resuspended in Parse Cell Buffer containing BSA. Cells were then fixed using the Parse Biosciences Cell Fixation (Parse Biosci-ences, ECF2001) kit and stored at −80°C.
Barcoding and library prep was performed with the Evercode Whole Transcriptome kit (Parse Biosciences, ECW02030). Samples were prepared with v1 kits, with the exception of SAG concentration series which was prepared with v2 kits. Samples were spread across 8 sublibraries and sequenced by Admera Health on a NovaSeq S4 (Illumina).

treatment_protocol: Molecules used: EGF (R&D Systems, 236-EG), FGF2 (R&D Systems, 233-FB), IWP-2 (STEMCELL Technologies, 72124), RA (Sigma, R2625), 2.5 µM DAPT (STEMCELL Technologies, 72082), brain-derived neurotrophic factor (BDNF; 20ng ml-1, PeproTech, 450-02), NT3 (20 ng ml-1, PeproTech, 450-03), FGF4 (PeproTech, 100-31), FGF8 (PeproTech, 100-25), SAG (Millipore, 5666660), CHIR 099021 (Selleckchem, S1263), BMP4 (PeproTech, 120-05ET), BMP7 (PeproTech, 120-03P), LDN-193189 (S7507, Selleckchem), Activin A (50 ng/mL, PeproTech, 120-14P), SR11237 (100 nM, Tocris, 3411), dorsomorphin (DM, Sigma Aldrich P5499), SB (Tocris, 3748), Y-27643 (Selleckchem), L-ascorbic acid (FUJIFILM Wako Chemical Corporation, 344-44822), cAMP (Millipore Sigma, D0627).

value: human fetal striatum

tag: differentiation time_point_(days)

tag: age (pcw)


title: OrganoidScreen; sample41; IWP2 switch CHIR

status: Public on May 30 2023

type: SRA

city: Stanford

state: CA

country: USA

institute: Stanford University

description:
Parse WT v1
OrganoidScreen_cellMetaData.csv
OrganoidScreen_geneInfo.csv
OrganoidScreen_counts.mtx
OrganoidScreen_processed_SeuratObject.rds

library_source: transcriptomic single cell

molecule: total RNA

organism: Homo sapiens

source_name: hPS-derived neural organoids

tag: tissue

value: hPS-derived neural organoids

tag: cell line

tag: treatment

value: IWP2 switch CHIR


title: OrganoidScreen; sample42; BMP4 SAG

value: BMP4 SAG


title: OrganoidScreen; sample40; CHIR switch IWP2

value: CHIR switch IWP2


title: Organoid.SAG.secondaryScreen; sample26; SAG_2uM

description:
Parse WT v2
Organoid.SAG.secondaryScreen_cellMetaData.csv
Organoid.SAG.secondaryScreen_geneInfo.csv
Organoid.SAG.secondaryScreen_counts.mtx
Organoid.SAG.secondaryScreen_processed_SeuratObject.rds


title: OrganoidScreen; sample43; BMP7 SAG

value: BMP7 SAG


title: Organoid.SAG.secondaryScreen; sample22; SAG_1uM


title: OrganoidScreen; sample48; BMP4 CHIR d11-16

value: BMP4 CHIR d11-16


title: Organoid.SAG.secondaryScreen; sample29; SAG_50nM


title: OrganoidScreen; sample39; CHIR-SAG-LDN

value: CHIR-SAG-LDN


title: C5ar1 WT macrophages_D2 pi_mouse 2

city: Bethesda

first: Behdad (Ben)

last: Afzali Khoshkbijari

state: MD

address: 10 Center Drive, 10/8D03

department: Kidney Diseases Branch

institute: National Institutes of Diabetes, Digestive, and Kidney Diseases

library_source: transcriptomic

data_processing:
Gene-level counts were filtered to remove low-expression genes (keeping genes with >15 counts in at least one sample). Differential gene expression was evaluated using limma (DOI: 10.1093/nar/gkv007). Differentially expressed genes were defined as genes with a fold change of at least ±2 in either direction at false discovery rate (FDR) less than 0.01. Pathway enrichment analysis for differentially expressed genes of C5ar1–/– versus C5ar1+/+ phagocytes was performed using GSEA.
Assembly: GRCm38 (mm10)
Supplementary files format and content: Processed reads displayed as log2(RPKM) values.

organism: Mus musculus

source_name: C5ar1 WT macrophages

extract_protocol: RNA was isolated from the sorted cells using the Trizol reagent (Thermo Fisher) and further processed using a clean-up and concentration micro-elute kit (RNA Clean-Up and Concentration Micro-Elute kit, Norgen Cat# 61000), as per manufacturer's instructions. cDNA was amplified using the SMART-Seq v4 UltraLow Input RNA kit (Takara Bio, San Jose, CA) and sequencing libraries were prepared using the Nextera XT (Illumina, San Diego, CA). Multiplexed libraries were sequenced on a HiSeq3000 instrument (Illumina, San Diego, CA) in 50-bp single-end mode and mapped using STAR (v2.5.2).

treatment_protocol: Single cell suspensions were prepared from WT and C5ar1–/– kidneys of mice infected with C. albicans at day 2 post-infection. Macrophages and neutrophils were stained using sterile anti-mouse antibodies against CD45 (clone 30-F11, Biolegend), Ly6G (clone 1A8, Biolegend), CD11b (clone M1/70, Biolegend), MHCII (clone M5/114.15.2, Thermo Fisher), F4/80 (clone BM8, Biolegend), CD3 (clone 145-2C11, Biolegend), CD19 (clone 1D3, Biolegend), and NKp46 (clone 29A1.4, Biolegend). Macrophages were defined as CD45+ CD3/CD19/NKp46/Ly6G- MHCIIhi F4/80+ CD11b+ , while and neutrophils were defined as CD45+ Ly6G+ CD11b+  and were FACS-sorted with purity of >90% into RPMI supplemented with 10% FBS and penicillin/streptomycin. The sorted cells were centrifuged at 300 xg for 5 minutes at 4 °C, resuspended in Trizol (QIAGEN, Germantown, MD) and and stored at -80 °C until further use.

tag: genotype/variation

value: wild type

tag: cell type

value: macrophages


title: OrganoidScreen; sample46; CHIR SAG FGF4

value: CHIR SAG FGF4


title: OrganoidScreen; sample36; CHIR-SAG-d16-21


title: OrganoidScreen; sample38; IWP2-SAG


title: OrganoidScreen; sample47; CHIR SAG FGF8

value: CHIR SAG FGF8


title: OrganoidScreen; sample37; SAG-CHIR-d16-21


title: OrganoidScreen; sample44; BMP4 CHIR

value: BMP4 CHIR


title: Organoid.SAG.secondaryScreen; sample27; SAG_1uM


title: Organoid.SAG.secondaryScreen; sample28; SAG_250nM


title: OrganoidScreen; sample45; BMP7 CHIR

value: BMP7 CHIR


title: OrganoidScreen; sample32; CHIR3-SAG1000


title: OrganoidScreen; sample33; CHIR1.5-SAG1000


title: OrganoidScreen; sample31; CHIR1.5-SAG250


title: OrganoidScreen; sample30; CHIR3-SAG250


title: OrganoidScreen; sample29; SAG-d16-21


title: OrganoidScreen; sample28; SAG-d11-16


title: OrganoidScreen; sample27; SAG-d6-11


title: OrganoidScreen; sample35; SAG-CHIRd10-21


title: OrganoidScreen; sample24; CHIR-d11-16


title: OrganoidScreen; sample23; CHIR-d6-11


title: OrganoidScreen; sample22; DAPT

value: DAPT


title: OrganoidScreen; sample13; FGF8


title: OrganoidScreen; sample15; CHIR1.5


title: OrganoidScreen; sample5; I/Activin/DAPT/SR11


title: OrganoidScreen; sample14; CHIR3


title: OrganoidScreen; sample6; C/S/BMP7/D


title: OrganoidScreen; sample19; RA10


title: OrganoidScreen; sample9; BMP7


title: OrganoidScreen; sample7; C/L/S/FGF8


title: OrganoidScreen; sample34; CHIR-SAGd10-21


title: OrganoidScreen; sample11; FGF2-50


title: OrganoidScreen; sample16; SAG250


title: OrganoidScreen; sample17; SAG1000


title: OrganoidScreen; sample18; RA100


title: Bio4_Healthy_Low

status: Public on May 31 2023

type: RNA

city: Québec

first: Antoine

last: BODEIN

state: QC

address: 2705 bd Laurier

country: Canada

institute: Université Laval

hyb_protocol: Affymetrix Pico protocol

scan_protocol: Affymetrix Pico protocol

data_processing: Bioconductor pipeline (oligo, affycoretools, pd.clariom.s.human packages)

label: Direct-zol RNA kit

source_name: Healthy patient, gingival fibroblasts and macrophages, co-cultured with 5 mM glucose

label_protocol: Affymetrix Pico protocol

growth_protocol: Immortalized HGF-1 were cultivated in DMEM supplemented with 10% heat-inactivated (HI)-FBS, 4 mM L-glutamine and 100 ug/ml penicillin/streptomycin + 0.25 ug/ml amphotericin B at 37C in 5% CO2. Human U937 monocytes (ATCC CRL1593.2) were cultivated in RPMI medium supplemented with 10% HI-FBS and 100 ug/ml penicillin/streptomycin + 0.25 ug/ml amphotericin B at 37C in 5% CO2. Two weeks prior the experiments, the cells were maintained in their culture media containing either 5 mM or 25 mM glucose. The coculture model was composed of immortalized HGF-1 overlaid with U937-macrophages-like cells. An arbitrary ratio of 10:1 macrophages to fibroblasts was used. U937 monocytes were differentiated into macrophages-like cells in presence of 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) for 72h. Macrophages-like cells were then washed to remove non-adherence cells, harvested by scraping, suspended in their culture medium and placed over the immortalized HGF-1. The coculture model was incubated overnight at 37C in 5% CO2 to allow macrophages adhesion prior to stimulation

extract_protocol: The cells were washed with ice-cold PBS and the RNA extracted from the coculture models using the Direct-zol RNA extraction kit from Zymo Research

treatment_protocol: The subgingival microbiomes samples were used to stimulate the gingival coculture model cultivated either in 5 mM or 25 mM glucose concentration for 24h. Untreated coculture models served as negative controls.

tag: glucose

value: Low

tag: group

value: Healthy


title: OrganoidScreen; sample12; FGF4


title: OrganoidScreen; sample4; C/S/D/FGF4


title: OrganoidScreen; sample3; S/I/E/FGF2


title: OrganoidScreen; sample2; C/S/R/E/FGF2/D


title: OrganoidScreen; sample1; FGF-20/EGF


title: OrganoidScreen; sample25; CHIR-d16-21


title: OrganoidScreen; sample21; LDN

value: LDN


title: OrganoidScreen; sample20; IWP2

value: hPS-derived neural organoids


title: OrganoidScreen; sample10; FGF2-20


title: Control2

city: Sydney

first: Gautam

last: Wali

address: Randwick

country: Australia

institute: Neuroscience Research Australia

data_processing:
Quality Control and Sequence Data Evaluation.
Reads Mapping
Transcriptome Assembly
Differential Expression
Assembly: hg38
Supplementary files format and content: The file 'gene_counts.txt' is a text matrix that consists of a first column of gene IDs, followed by a column of read counts for each of the samples. The first row shows the sample name for each of the columns. The read counts are the number of reads for the sample denoted by the column that mapped to the gene denoted by the row.

source_name: induced pluripotent stem cell derived cortical neurons

extract_protocol:
RNA was extracted using the RNeasy Mini Kit (Qiagen). 1.0 ug of total RNA was used for the construction of sequencing libraries.
Illumina's TruSeq stranded RNA sample preparation protocol was used for processing the samples.


title: Control3

value: induced pluripotent stem cell derived cortical neurons


title: Control1


title: SPG7 Patient1


title: SPG7 Patient3


title: SPG7 Patient2


title: WT, DMSO, rep1

first: Seungsoo

last: Kim

address: 265 Campus Dr

department: Chemical and Systems Biology

data_processing:
Newly generated sequencing data (read 1 only) were trimmed of adapters and low-quality bases then poly A strings using skewer v0.2.2.
Trimmed reads were mapped to the hg38 analysis set reference genome and counted over Ensembl GRCh38 v97 protein coding transcript 3' UTRs using slamdunk v0.4.3 with map options -n 100 -5 0 -ss with -q for plain RNA-seq (not SLAM-seq) libraries.
Assembly: hg38
Supplementary files format and content: csv files contain slamdunk output collapsed by gene, including T->C converted and total read counts and CPM values.

molecule: polyA RNA

source_name: Human cranial neural crest cells

growth_protocol: H9 cells (WiCell, WA09, RRID:CVCL_9773) were cultured in feeder-free conditions, in mTeSR1 medium (Stem Cell Technologies, 85850) on Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning, 356231) and passaged using ReLeSR (Stem Cell Technologies, 05872) every 4-6 days. Cells were switched to mTeSR Plus medium (Stem Cell Technologies, 100-0276) prior to and during genome editing and clonal expansion, but switched back to mTeSR1 before differentiation to CNCC. hESCs were differentiated to CNCC as previously described. Briefly, hESC colonies were partially detached from the plate with collagenase IV (Gibco, 17104019) in Knockout DMEM medium (Gibco, 10829018) for 30-60 min and scraped to break up large colonies, and then cultured in Neural Crest Differentiation Medium (50%-50% v/v mixture of DMEM/F12 1:1 medium with L-glutamine, without HEPES (Cytiva, SH30271.FS) and Neurobasal medium (Gibco, 21103049) with 0.5x N2 NeuroPlex (Gemini Bio, 400-163) and Gem21 NeuroPlex (Gemini Bio, 400-160) supplements and GlutaMAX (Gibco, 35050061), and 1x antibiotic/antimycotic, and 20 ng/ml EGF (Peprotech, AF-100-15), 20 ng/ml bFGF (Peprotech, 100-18B), and 5 ug/ml bovine insulin (Gemini Bio, 700-112P)) for 11 days in bacterial-grade petri dishes, changing the plate to prevent attachment for 4 days and then leaving the cells unfed for two days to allow attachment, and then fed as needed at least every other day. At day 11, cells (now called ‘early hCNCC’) were harvested by treatment with Accutase (Sigma-Aldrich, A6964-100ML), strained to remove residual neuroectodermal spheres, and plated onto plates coated with 7.5 ug/ml human fibronectin (Millipore, FC010-10MG) and cultured in Neural Crest Maintenance Medium (Neural Crest Differentiation Medium with bovine insulin replaced by 1 mg/ml BSA (Gemini Bio, 700-104P)). These hCNCC were then passaged every 2-3 days upon reaching confluency, with cells in the third or subsequent passages defined as ‘late hCNCC’ and cultured with added 50 pg/ml BMP2 (Peprotech, 120-02) and 3 uM CHIR-99021 (Selleck, S2924). dTAGV-1 (Tocris, 6914/5) was dissolved in DMSO at 5 mM and then diluted to 250 uM in 60% DMSO/40% water (v/v) before dilution to 500 nM for acute depletions (up to 1 day) or diluted directly from the 5 mM stock for long-term depletions.

extract_protocol:
Cells were harvested by TRIzol (Invitrogen, 15596018) and stored at -80°C until processing. Chloroform was added to TRIzol lysate and separated into aqueous and organic phases by centrifugation per manufacturer instructions, and then the aqueous fraction was extracted using the RNA Clean & Concentrator-5 (Zymo, R1013) with on-column DNase I digestion. RNA was checked for purity by Nanodrop and was quantified by Qubit RNA broad range assay (Invitrogen, Q10210).
For acute depletions, SLAM-seq96 was performed as described with minor modifications, with 4-thiouridine labeling of nascent transcription for the last 2 h prior to harvest. Briefly, RNA was extracted using the Direct-zol RNA miniprep kit (Zymo, R2052), modified to include 0.1 mM DTT in wash buffers and 1 mM DTT to the water for elution, with protection from light. Four ug of RNA was then alkylated with 10 mM iodoacetamide (G Biosciences, 786-078) dissolved in ethanol at 100 mM, in 50% v/v DMSO, 50 mM NaPO4 pH 8 for 15 min at 50°C. Alkylation was quenched by addition of 1 ul of 1 M DTT, and alkylated RNA was extracted by RNA Clean & Concentrator-5 kit.
RNA-seq libraries were prepared using the QuantSeq 3’ mRNA-Seq Library Prep FWD kit (Lexogen, 113.96) using 500 ng of input RNA and ~15 cycles of amplification, with unique dual indices.

treatment_protocol: DMSO or dTAGV-1 (500 nM) were added by dilution into media before changing media. 4-thiouridine (100 uM) was added 2 h before harvest.

value: Human cranial neural crest cells

tag: genotype

value: WT

tag: clone

tag: time

value: long-term

tag: library type

value: RNA-seq


title: WT, DMSO, rep2


title: WT, dTAG, rep3

value: dTAGV-1 (500 nM)


title: ALX1FV ALX4-, DMSO, rep2

value: ALX1FV ALX4-


title: ALX1FV ALX4-, dTAG, rep3


title: ALX1FV ALX4-, dTAG, rep1


title: MSX1FNV, dTAG, rep2


title: MSX1FNV, dTAG, rep1


title: WT, dTAG, rep1


title: MSX1FNV, DMSO, rep2


title: MSX1FNV, dTAG, rep3


title: WT, DMSO, rep3


title: WT, dTAG, rep2


title: ALX1FV ALX4-, DMSO, rep1


title: ALX1FV ALX4-, dTAG, rep2


title: PRRX1FV, dTAG, rep2


title: MSX1FNV, DMSO, rep3


title: PRRX1FV, DMSO, rep3


title: ALX1FV ALX4-, DMSO, rep3


title: PRRX1FV, DMSO, rep2


title: PRRX1FV, DMSO, rep1


title: PRRX1FV, dTAG, rep1


title: ALX1FV, dTAG, rep3


title: ALX1FV, DMSO, rep3


title: PRRX1FV, dTAG, rep3


title: MSX1FNV, DMSO, rep1


title: ALX1FV, dTAG, rep2


title: ALX1FV, dTAG, rep1


title: ALX1FV, DMSO, rep2


title: ALX1FV, DMSO, rep1


title: TWIST1FV, dTAG, rep2


title: TWIST1FV, DMSO, rep2


title: MSX1FNV, 24h dTAG, rep3

value: SLAM-seq


title: ALX1FV ALX4-, 24h dTAG, rep1


title: ALX1FV ALX4-, 0h dTAG, rep3


title: ALX1FV ALX4-, 0h dTAG, rep1


title: ALX1FV ALX4-, 0h dTAG, rep2


title: TWIST1FV, dTAG, rep3


title: ALX1FV ALX4-, 24h dTAG, rep2


title: ALX1FV ALX4-, 3h dTAG, rep3


title: ALX1FV ALX4-, 24h dTAG, rep3


title: ALX1FV ALX4-, 3h dTAG, rep1


title: MSX1FNV, 3h dTAG, rep3


title: TWIST1FV, dTAG, rep1


title: MSX1FNV, 0h dTAG, rep3


title: MSX1FNV, 24h dTAG, rep2


title: MSX1FNV, 3h dTAG, rep2


title: MSX1FNV, 24h dTAG, rep1


title: MSX1FNV, 0h dTAG, rep2


title: MSX1FNV, 3h dTAG, rep1


title: PRRX1FV, 3h dTAG, rep3


title: PRRX1FV, 24h dTAG, rep3


title: MSX1FNV, 0h dTAG, rep1


title: PRRX1FV, 0h dTAG, rep3


title: PRRX1FV, 3h dTAG, rep2


title: PRRX1FV, 0h dTAG, rep2


title: TWIST1FV, DMSO, rep1


title: ALX1FV ALX4-, 3h dTAG, rep2


title: TWIST1FV, DMSO, rep3


title: PRRX1FV, 24h dTAG, rep1


title: PRRX1FV, 0h dTAG, rep1


title: ALX1FV, 24h dTAG, rep3


title: ALX1FV, 0h dTAG, rep2


title: ALX1FV, 3h dTAG, rep3


title: ALX1FV, 3h dTAG, rep2


title: PRRX1FV, 3h dTAG, rep1


title: ALX1FV, 0h dTAG, rep1


title: ALX1FV, 3h dTAG, rep1


title: ALX1FV, 0h dTAG, rep3


title: ALX1FV, 24h dTAG, rep2


title: ALX1FV, 24h dTAG, rep1


title: WT, 24h dTAG, rep1


title: WT, 3h dTAG, rep2


title: WT, 24h dTAG, rep2


title: WT, 0h dTAG, rep1


title: TWIST1FV, 24h dTAG, rep3


title: TWIST1FV, 3h dTAG, rep3


title: PRRX1FV, 24h dTAG, rep2


title: WT, 3h dTAG, rep1


title: TWIST1FV, 24h dTAG, rep2


title: TWIST1FV, 0h dTAG, rep3


title: TWIST1FV, 3h dTAG, rep2


title: TWIST1FV, 24h dTAG, rep1


title: TWIST1FV, 0h dTAG, rep2


title: TWIST1FV, 3h dTAG, rep1


title: TWIST1FV, 0h dTAG, rep1


title: TWIST1FV, 1h dTAG, CUT&RUN, IgG

library_source: genomic

data_processing:
Reads were trimmed of Truseq adapter sequences and low-quality bases (-Q 10) using skewer v0.2.2.
Trimmed reads were mapped to a combined hg38 analysis set + E. coli K12 (substr. MG1655) reference genome using Bowtie2 v2.4.1 with the options --very-sensitive -X 2000.
Reads were deduplicated with samtools v1.10 markdup and uniquely mapped reads (-q 20) mapped to the main chromosomes (excluding mitochondria and unplaced contigs) were retained using samtools view.
MACS2 v2.2.7.1 was used to call peaks using the IgG sample as a negative control, with options -f BAMPE --nomodel --keep-dup all --call-summits -g hs.
bigWig tracks were generated with deeptools v3.5.0 bamCoverage -bs 10 --normalizeUsing RPGC --samFlagInclude 64 --samFlagExclude 8 --extendReads with 1h dTAG samples normalized by the fraction of reads mapping to the E. coli genome.
Assembly: hg38
Supplementary files format and content: bigWig, narrowPeak (except for IgG samples)
Library strategy: CUT&RUN

molecule: genomic DNA

growth_protocol: H9 cells (WiCell, WA09, RRID:CVCL_9773) were cultured in feeder-free conditions, in mTeSR1 medium (Stem Cell Technologies, 85850) on Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning, 356231) and passaged using ReLeSR (Stem Cell Technologies, 05872) every 4-6 days. Cells were switched to mTeSR Plus medium (Stem Cell Technologies, 100-0276) prior to and during genome editing and clonal expansion, but switched back to mTeSR1 before differentiation to CNCC. hESCs were differentiated to CNCC as previously described. Briefly, hESC colonies were partially detached from the plate with collagenase IV (Gibco, 17104019) in Knockout DMEM medium (Gibco, 10829018) for 30-60 min and scraped to break up large colonies, and then cultured in Neural Crest Differentiation Medium (50%-50% v/v mixture of DMEM/F12 1:1 medium with L-glutamine, without HEPES (Cytiva, SH30271.FS) and Neurobasal medium (Gibco, 21103049) with 0.5x N2 NeuroPlex (Gemini Bio, 400-163) and Gem21 NeuroPlex (Gemini Bio, 400-160) supplements and GlutaMAX (Gibco, 35050061), and 1x antibiotic/antimycotic, and 20 ng/ml EGF (Peprotech, AF-100-15), 20 ng/ml bFGF (Peprotech, 100-18B), and 5 ug/ml bovine insulin (Gemini Bio, 700-112P)) for 11 days in bacterial-grade petri dishes, changing the plate to prevent attachment for 4 days and then leaving the cells unfed for two days to allow attachment, and then fed as needed at least every other day. At day 11, cells (now called ‘early hCNCC’) were harvested by treatment with Accutase (Sigma-Aldrich, A6964-100ML), strained to remove residual neuroectodermal spheres, and plated onto plates coated with 7.5 ug/ml human fibronectin (Millipore, FC010-10MG) and cultured in Neural Crest Maintenance Medium (Neural Crest Differentiation Medium with bovine insulin replaced by 1 mg/ml BSA (Gemini Bio, 700-104P)). These hCNCC were then passaged every 2-3 days upon reaching confluency, with cells in the third or subsequent passages defined as ‘late hCNCC’ and cultured with added 50 pg/ml BMP2 (Peprotech, 120-02) and 3 uM CHIR-99021 (Selleck, S2924).

extract_protocol:
The CUTANA CUT&RUN (Epicypher) protocol v1.9 and reagents (concanavalin A beads, 21-1401 and pAG-MNase, 15-1016) were used with minor modifications: digestion was performed for 30 min on ice, and digestion supernatant was treated with 0.1% w/v SDS and 0.25 mg/ml proteinase K at 50°C for 1 h and then phenol-chloroform extraction was performed to extract DNA. Primary antibody incubations were performed overnight, and secondary antibody was used for mouse antibodies (TWIST1, ALX4, TCF3).
Library prep was performed with modifications to the NEBNext Ultra II DNA kit as in dx.doi.org/10.17504/protocols.io.bagaibse.

treatment_protocol: Media was changed 1 h before harvest, with added DMSO (0.12%) or dTAGV-1 (500 nM in 0.12% DMSO final). dTAGV-1 (Tocris, 6914/5) was dissolved in DMSO at 5 mM and then diluted to 250 uM in 60% DMSO/40% water (v/v) before dilution to 500 nM

value: Human cranial neural crest cell

tag: spike-in cell_type

value: E. coli

tag: spike-in cell_line

tag: antibody

value: Rabbit anti-mouse IgG (Abcam #ab206566)


title: TWIST1FV, 1h dTAG, CUT&RUN, V5


title: TWIST1FV, 1h dTAG, CUT&RUN, AP2a

value: AP2a (Cell Signaling #3215)


title: TWIST1FV, 1h dTAG, CUT&RUN, TCF3

value: TCF3 (Santa Cruz #sc-133074)


title: WT, 0h dTAG, rep2


title: TWIST1FV, 1h DMSO, CUT&RUN, IgG


title: TWIST1FV, 1h dTAG, CUT&RUN, ALX4

value: ALX4 (Novus Bio, #NBP2-45490)


title: TWIST1FV, 1h DMSO, CUT&RUN, V5


title: TWIST1FV, 1h dTAG, CUT&RUN, TWIST1

value: TWIST1 (Abcam, #ab50887)


title: TWIST1FV, 1h dTAG, CUT&RUN, CTCF

value: CTCF (Cell Signaling #2899)


title: TWIST1FV, 1h DMSO, CUT&RUN, ALX4


title: TWIST1FV, 1h DMSO, CUT&RUN, TWIST1


title: TWIST1FV, 1h DMSO, CUT&RUN, TCF3


title: TWIST1FV, 1h DMSO, CUT&RUN, AP2a


title: TWIST1FV, 1h DMSO, CUT&RUN, CTCF


title: HCA_trmB_0.1glu_B_IP

city: Durham

first: Amy

middle: K

last: Schmid

state: NC

address: 124 Science Dr

institute: Duke University

description: TrmB:HA translational fusion. Replicate B. HhCA media supplemented with 0.1% glucose (w/v). Immuno-precipitated sample.

data_processing:
Adapter sequences were removed with Trim_galore!  0.4.3, cutadapt 2.3, and and sample quality assessed with FastQC 0.11.7 using default parameters.
Reads were aligned to the Haloarcula hispanica ATCC33960 reference genome (GCF_000223905.1, accessed 2018-11-13) with Bowtie2 2.3.4.3 in single-end mode.
Aligned sequence files were converted to binary format, sorted, and indexed using samtools 1.9.
Aligned and sorted BAM files were imported in to R coding environment and fragment length was optimized for each IP and input (WCE) sample using ChIPQC 1.30.
Peaks were called using Mosaics 2.32 in R 4.1.2 using the estimated fragment lengths from ChIPQC and FDR < 0.01.
For per base coverage plots, BAM files were extended in the 3' direction to 150 bps with samtools 1.10 and converted to BEDGRAPH format using bedtools 2.30.0 as described in Grünberger et al. 2020.
Assembly: GCF_000223905.1
Supplementary files format and content: BED files with mosaics called peaks (except for input (WCE) samples)
Supplementary files format and content: bedgraph files with per base read coverage

organism: Haloarcula hispanica ATCC 33960

source_name: delta-pyrF trmB::HA cell culture

growth_protocol: Strains DF60 and AKS155 were struck from freezer stocks onto YPC23+uracil plates and incubated at 37 C for 8 days. Independent colonies were used to start 2 cultures of DF60 and 4 cultures AKS155 in 10 mL YPC23+uracil. Precultures were grown for 29 hours at 37C with 250 rpm shaking (average optical density at 600nm = 2) then collected at 6000 rpm for 2 min and washed twice with Hh-CA+uracil. AKS155 pellets were resuspended in either Hh-CA+uracil or Hh-CA+uracil + 0.1% glucose and grown to mid-log phase (average OD = 0.35) at 37C with shaking.

extract_protocol:
Samples were processed as described in Wilbanks et al. (2012) using anti-HA polyclonal antibody (Abcam #ab9110) to immunoprecipitate cross-linked fragments.
Libraries were constructed by the Center for Genomic and Computational Biology at Duke University (Durham, NC) using KAPA Hyper Prep kit and Illumina TruSeq adapters, and the 50 bp, single-ended libraries were sequenced on an Illumina HiSeq 4000

tag: strain

value: delta-pyrF trmB::HA

value: delta-pyrF trmB::HA cell culture

value: Hh-CA + uracil + 0.1% glucose (w/v)

tag: chip antibody

value: Abcam #ab9110


title: HCA_trmB_0glu_D_IP

description: TrmB:HA translational fusion. Replicate C. HhCA media. Immuno-precipitated sample.

value: Hh-CA + uracil


title: HCA_trmB_0glu_B_IP

description: TrmB:HA translational fusion. Replicate B. HhCA media. Immuno-precipitated sample.


title: HCA_trmB_0.1glu_A_IP

description: TrmB:HA translational fusion. Replicate A. HhCA media supplemented with 0.1% glucose (w/v). Immuno-precipitated sample.


title: HCA_WT_0glu_B_IP

description: Parental, no-tag control. Replicate A. Immuno-precipitated sample.

source_name: delta-pyrF cell culture

value: delta-pyrF

value: delta-pyrF cell culture


title: HEK293, pcDNA3.1_V5-NEUROD1 and pcDNA3.1_ALX4-Flag, input

data_processing:
Reads were trimmed of Truseq adapter sequences and low-quality bases (-Q 10) using skewer v0.2.2.
Trimmed reads were mapped to the hg38 analysis set (human), mm39 (mouse), or a combined reference genome using Bowtie2 v2.4.1 with the options --very-sensitive -X 2000.
Reads were deduplicated with samtools v1.10 markdup and uniquely mapped reads (-q 20) mapped to the main chromosomes (excluding mitochondria and unplaced contigs) were retained using samtools view.
MACS2 v2.2.7.1 was used to call peaks using the IgG sample as a negative control, with options -f BAMPE --nomodel --keep-dup all --call-summits -g hs (human) or mm (mouse) with the -B option for H3K27ac.
bigWig tracks were generated with deeptools v3.5.0 bamCoverage -bs 10 --normalizeUsing RPGC --samFlagInclude 64 --samFlagExclude 8 --extendReads with 1h dTAG samples normalized by the fraction of reads mapping to the E. coli genome.
H3K27ac ChIP reads were counted over merged ATAC peaks using deeptools v3.5.0 multiBamSummary with options -e --outRawCounts.
Assembly: hg38 or mm39
Supplementary files format and content: bigWig, narrowPeak (except for input samples, H3K27ac ChIPs, and V5 ChIPs of dTAG-treated samples) for each sample, and tab-delimited text (.txt) files with read counts of H3K27ac ChIPs over merged ATAC peaks

growth_protocol: H9 cells (WiCell, WA09, RRID:CVCL_9773) were cultured in feeder-free conditions, in mTeSR1 medium (Stem Cell Technologies, 85850) on Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning, 356231) and passaged using ReLeSR (Stem Cell Technologies, 05872) every 4-6 days. Cells were switched to mTeSR Plus medium (Stem Cell Technologies, 100-0276) prior to and during genome editing and clonal expansion, but switched back to mTeSR1 before differentiation to CNCC. hESCs were differentiated to CNCC as previously described. Briefly, hESC colonies were partially detached from the plate with collagenase IV (Gibco, 17104019) in Knockout DMEM medium (Gibco, 10829018) for 30-60 min and scraped to break up large colonies, and then cultured in Neural Crest Differentiation Medium (50%-50% v/v mixture of DMEM/F12 1:1 medium with L-glutamine, without HEPES (Cytiva, SH30271.FS) and Neurobasal medium (Gibco, 21103049) with 0.5x N2 NeuroPlex (Gemini Bio, 400-163) and Gem21 NeuroPlex (Gemini Bio, 400-160) supplements and GlutaMAX (Gibco, 35050061), and 1x antibiotic/antimycotic, and 20 ng/ml EGF (Peprotech, AF-100-15), 20 ng/ml bFGF (Peprotech, 100-18B), and 5 ug/ml bovine insulin (Gemini Bio, 700-112P)) for 11 days in bacterial-grade petri dishes, changing the plate to prevent attachment for 4 days and then leaving the cells unfed for two days to allow attachment, and then fed as needed at least every other day. At day 11, cells (now called ‘early hCNCC’) were harvested by treatment with Accutase (Sigma-Aldrich, A6964-100ML), strained to remove residual neuroectodermal spheres, and plated onto plates coated with 7.5 ug/ml human fibronectin (Millipore, FC010-10MG) and cultured in Neural Crest Maintenance Medium (Neural Crest Differentiation Medium with bovine insulin replaced by 1 mg/ml BSA (Gemini Bio, 700-104P)). These hCNCC were then passaged every 2-3 days upon reaching confluency, with cells in the third or subsequent passages defined as ‘late hCNCC’ and cultured with added 50 pg/ml BMP2 (Peprotech, 120-02) and 3 uM CHIR-99021 (Selleck, S2924). dTAGV-1 (Tocris, 6914/5) was dissolved in DMSO at 5 mM and then diluted to 250 uM in 60% DMSO/40% water (v/v) before dilution to 500 nM for acute depletions (up to 1 day) or diluted directly from the 5 mM stock for long-term depletions.  RS4;11 cells (ATCC, CRL-1873, RRID:CVCL_0093) were cultured in RPMI-1640 medium (Gibco, 11875093) supplemented with 10% v/v FBS and 1x antibiotic/antimycotic.  HEK293 cells (ATCC, CRL-1573, RRID:CVCL_0045) and 293FT cells (Invitrogen, R70007, RRID:CVCL_6911) were cultured in DMEM high glucose medium with sodium pyruvate and L-glutamine, supplemented with 10% v/v FBS and 1x GlutaMAX, non-essential amino acids, and antibiotic/antimycotic.  O9-1 cells (Millipore, SCC049, RRID:CVCL_GS42) used for spike-in controls for ChIPs of TWIST1 depletions were cultured in Complete ES Cell Medium with 15% FBS (Millipore, ES-101-B), 25 ng/ml bFGF, and mLIF (Millipore, ESG1107).

extract_protocol:
Cells (about 1 confluent 10-cm plate or ~10-20 million cells) were crosslinked with 1% methanol-free formaldehyde (Pierce, 28908) in PBS for 10 min at room temperature and then quenched by adding 2.5 M glycine to 125 mM final concentration and incubating for 10 min. Cells were washed in PBS with 0.001% v/v Triton X-100, harvested by scraping, and collected by centrifugation for 5 min at 4°C. Cells were washed with PBS and flash frozen for storage at -80°C. Cell pellets were later thawed on ice for 30 min, and then sequentially resuspended in lysis buffer 1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Igepal CA-630, 0.25% Triton X-100, 1x cOmplete EDTA-free protease inhibitor cocktail (PIC), 1 mM PMSF), lysis buffer 2 (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x cOmplete EDTA-free protease inhibitor cocktail, 1 mM PMSF), and lysis buffer 3 (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine, 1x PIC, 1 mM PMSF), with 10 min incubations in each buffer, with rotation. Lysates were sonicated for 10-15 cycles of 30s ON/30s OFF on high power using the Bioruptor Plus (Diagenode), then diluted in additional lysis buffer 3 and clarified by centrifugation for 10 min at max speed at 4°C. Triton X-100 was added to 1%, and a small aliquot was used to extract DNA to check chromatin yield and size distribution, by dilution in elution buffer (1% w/v SDS and 100 mM NaHCO3) and incubation with 200 mM NaCl and RNase A (Thermo, EN0531) at 65°C for 1 h, then proteinase K (Thermo, EO0491) at 65°C for 1 h, and clean up with the ChIP DNA Clean & Concentrator-5 kit (Zymo, D5205). DNA was quantified by Qubit dsDNA high sensitivity kit, and the remaining chromatin was then normalized for immunoprecipitations. For TWIST1 acute depletions, chromatin from O9-1 mouse CNCCs were added prior to ChIP at ~10% of the total chromatin as a spike-in control. For H3K27ac, 5 ug of antibody was used per ChIP; for TFs, 9 ug of antibody was used per ChIP, except for dissected mouse embryos where 4.5 ug was used in half of the total ChIP volume. ChIPs were incubated overnight, then incubated for 4-6h with 100 ul Dynabeads Protein A (Invitrogen, 10002D) or Protein G (Invitrogen, 10004D) prewashed with 0.1% w/v BSA in PBS, then washed 5x with RIPA wash buffer (50 mM HEPES-KOH pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% Igepal CA-630, 0.7% w/v sodium deoxycholate), once with 50 mM Tris-HCl pH 8, 10 mM EDTA, 50 mM NaCl, and eluted in elution buffer at 65°C for 30 min. Eluate was then reverse crosslinked and treated with RNase A and proteinase K, and then DNA was extracted with the ChIP DNA Clean & Concentrator-5 kit.
Libraries were prepared using the NEBNext Ultra II DNA kit (New England Biolabs, E7645S) using up to 50 ng of input or ChIP DNA, with ~4-8 cycles of amplification, with no pre-PCR size selection but a post-PCR double-sided 0.5x/0.9x Ampure XP bead clean-up.

treatment_protocol: DMSO or dTAGV-1 (500 nM) were added by dilution into media before changing media. HEK293 cells were transfected with Lipofectamine 2000 (Invitrogen, 11668019) at a ratio of 2.8 ul lipofectamine per ug of DNA, diluted in Opti-MEM. Cells were transfected with 2.5 ug DNA per well of a 6-well plate or 15 ug DNA per 10-cm plate 1-2 days after seeding, when they reached 70-90% confluency. Media was replaced 4-6h after transfection, and then cells were harvested for Western blot or chromatin immunoprecipitation at 24 h after transfection.

value: Human embryonic kidney

value: WT

value: transfection with pcDNA3.1_V5-NEUROD1 and pcDNA3.1_ALX4-Flag

tag: reference genome_for_alignment

tag: reference genome_for_processed_files


title: HEK293, pcDNA3.1_V5-NEUROD1, input

tag: cell line

value: transfection with pcDNA3.1_V5-NEUROD1


title: HEK293, pcDNA3.1_V5-TWIST1(WT) and pcDNA3.1_ALX4-Flag, input

tag: treatment

value: transfection with pcDNA3.1_V5-TWIST1(WT) and pcDNA3.1_ALX4-Flag


title: HEK293, pcDNA3.1_V5-TWIST1(NEUROD1 loop) and pcDNA3.1_ALX4-Flag, input

value: transfection with pcDNA3.1_V5-TWIST1(NEUROD1 loop) and pcDNA3.1_ALX4-Flag


title: HEK293, pUC19, input

value: transfection with pUC19


title: HEK293, pcDNA3.1_V5-TWIST1(NEUROD1 loop), input

value: transfection with pcDNA3.1_V5-TWIST1(NEUROD1 loop)


title: HCA_trmB_0glu_A_IP

description: TrmB:HA translational fusion. Replicate A. HhCA media. Immuno-precipitated sample.


title: HCA_WT_0glu_A_IP


title: HCA_trmB_0glu_D_WCE

description: TrmB:HA translational fusion. Replicate C. HhCA media. Input sample.


title: HEK293, pUC19, V5


title: HCA_trmB_0glu_C_WCE


title: HEK293, pcDNA3.1_V5-TWIST1(WT), input


title: HEK293, pcDNA3.1_V5-NEUROD1 and pcDNA3.1_ALX4-Flag, V5


title: HEK293, pcDNA3.1_V5-TWIST1(NEUROD1 loop) and pcDNA3.1_ALX4-Flag, V5


title: HEK293, pcDNA3.1_V5-NEUROD1, V5


title: HEK293, pcDNA3.1_V5-TWIST1(WT) and pcDNA3.1_ALX4-Flag, V5


title: HCA_trmB_0glu_B_WCE

description: TrmB:HA translational fusion. Replicate B. HhCA media. Input sample.


title: HCA_trmB_0.1glu_B_WCE

description: TrmB:HA translational fusion. Replicate B. HhCA media supplemented with 0.1% glucose (w/v). Input sample.


title: HCA_trmB_0glu_C_IP


title: HCA_trmB_0glu_A_WCE

description: TrmB:HA translational fusion. Replicate A. HhCA media. Input sample.


title: HCA_trmB_0.1glu_A_WCE

description: TrmB:HA translational fusion. Replicate A. HhCA media supplemented with 0.1% glucose (w/v). Input sample.


title: HEK293, pcDNA3.1_V5-TWIST1(NEUROD1 loop), V5


title: HCA_WT_0glu_B_WCE

description: Parental, no-tag control. Replicate A. Input sample.


title: HCA_WT_0glu_A_WCE


title: HEK293, pcDNA3.1_V5-TWIST1 and pcDNA3.1_PHOX2A-Flag, input

value: transfection with pcDNA3.1_V5-TWIST1 and pcDNA3.1_PHOX2A-Flag


title: HEK293, pcDNA3.1_V5-TWIST1(WT), V5


title: HEK293, pcDNA3.1_V5-TWIST1 and pcDNA3.1_MSX1-Flag, input

value: transfection with pcDNA3.1_V5-TWIST1 and pcDNA3.1_MSX1-Flag


title: HEK293, pcDNA3.1_V5-TWIST1 and pcDNA3.1_PRRX1b-Flag, input

value: transfection with pcDNA3.1_V5-TWIST1 and pcDNA3.1_PRRX1b-Flag


title: LNCaP cells exposed to BPF 5nM Biol Rep 2

city: MEXICO CITY

first: Mauricio

last: Rodriguez Dorantes

state: Distrito Federal, Mexico

address: Periferico Sur 4809, Arenal Tepepan, Tlalpan,  Ciudad de México, CDMX, México

country: Mexico

institute: INMEGEN

description: Gene expression data from prostate cancer cells exposed to bisphenols  F at 5 nM concentration

hyb_protocol: We followed the manufacturer’s protocol for sample preprocessing, labeling, hybridization, and microarray imaging

data_processing: Data were analyzed using the Thermo-Fisher Scientific’s Transcriptome Analysis Console (TAC 4.0.2). Raw data were processed for normalization and gene level analysis using the robust multi-array average (RMA) included in the TAC software. Each experimental con-dition has three independent microarray replicates. Differential expressed transcripts were calculated using the means of the gene expression signals, to obtain a Fold Change (FC) of the expression values. Statistical analysis was done using ebayes limma method included in the TAC software. Those FC values ≥ 2 , ≤ -2, and with a Gene-Level-p-value ≤0.05 were considered significant. Volcano plots, and clustergrams (Heatmaps) were ob-tained from the TAC software

label: biotin

source_name: LNCaP prostate cancer cells exposed to BPF 5nM Biol Rep 2

growth_protocol: Prostate cancer cell lines LNCaP (CRL-1740) & PC-3 (CRL-1435) were acquired from the American Type Culture Collection (ATCC, Inc.) All cells were cultured with RPMI 1640 media (pH= 7.4) (Sigma-Aldrich, Inc. R8758) with 10% of fetal bovine serum (FBS),and incubated under standard conditions (5% CO2, 37°C).

extract_protocol: RNA purification was performed by using TRI reagent (Sigma-Aldrich, Inc.)[74], following the manufacturer’s protocol for cells cultured in monolayer. Once extracted, the RNA concentration and purity was measured using a NanoDrop spectrophotometer (Ther-mo-Fisher Scientific, Inc.). determining the sample absorbance at a wavelength of 260 nm, and for purity evaluation we measured the absorbance ratio at 260/A280 nm & 260/A230 nm. We considered that a sample as relatively pure when the 260/A280 nm ratio was ~2.0, and the 260/230 ratio was between 2.0-2.2. Then the RNA quality was determined by automated electrophoresis, using the Agilent, Inc. 2100 Bioanalyzer Instrument and following the manufacturer’s indications. Samples with an RNA integrity number (RIN) ≥9.0 were considered suitable for following tests.

treatment_protocol: LNCaP & PC-3 cells were seeded into flat bottom plates of 25 mm2. Twenty-four hours after seeding, cells were hormone depleted using RPMI 1640 media without phenol red and FBS charcoal stripped (1%). Low concentration of FBS is crucial to perform this assay to starve cells avoiding cell proliferation.  After hormone depletion and starving, cells were treated with bisphenols A, S & F added to their cell culture media to reach final concentration of 5nM (Vehicle final concentration was 1% v/v). Forty-eight hours after treatment, cell culture media was removed and cells were washed with phosphate-buffered saline (PBS) 1X, then RNA extraction was performed

value: LNCaP

value: Bisphenol F

tag: androgen receptor

value: AR+


title: LNCaP cells exposed to BPF 5nM Biol Rep 3

scan_protocol: We followed the manufacturer’s protocol for sample preprocessing, labeling, hybridization, and microarray imaging

source_name: LNCaP prostate cancer cells exposed to BPF 5nM Biol Rep 3


title: LNCaP cells exposed  to BPA 5 nM Biol Rep 2

description: Gene expression data from prostate cancer cells exposed to bisphenols A at 5 nM concentration

source_name: LNCaP prostate cancer cells exposed  to BPA 5 nM Biol Rep 2

label_protocol: We followed the manufacturer’s protocol for sample preprocessing, labeling, hybridization, and microarray imaging

value: Bisphenol A


title: LNCaP cells exposed  to BPA 5 nM Biol Rep 3

source_name: LNCaP prostate cancercells exposed  to BPA 5 nM Biol Rep 3


title: LNCaP cells exposed to BPF 5nM Biol Rep 1

source_name: LNCaP prostate cancer cells exposed to BPF 5nM Biol Rep 1


title: LNCaP cells exposed  to BPS 5 nM Biol Rep 2

description: Gene expression data from prostate cancer cells exposed to bisphenols S  at 5 nM concentration

source_name: LNCaP prostate cancer cells exposed  to BPS 5 nM Biol Rep 2

value: Bisphenol S


title: HEK293, pcDNA3.1_V5-TWIST1 and pcDNA3.1_PRRX1a-Flag, input

tag: cell type

value: transfection with pcDNA3.1_V5-TWIST1 and pcDNA3.1_PRRX1a-Flag


title: HEK293, pcDNA3.1_V5-TWIST1 and pcDNA3.1_ALX4-Flag, input

value: Human embryonic kidney

value: transfection with pcDNA3.1_V5-TWIST1 and pcDNA3.1_ALX4-Flag


title: LNCaP cells exposed  to BPS 5 nM Biol Rep 3

description: Gene expression data from prostate cancer cells exposed to bisphenols  S at 5 nM concentration

source_name: LNCaP prostate cancer cells exposed  to BPS 5 nM Biol Rep 3


title: LNCaP cells exposed  to BPS 5 nM Biol Rep 1

source_name: LNCaP prostate cancer cells exposed  to BPS 5 nM Biol Rep 1


title: HEK293, pcDNA3.1_V5-TWIST1 and pcDNA3.1_PHOX2A-Flag, TWIST1

value: WT

value: TWIST1 (Abcam, #ab50887)


title: HEK293, pcDNA3.1_V5-TWIST1 and pcDNA3.1_MSX1-Flag, TWIST1


title: HEK293, pcDNA3.1_V5-TWIST1 and pcDNA3.1_PRRX1b-Flag, TWIST1


title: LNCaP cells exposed  to BPA 5 nM Biol Rep 1

source_name: LNCaP prostate cancer cells exposed  to BPA 5 nM Biol Rep 1


title: LNCaP cells exposed toVehicle (EtOH) Biol Rep 3

description: Gene expression data from prostate cancer cells exposed to Ethanol 1%

source_name: LNCaP prostate cancer cells exposed toVehicle (EtOH) Biol Rep 3

value: Ethanol


title: HEK293, pcDNA3.1_V5-TWIST1 and pcDNA3.1_PRRX1a-Flag, TWIST1


title: LNCaP cells exposed to Vehicle (EtOH) Biol Rep 2

source_name: LNCaP prostate cancer cells exposed to Vehicle (EtOH) Biol Rep 2


title: HEK293, pcDNA3.1_V5-TWIST1, TWIST1

value: transfection with pcDNA3.1_V5-TWIST1


title: HEK293, pcDNA3.1_V5-TWIST1, input


title: RS4;11, input

value: Human lymphoblast


title: LNCaP cells exposed to Vehicle (EtOH) Biol Rep 1

source_name: LNCaP  prostate cancer cells exposed to Vehicle (EtOH) Biol Rep 1


title: PC-3 cells exposed to BPF 5nM Biol Rep 3

source_name: PC-3 prostate cancer cells exposed to BPF 5nM Biol Rep 3

value: AR-


title: PC-3 cells exposed to BPF 5nM Biol Rep 2

source_name: PC-3 prostate cancer cells exposed to BPF 5nM Biol Rep 2


title: hCNCC, input

value: Human cranial neural crest cell


title: RS4;11, TWIST1


title: PC-3 cells exposed to BPF 5nM Biol Rep 1

source_name: PC-3 prostate cancer cells exposed to BPF 5nM Biol Rep 1


title: hCNCC, TWIST1, Abcam


title: PC-3 cells exposed  to BPS 5 nM Biol Rep 3

source_name: PC-3 prostate cancer cells exposed  to BPS 5 nM Biol Rep 3


title: hCNCC, AP2a, Cell Signaling

value: AP2a (Cell Signaling #3215)


title: PC-3 cells exposed  to BPS 5 nM Biol Rep 2

source_name: PC-3 prostate cancer cells exposed  to BPS 5 nM Biol Rep 2


title: PC-3 cells exposed  to BPS 5 nM Biol Rep 1

source_name: PC-3 prostate cancer cells exposed  to BPS 5 nM Biol Rep 1


title: hCNCC, AP2a, Novus Bio


title: PC-3 cells exposed  to BPA 5 nM Biol Rep 2

source_name: PC-3 prostate cancer cells exposed  to BPA 5 nM Biol Rep 2


title: ALX1FV ALX4-, 0h dTAG, rep2, TWIST1

value: ALX1FV ALX4-

value: dTAGV-1 (500 nM)


title: ALX1FV ALX4-, 3h dTAG, rep2, V5


title: ALX1FV ALX4-, 0h dTAG, rep1, TWIST1


title: PC-3 cells exposed toVehicle (EtOH) Biol Rep 3

source_name: PC-3 prostate cancer cells exposed toVehicle (EtOH) Biol Rep 3


title: PC-3 cells exposed  to BPA 5 nM Biol Rep 1

source_name: PC-3 prostate cancer cells exposed  to BPA 5 nM Biol Rep 1


title: PC-3 cells exposed  to BPA 5 nM Biol Rep 3

source_name: PC-3 prostate cancercells exposed  to BPA 5 nM Biol Rep 3


title: ALX1FV ALX4-, 3h dTAG, rep1, V5


title: ALX1FV ALX4-, 0h dTAG, rep2, V5


title: HEK293, pcDNA3.1_V5-TWIST1 and pcDNA3.1_ALX4-Flag, TWIST1


title: ALX1FV ALX4-, 0h dTAG, rep1, V5


title: PC-3 cells exposed to Vehicle (EtOH) Biol Rep 1

source_name: PC-3  prostate cancer cells exposed to Vehicle (EtOH) Biol Rep 1


title: ALX1FV ALX4-, 0h dTAG, rep2, input


title: ALX1FV ALX4-, 24h dTAG, rep2, input


title: PC-3 cells exposed to Vehicle (EtOH) Biol Rep 2

source_name: PC-3 prostate cancer cells exposed to Vehicle (EtOH) Biol Rep 2


title: ALX1FV ALX4-, 24h dTAG, rep1, input


title: Pig 6, Myocardial infarction only_RZ

city: Tucson

first: Marissa

middle: Anne

last: Lopez-Pier

state: AZ

address: 1656 E. Mabel St.

department: Physiology

institute: The University of Arizona

data_processing:
The clustering of the index-coded samples was performed on a cBot Cluster Generation Systemusing PECluster KitcBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparationsweresequenced on an Illumina platform and 125 bp/150 bp paired-end reads were generated
Raw data (raw reads) of fastq format were firstly processed. In this step, clean data (clean reads) were obtainedbyremoving reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the sametime, Q20,Q30 and GC content the clean data were calculated. All the downstream analyses were based on the cleandatawithhighquality
Reference genome and gene model annotation files were downloaded from genome website directly. Indexof thereference genome was built using hisat2 2.1.0 and paired-end clean reads were aligned to the reference genomeusingHISAT2. We selected HISAT2 as the mapping tool for that Hisat2 can generate a database of splice junctions basedon the gene model annotation file and thus a better mapping result than other non-splice mapping tools, whichis afast andsensitive alignment program for mapping next-generation sequencing reads against the general human population.
FeatureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene(Liao Y et al., 2013). AndthenFPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currentlythemostcommonly used method for estimating gene expression levels (Trapnell, Cole, et al., 2010)
(For DESeq2 with biological replicates) Differential expression analysis between two conditions/groups(two biological replicates per condition) was performed using the DESeq2 R package (1.14.1). DESeq2provide statistical routines for determining differential expression in digital gene expressiondatausingamodel based on the negative binomial distribution. The resulting P-values were adjusted usingtheBenjamini and Hochberg’s approach for controlling the False Discovery Rate(FDR). Genes withanadjustedP-value
Gene Ontology (GO) enrichment analysis of differentially expressed genes was implementedbytheclusterProfiler R package, in which gene length bias was corrected. GO terms with correctedPvaluelessthan 0.05 were considered significantly enriched by differential expressed genes. KEGG is a database resource for understanding high-level functions and utilities of the biological system,such as the cell, the organism and the ecosystem, from molecular level information, especiallylarge-scalemolecular datasets generated by genome sequencing and other high-through put experimental technologies(http://www.genome.jp/kegg/). We used clusterProfiler R package to test the statistical enrichmentofdifferential expression genes in KEGG pathways.
PPI analysis of differentially expressed genes was based on the STRING database, which  known and predicted Protein-Protein Interactions. For the species existing in the database, we construct the networks by extract the target gene list from the database; Otherwise, Blastx (v2.2.28) was used to align the target gene sequences to the selected reference  protein sequences, and then the networks was built according to the known interaction of  selected reference species. Novel transcripts prediction and alternative splicing analysis  The Cufflinks v2.1.1 Reference Annotation Based Transcript (RABT) assembly method  was used to construct and identify both known and novel transcripts from TopHat  alignment results. Alternative splicing events were classified to 12 basic types by the  software Asprofile v1.0. The number of AS events in each sample was estimated,  separately
Picard tools(v1.111) and samtools v0.1.18 were used to sort, mark duplicated reads and reorder the bam alignment results of each sample. GATK4.1 software was used to perform SNP calling
References  Liao Y, Smyth GK and Shi W. featureCounts: an efficient general-purpose program for assigning sequence readstogenomic features. Bioinformatics, 2013. doi: 10.1093/bioinformatics/btt656(featureCounts)  Anders S, Huber W. (2010).Differential expression analysis for sequence count data.Genome  Biology,doi:10.1186/gb-2010-11-10-r106. (DESeq2)  Robinson M D, McCarthy D J, Smyth G K. edgeR: a Bioconductor package for differential expressionanalysisof digital gene expression data[J]. Bioinformatics, 2010, 26(1): 139-140. (edgeR)  Kanehisa, M., M. Araki, et al. (2008). KEGG for linking genomes to life and the environment. Nucleic acids research.(KEGG)  Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. (2009). Ultrafast and memory‐efficient  alignment of short DNA sequences to the human genome. Genome Biol.(Bowtie)  Langmead, B. and S. L. Salzberg (2012). Fast gapped‐read alignment with Bowtie 2. Nature  methods.(Bowtie 2)  Mao, X., Cai, T., Olyarchuk, J.G., Wei, L. (2005). Automated genome annotation and pathway  identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinformatics.(KOBAS)  Marioni, J. C., C. E. Mason, et al. (2008). RNA‐seq: an assessment of technical reproducibility  and comparison with gene expression arrays. Genome research. Van der Auwera et al. (2013). From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best  practices pipeline. Current protocols in bioinformatics. (GATK)  Mortazavi, A., B. A. Williams, et al. (2008). Mapping and quantifying mammalian transcriptomes by RNA0Seq. Naturemethods. Shannon et al. (2003). Cytoscape: a software environment for integrated models of biomolecular interactionnetworks.(Cytoscape)  Trapnell, C. et al. (2010). Transcript assembly and quantification by RNA0 seq reveals unannotated transcripts andisoform switching during cell differentiation. Nat. Biotechnol.(Cufflinks)  Daehwan K.,Ben L., et al. (2008).HISAT: Hierarchical Indexing for Spliced Alignment of Transcripts.(HISAT2)  Trapnell, C., A. Roberts, et al. (2012). Differential gene and transcriptexpression analysis of RNA0 seq experimentswith TopHat and Cufflinks. nature protocols.(Tophat & Cufflinks) Wang, L.Feng, Z.Wang, X.Zhang, X. (2010). DEGseq:an R package for identifying differentially expressed genes from RNA0 seq data. Bioinformatics.(DEGseq) Wang, Z.,M.Gerstein, et al. (2009). RNA0 Seq: a revolutionary tool for transcriptomics. Nature Reviews Genetics. Young, M. D., Wakefield, M. J., Smyth, G. K., and Oshlack, A. (2010).Gene ontology analysis for RNA0seq:accounting for selection bias. Genome
Assembly: Sus scrofa version 10.2
Supplementary files format and content: tab-delimited text files include RPKM values for each sample

organism: Sus scrofa domesticus

source_name: cardiac tissue

extract_protocol:
Following excision of LV core from IZ, BZ, and RZ, samples were rinsed in 1X PBS (10x PBS Solution, 1.37M NaCl, 0.027M KCl and 0.119M phosphates, pH 7.4, Fisher BioReagents, Ref: BP3994) to remove any residual blood. Tissue was submerged in RNAlater® (Sigma-Aldrich, St. Louis, MO, Ref: R0901-500ML) and placed in 4ºC for 48 hours prior to storing in -80ºC.   The epicardial surface of the LV core biopsy (1/3 of the tissue) was utilized for RNA extraction. Tissue samples were submerged in 400μL of Trizol reagent (ThermoFisher, Carlsbad, CA, Ref:15596026) and ground for 30 seconds in a 2mL flat centrifuge tube using an OMNI THQ digital rotor-stator (OMNI, Kennesaw, GA, Ref:12-500) at 15,000RPM and hard tissue OMNI TipTM (OMNI, Kennesaw, GA, Ref: 30750H). Each sample was ground using a fresh tip to prevent cross contamination. Tubes were gently rocked back and forth to mix contents and were left undisturbed at room temperature for 10 minutes. Thereafter, 100μL of chloroform (Millipore-Sigma, Milwaukee, WI, Ref:288306) was added, mixed, and centrifuged (Eppendorf 5424R, Hamburg, Germany) at 15,000RPM for 5 minutes. The supernatant was transferred to a new tube, to which lysis buffer from the Qiagen RNEasy Plus Mini kit (Qiagen, Hilden, Germany, Ref: 74134) containing BME was added to bring samples up to a volume of 650μL. The total volume of sample was then transferred to a gDNA eliminator column from the Qiagen RNEasy Plus Mini kit and centrifuged in the Eppendorf at 8,000RPM for 30 seconds. The flow-through from the centrifugation step was then mixed in a 1:1 volume ratio of 70% EtOH, resulting in a final concentration of 35% EtOH by volume of the total sample volume. The subsequent steps were followed according to kit manufacturer’s protocol.    RNA samples were assessed for quality with an Advanced Analytics Fragment Analyzer (High Sensitivity RNA Analysis Kit – Ref: DNF-471-0500) and quantity with a Qubit RNA quantification kit (Qubit® RNA HS Assay KitAssay Kit – Ref: Q32852). RNA Quality Number (RQN) of samples ranged from 5.2 to 9.1.
Given satisfactory quality and quantity, samples were used for library builds. A total amount of 1μg RNA per sample was used as input material for the RNA sample preparations. Libraries were constructed using NEBNext®UltraTMRNA Library Prep Kit for Illumina® (NEB, USA) according to manufacturer’s recommendations. Index codes were added to attribute sequences to each sample. Poly-T oligo-attached magnetic beads were used to purify mRNA from the total RNA sample, and fragmentation was achieved using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). Subsequently, second strand cDNA synthesis was performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. To prepare for hybridization, NEBNext Adaptor with hairpin loop structure were ligated following 3’ end adenylation. The AMPure XP system (Beckman Coulter, Beverly, USA) was used to isolate cDNA fragments of approximately 150-200 base pairs long. PCR was performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer following addition of 3μl USER Enzyme (NEB, USA) with size-selected, adaptor-ligated cDNA at 37°C for 15 minutes followed by 5 minutes at 95°C. Lastly, PCR products were purified (AMPure XP system), and library quality was assessed on the Agilent Bioanalyzer 2100 system.
A total amount of 1μg RNA per sample was used as input material for the RNA sample preparations. Libraries were constructed using NEBNext®UltraTMRNA Library Prep Kit for Illumina® (NEB, USA) according to manufacturer’s recommendations

tag: tissue

value: cardiac tissue

tag: tissue region

value: remote zone, or  LV tissue far away from infarcted area.

value: wild type

tag: disease state

value: Myocardial infarction


title: Pig 6, Myocardial infarction only_IZ

value: infarct region of cardiac LV tissue

tag: genotype


title: ALX1FV ALX4-, 3h dTAG, rep1, input

tag: clone

tag: time


title: Pig 6, Myocardial infarction only_BZ

value: border of infarcted cardiac  LV tissue to non injured tissue


title: ALX1FV ALX4-, 0h dTAG, rep1, input

tag: spike-in cell_type


title: ALX1FV ALX4-, 24h dTAG, rep2, H3K27ac

tag: spike-in cell_line

value: H3K27ac (Active Motif #39133)


title: Pig 5, Myocardial infarction only_RZ


title: ALX1FV ALX4-, 3h dTAG, rep2, H3K27ac

tag: antibody


title: Pig 5, Myocardial infarction only_IZ


title: ALX1FV ALX4-, 0h dTAG, rep2, H3K27ac


title: Pig 5, Myocardial infarction only_BZ


title: ALX1FV ALX4-, 3h dTAG, rep2, input


title: ALX1FV ALX4-, 3h dTAG, rep1, H3K27ac


title: ALX1FV ALX4-, 24h dTAG, rep1, H3K27ac


title: ALX1FV ALX4-, 0h dTAG, rep1, H3K27ac


title: Pig 4, Myocardial infarction and HPAC treatment_RZ

value: HPAC


title: Pig 1, Myocardial infarction only_BZ


title: Pig 7, Myocardial infarction and HPAC treatment_IZ


title: Pig 7, Myocardial infarction and HPAC treatment_BZ


title: MSX1FNV, 3h dTAG, rep2, V5


title: MSX1FNV, 3h dTAG, rep1, V5


title: Pig 4, Myocardial infarction and HPAC treatment_IZ


title: Pig 1, Myocardial infarction only_RZ


title: Pig 7, Myocardial infarction and HPAC treatment_RZ


title: MSX1FNV, 0h dTAG, rep1, V5


title: Pig 2, Myocardial infarction and HPAC treatment_IZ


title: MSX1FNV, 3h dTAG, rep2, input


title: MSX1FNV, 24h dTAG, rep2, input


title: Pig 2, Myocardial infarction and HPAC treatment_BZ


title: Pig 1, Myocardial infarction only_IZ


title: Pig 11, control/sham_RZ

value: healthy


title: MSX1FNV, 0h dTAG, rep2, input


title: MSX1FNV, 0h dTAG, rep2, V5


title: Pig 11, control/sham_IZ


title: Pig 11, control/sham_BZ


title: Pig 10, control/sham_BZ


title: Pig 10, control/sham_RZ


title: Pig 10, control/sham_IZ


title: Pig 4, Myocardial infarction and HPAC treatment_BZ


title: ChIP anti-H3.3 – siRNA anti-PML – IFN treated

city: Lyon

first: Thomas

last: Simonet

address: Faculté de Médecine Lyon Est, 8 Avenue Rockefeller

country: France

institute: Université de Lyon

data_processing:
Sequencing and Base-calling performed by BGI
Adaptor sequence trimming and low quality base filtering using Trimmomatic SE (v0.39), with parameters : ILLUMINACLIP:TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:20
Mapping to the hg38 human genome using bwa (v0.7.17-r1188), followed by genomic coordinates sorting and indexing, with sambamba (v0.7.0)
Marking PCR duplicates, with Picard MarkDuplicates (v2.20.8)
Filtering out unmapped reads (0x4), not primary alignments (0x100), reads failing platform/vendor quality checks (0x200), PCR or optical duplicates (0x400), supplementary alignments (0x800): samtools -F 3844 (v1.9)
Broad Peak Calling using MACS2 (2.2.7.1) : macs2 callpeak --treatment ChIP.bam --control input.bam --format BAM -g hs-q 0.01 --extsize 250 --broad --broad-cutoff 0.05 --bdg –SPMR
Mapped read ends extended to 250bp, in bed format:  `bedtools bamtobed -i bam | awk '$1!~/[_\.]/' | awk 'BEGIN {OFS="\t"} { if ($6=="+") {start=$2; end=$2+250} else {start=$3-250; end=$3}; print $1,start,end,".",$5,$6 }'`
Normalized read counts (CPM) within TES of IFN dependant genes (McFarlane et al 2019, PMID: 30901352): bedtools intersect -c -sorted -g genome.chrLen.txt -a TES.bed -b SampleX.ext250.bed | awk -v norm=$total_mapped_reads 'BEGIN{OFS="\t"}{print $1,$2,$3,$4,$5,$6,$7,($7*1000000/norm)}' ; CPM from inputs subtracted from ChIP CPM.
Genome_build: hg38 human genome assembly containing Decoy sequences (https://local-run-manager.s3.amazonaws.com/genomes/Homo_sapiens_NCBI_GRCh38Decoy.zip)
Supplementary_files_format_and_content: bigwig files were generated using MACS2: macs2 bdgcmp -t treat_pileup.bdg -c control_lambda.bdg -m FE → bedClip → LC_COLLATE=C sort -k1,1 -k2,2n → bedGraphToBigWig ; Scores represent fold enrichment profiles of ChIP DNA over input DNA
Supplementary_files_format_and_content: peak files in bigbed format
Supplementary_files_format_and_content: normalized ChIP read counts at TES +/- 500bp

source_name: Human primary fibroblasts

growth_protocol: Human BJ primary foreskin fibroblasts (ATCC, CRL-2522) were cultivated in DMEM medium (Sigma-Aldrich, D6429) containing 10% of fetal calf serum (FCS) (Sigma-Aldrich, F7524), 1% of penicillin/streptomycin (Sigma-Aldrich, P4458), at 37°C under 5% CO2 and humid atmosphere.

extract_protocol:
Cells were crosslinked directly in the culture dishes by adding fixing solution (11% formaldehyde, 50mM HEPES-KOH pH 7.5, 100mM NaCl, 1mM EDTA, 0,5mM EGTA) to the medium to have 1% formaldehyde final concentration and incubated with agitation for 5 minutes at RT°C. Crosslinking was quenched by adding 125mM glycine and cells were incubated with agitation for 5 minutes at RT°C. Cells were washed with ice-cold PBS, scraped and collected in 15mL tubes. They were washed twice with ice-cold PBS and pelleted at 200g for 4 minutes at 4°C. Cell pellets were snap-frozen in liquid nitrogen and stored at -80°C before immunoprecipitation. Cells were de-frozen on ice and lysed in 1X Lysis buffer (50mM HEPES-KOH pH 7.5, 140mM NaCl, 10% Glycerol, 1mM EDTA, 0,5% NP-40, 0,25% Triton X-100) supplemented with protease inhibitors (0,2mM PMSF and 1X PIC) for 10 minutes on a rotating wheel at 4°C. Intact nuclei were pelleted by centrifugation at 1700g for 5 minutes at 4°C.  Nuclei were washed with 1X Wash buffer (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0,5mM EGTA) supplemented with protease inhibitors for 10 minutes on a rotating wheel at 4°C. Intact nuclei were pelleted by centrifugation at 1700g for 5 minutes at 4°C. Nuclei were washed twice in 1X Shearing buffer (10mM Tris-HCl pH 8.0, 1mM EDTA, 0,1% SDS) supplemented with protease inhibitors, pelleted by centrifugation at 1700g for 5 minutes at 4°C and resuspended in 1X Shearing buffer with inhibitors for sonication. We used the Covaris M220 Focused-ultrasonicator to shear through chromatin (7 minutes at 140W, Duty off 10%, Burst cycles 200). After shearing, samples were supplemented with 1% Triton X-100 and 150mM NaCl. Samples were centrifuged at maximal speed for 10 minutes at 4°C to get rid of nuclear debris. The desired mass of chromatin was aliquoted into low-retention 1,5mL tubes (Axygen, MCT-150-L-C) and volume was completed to 1mL with IP buffer (10mM Tris-HCl pH 8.0, 150mM NaCl, 1mM EDTA, 0,1% SDS, 1% Triton X-100) with protease inhibitors. A separate aliquot of chromatin was reserved as the input sample and store at -20°C. Dynabeads protein A magnetic beads (Invitrogen, 10001D) were used for immunoprecipitation with rabbit primary antibodies. Dynabeads were washed twice with ice-cold PBS and resuspended in PBS 0,5% BSA supplemented with primary antibodies (anti-H3.3 (Diagenode, C15210011), anti-panH3 (Abcam, ab1791), rabbit IgG (Diagenode, C15410206)). We used 2μg of antibody for 20μL of dynabeads. Dynabeads and antibodies were incubated on a rotating wheel for 6 hours at 4°C to allow antibody conjugation. Antibody-conjugated dynabeads were then washed twice with IP buffer and resuspended in the 1mL of chromatin. ChIP samples were incubated on a rotating wheel O/N at 4°C. Using a magnetic rack, the unbound lysates were aspirated and dynabeads were washed five times with ice-cold ChIP RIPA Buffer (50mM HEPES pH 7.5, 500mM LiCl, 1mM EDTA, 1% NP-40, 0,7% Na-deoxycholate) and once with ice-cold TE buffer (10mM Tris-HCl pH 8.0, 1mM EDTA). Chromatin was then eluted in 200uL of ChIP Elution buffer (Tris-HCl pH 8.0, 10mM EDTA, 1% SDS) for 30 minutes at 65°C with shaking and the eluates were transferred to a new tube. Reserved input samples were diluted at least 3-fold in ChIP Elution buffer. To purify DNA, ChIP eluates and input samples were decrosslinked by heating O/N at 65°C. Samples were diluted with 200μL of TE buffer and treated for 2 hours with RNase A (200g/mL) (SIGMA R6513) at 37°C followed by 2 hours with recombinant Proteinase K (200g/mL) (Thermo Scientific, 10181030) at 55°C. DNA was purified by phenol-chloroform-isoamyl alcohol extraction. The extracted aqueous phase was supplemented with 200mM of NaCl and 20μg of glycogen. DNA was precipitated with EtOH for 2 hours at -20°C and pelleted at 20 000g for 20 minutes at 4°C. DNA pellets were resuspended in ddH20 and stored at -20°C before sequencing analysis.
Library construction was made by BGI sequencing services according to their internal protocol

treatment_protocol: BJ cells were treated with the indicated siRNAs for 72h and with IFNβ at 1000U/mL for the last 24h of siRNAs treatment

value: BJ cells

tag: passages

value: H3.3 Rabbit monoclonal Antibody Diagenode #C15210011

tag: sirna

value: targeting PML

value: Interferon β

value: Human primary fibroblasts


title: Pig 2, Myocardial infarction and HPAC treatment_RZ


title: Input DNA – siRNA anti-PML – IFN treated

tag: chip antibody


title: ChIP anti-H3.3 – siRNA anti-HIRA – IFN treated

value: targeting HIRA


title: Input DNA – siRNA anti-HIRA – IFN treated


title: MSX1FNV, 3h dTAG, rep1, input

value: Human cranial neural crest cell

tag: reference genome_for_alignment

tag: reference genome_for_processed_files


title: PRRX1FV, 3h dTAG, rep2, V5

tag: cell line

tag: treatment

value: dTAGV-1 (500 nM)


title: MSX1FNV, 24h dTAG, rep1, H3K27ac

tag: cell type


title: MSX1FNV, 0h dTAG, rep2, H3K27ac


title: MSX1FNV, 0h dTAG, rep1, H3K27ac


title: PRRX1FV, 0h dTAG, rep2, V5


title: MSX1FNV, 24h dTAG, rep1, input


title: MSX1FNV, 3h dTAG, rep1, H3K27ac


title: Pig 9, control/sham_RZ

tag: tissue

value: cardiac tissue


title: ChIP anti-H3.3 – siRNA anti-Luc – IFN treated

value: targeting Luciferase


title: MSX1FNV, 0h dTAG, rep1, input


title: Pig 9, control/sham_BZ

tag: tissue region

value: wild type


title: MSX1FNV, 3h dTAG, rep2, H3K27ac


title: MSX1FNV, 24h dTAG, rep2, H3K27ac


title: Pig 9, control/sham_IZ

tag: disease state


title: PRRX1FV, 3h dTAG, rep1, V5

tag: genotype


title: PRRX1FV, 3h dTAG, rep2, input

tag: clone


title: Input DNA – siRNA anti-Luc – IFN treated


title: ChIP anti-H3.3 – siRNA anti-Luc – No treatment


title: Input DNA – siRNA anti-Luc – No treatment


title: PRRX1FV, 0h dTAG, rep2, input

tag: time


title: PRRX1FV, 3h dTAG, rep1, input

tag: spike-in cell_type


title: PRRX1FV, 0h dTAG, rep1, V5

tag: spike-in cell_line


title: N T3 RNA 2

city: Heraklion

first: Christos

last: Delidakis

address: 100 N Plastira St

country: Greece

institute: FORTH-IMBB

data_processing:
Quality control checks on raw fastq sequence data was performed by FastQC (Version 0.11.9)(1. Andrews, S. (2010), FASTQC. A quality control tool for high throughput sequence data).
Generated fastq files were aligned to the Drosophila melanogaster reference genome (Genome assembly: BDGP6.32) using STAR2 (Version 2.7.5), (https://doi.org/10.1093/bioinformatics/bts635)
Raw counts were created using the featureCounts3 (Version 2.0.1) (https://doi.org/10.1093/bioinformatics/btt656 ).
Downstream analysis of the resulted counts was performed in R4 (Version 4.0.2 (https://www.R-project.org/) using the standard DESeq25 (Version 1.30.1) package pipeline (5. Love, M.I., Huber, W., Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology 15(12):550 (2014)).
dm6
Supplementary files format and content: supplementary files contain Matrix tables with  Fold change(FC), log2FC, p value and p.adj for zll genes for the indicated comparisons of conditions

organism: Drosophila melanogaster

extract_protocol:
150 larval brains per replica carrying GFP positive actin>F/O clones overexpressing UAS-NΔecd were hand dissected, dissociated with collagenase and GFP positive cells were purified by FACS sorting and stored in Trizol. In parallel, larval brains of the same genotype were allografted into adult w1118 host flies and grown for 8-10 days. 25 T0 host flies were sacrificed per replica and the GFP tumour masses were collected in Trizol. Some tumour masses were used to generate more advanced allograft stages. In the forth allograft stage (T3), 25 host flies were again sacrificed per replica 6-8 days post transplantation and GFP tumour masses were stored in Trizol.  RNA was extracted according to standard protocols.
NGS libraries were generated using total RNA as input with polyA mRNA magnetic isolation kit (NEB) and the NEB Ultra II RNA library kit for Illumina kit according to manufactures protocol, using 13 cycles of amplification. Libraries were sequenced on Illumina Nextseq 500 on 1 x 75 High flowcell.

value: 4th allograft Ν tumour (T3) from w1118 adult hosts

value: Allograft tumour T3

value: Τ3 allograft Ν tumours grown for 6-8 days post 4rth transplantation in w1118 adult host flies


title: N T3 RNA 4


title: PRRX1FV, 0h dTAG, rep1, input

tag: antibody


title: PRRX1FV, 24h dTAG, rep1, input


title: PRRX1FV, 24h dTAG, rep2, H3K27ac

value: H3K27ac (Active Motif #39133)


title: PRRX1FV, 24h dTAG, rep2, input


title: N T3 RNA 1


title: N T0 RNA 4

value: 1st allograft Ν tumour (T0) from w1118 adult hosts

value: Allograft tumour T0

value: Τ0 allograft Ν tumours grown for 8-10 days post 1st transplantation in w1118 adult host flies


title: N T0 RNA 3


title: N FACs RNA4

value: larval CNS(FACs sorted GFP positive Ν cells)

value: Larval CNS

value: 3rd instar larvae, 4 days post actinF/O>Ν clone induction


title: N FACs RNA1


title: N FACs RNA2


title: PRRX1FV, 3h dTAG, rep2, H3K27ac


title: PRRX1FV, 0h dTAG, rep2, H3K27ac


title: ALX1FV, 3h dTAG, rep1, V5


title: NPC_KD-3

city: Shanghai

first: Xianhua

last: Chen

address: 131 Dongan road,New Research Building, Room B5015

country: China

institute: Fudan University

data_processing:
Reads filtering under criteria removing reads with 20% of the base quality lower than 13
HISAT2.1.0 mapping to reference genome
count counting
Assembly: mm10
Supplementary files format and content: TXT file contain counts for each sample

source_name: cortex

growth_protocol: The neural progenitor cells were cultured in DMEM/F12 medium containing 20 ng/μl bFGF, 20 ng/μl EGF, 5 μg/mL heparin, B27 supplement, GlutaMax, and penicillin/streptomycin.

extract_protocol:
Total RNA was extracted from cell or animal tissue by Trizol reagent (Invitrogen) separately, The RNA quality was checked by Bioanalyzer 2200 (Aligent) and kept at -80℃.The RNA with RIN >8.0 is right for cDNA library construction.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: The suspension cultures of neural progenitor cells were infected with lentivirus for SRSF10 knockdown. 12 h after cell inoculation, the culture mediums were replaced by the mixtures of lentivirus, infection reagents and medium for lentivirus infection with MOI of 30. 24h after lentivirus infection, the medium containing lentivirus was replaced by fresh lentivirus-free ones. Follow-up RNA extractions were performed 72 h after infection. At least 10^6 cells were used for RNA extraction for each sample.

tag: strain

value: cortex

value: primary cultured cell

value: neural progenitor cells

value: SRSF10 knockdown


title: ALX1FV, 0h dTAG, rep2, input

value: Human cranial neural crest cell

value: dTAGV-1 (500 nM)


title: NPC_KD-2


title: ALX1FV, 3h dTAG, rep2, V5


title: ALX1FV, 0h dTAG, rep2, V5


title: NPC_KD-1


title: PRRX1FV, 0h dTAG, rep1, H3K27ac


title: ALX1FV, 3h dTAG, rep2, input


title: PRRX1FV, 3h dTAG, rep1, H3K27ac


title: ALX1FV, 24h dTAG, rep2, input


title: NPC_Ctrl-3

value: WT


title: NPC_Ctrl-1


title: NPC_Ctrl-2


title: cKO-2

value: Srsf10 flox /flox Nestin-Cre+


title: PRRX1FV, 24h dTAG, rep1, H3K27ac


title: ALX1FV, 24h dTAG, rep1, input


title: N T0 RNA 2


title: ALX1FV, 24h dTAG, rep2, H3K27ac


title: ALX1FV, 0h dTAG, rep1, input


title: ALX1FV, 3h dTAG, rep1, input


title: cKO-1


title: ALX1FV, 0h dTAG, rep1, V5


title: ALX1FV, 3h dTAG, rep2, H3K27ac


title: Ctrl-2

value: Srsf10 flox /flox Nestin-Cre-


title: Ctrl-1


title: Exp3 POI mouse ovary + BM-Exo 1X #3

city: Chicago

first: Hang-soo

last: Park

state: IL

address: 5821 S Drexel Ave

institute: University of Chicago

data_processing:
Quasi-mapping and quantification: salmon 1.5.2
Assembly: GRCm38.primary_GencodeM25
Supplementary files format and content: gene level raw abdunance in TSV format

source_name: Ovary

growth_protocol: C57/BL6 mice were purchased from charles river. For POI disease model, female C57BL/6 mice received an intraperitoneal injection of busulfan (30 mg/kg) and cyclophosphamide (120 mg/kg).

extract_protocol:
RNA isolation was performed using an RNeaxy Micro kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The RNA concentration was quantiﬁed by spectrophotometry at 260 nm using a Nanodrop 2000 (Thermo Fisher Scientiﬁc). Approximately 1 µg to 1.5 µg of RNA sample was sent to the core facility (University of Chicago Genomics Facility) for further processing.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: After 7 days of chemotherapy, hBM-MSCs were transplanted into the ovary via intravenous injection. All mice were anesthetized using 1-4% isoflurane inhalation. A total of 1×104 to 1×106 hBM-MSCs were resuspended in 100 µl of PBS and injected through the retro-orbital sinus. For exosome injection, 1.5×107 to 1.5×109 exosomal particles were resuspended in 100 µl of PBS and injected through the same route. For the control and untreated POI groups, each mouse was injected with 100 µl of PBS through intravenous injection.

value: Ovary

tag: disease

value: POI

value: MSC derived exosome


title: Exp3 POI mouse ovary + BM-Exo 1X #2


title: Exp3 POI mouse ovary + BM-Exo 10X #3


title: ALX1FV, 3h dTAG, rep1, H3K27ac

value: H3K27ac (Active Motif #39133)


title: cKO-3


title: Exp3 POI mouse ovary + BM-Exo 10X #2


title: Exp3 POI mouse ovary + BM-Exo 10X #1


title: Exp3 POI mouse ovary + BM-Exo 100X #3


title: Exp3 POI mouse ovary + BM-Exo 100X #1


title: Exp3 POI mouse ovary + BM-Exo 100X #2


title: Exp3 POI mouse ovary #1

value: saline


title: Exp3 Control mouse ovary #3

value: healthy


title: WT, long-term DMSO, rep2

data_processing:
Reads were trimmed of Nextera adapter sequences and low-quality bases (-Q 10) using skewer v0.2.2.
Trimmed reads were mapped to the hg38 analysis set reference genome using Bowtie2 v2.4.1 with the options --very-sensitive -X 2000.
Reads were deduplicated with samtools v1.10 markdup and uniquely mapped reads (-q 20) mapped to the main chromosomes (excluding mitochondria and unplaced contigs) were retained using samtools view.
Read ends were shifted inward 5 bp (+5 bp on + strand, -5 bp on - strand) for each fragment.
MACS2 v2.2.7.1 was used to call peaks and generate coverage tracks using the IgG sample as a negative control, with options --shift -100 --extsize 200 -f BED --nomodel --keep-dup all --call-summits -g hs --SPMR.
bedgraph tracks from MACS2 were converted to bigWig format using UCSC bedGraphToBigWig
Reads were counted over merged peaks using bedtools intersect.
Assembly: hg38
Supplementary files format and content: bigWig, narrowPeak for each sample, tab-delimited text files (.txt) with counts of samples for each comparison (across all replicates), and bed files with coordinates of merged peaks or the top summit from each merged peak with the -log10(pvalue) from MACS2

extract_protocol:
DNase-treated cells were harvested by Accutase, counted using the Countess II (Invitrogen), and 50,000 cells were collected by centrifugation at 500g for 5 min at 4°C. Cells were resuspended in lysis buffer (resuspension buffer (RSB), or 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2, with 0.1% Igepal CA-630, 0.1% Tween-20, and 0.01% digitonin) for 3 min, then quenched by dilution with RSB with 0.1% Tween-20. Lysate was centrifuged for 10 min at 500g at 4°C and then resuspended in transposition buffer (25 ul TD buffer and 2.5 ul TD enzyme (Illumina, 20034197), 16.5 ul PBS, 0.01% digitonin, 0.1% Tween-20, and water up to 50 ul). Transposition reactions were performed for 30 min at 37°C, and then cleaned up with the DNA Clean & Concentrator-5 kit (Zymo, D4013) and eluted in 21 ul 10 mM Tris-HCl pH 8.
Transposed DNA was pre-amplified 5 cycles with NEBNext Ultra II Q5 master mix (New England Biolabs, M0544) with a cycling protocol of 72°C for 5 min, 98°C for 30s, and 5 cycles of 98°C for 10s, 63°C for 30s, 72°C for 1 min. Then 5 ul of the 50 ul reaction was used to run a qPCR reaction (with the same cycling protocol except the initial 72°C incubation) to determine the optimal number of PCR cycles for each sample. The remaining portion of the reaction was then amplified the appropriate number of cycles, and then subjected to two rounds of double-sided Ampure XP bead cleanup, with 0.5x/1.3x and 0.5x/1.0x bead ratios (numbers indicate bead ratios added in first and second steps). Libraries were quantified by Qubit dsDNA high sensitivity assay (Invitrogen, Q33231), run on a 5% polyacrylamide TBE gel to check the size distribution, and then pooled for sequencing.

treatment_protocol: DMSO or dTAGV-1 (500 nM) were added by dilution into media before changing media. Cells were treated for 30 min with 200 U/ml DNase I (Worthington, LS006331) at 37°C prior to harvesting cells.

value: Human cranial neural crest cells

value: long-term


title: WT, long-term dTAG, rep2


title: WT, long-term dTAG, rep3


title: WT, long-term dTAG, rep1


title: ALX1FV ALX4-, long-term dTAG, rep2

value: ALX1FV ALX4-


title: ALX1FV ALX4-, long-term dTAG, rep3


title: ALX1FV ALX4-, long-term DMSO, rep3


title: ALX1FV ALX4-, long-term dTAG, rep1


title: ALX1FV ALX4-, long-term DMSO, rep2


title: ALX1FV, 0h dTAG, rep2, H3K27ac


title: Exp3 Control mouse ovary #2


title: WT, long-term DMSO, rep1


title: Exp3 Control mouse ovary #1


title: Ctrl-3

value: cortex


title: ALX1FV, 24h dTAG, rep1, H3K27ac


title: ALX1FV, 0h dTAG, rep1, H3K27ac


title: ALX1FV ALX4-, long-term DMSO, rep1


title: MSX1FNV, long-term dTAG, rep3


title: Exp3 POI mouse ovary + BM-Exo 1X #1


title: Exp2 POI mouse ovary + UC-Exo 1X #3


title: WT, long-term DMSO, rep3


title: Exp2 POI mouse ovary + UC-Exo 1X #1


title: Exp2 POI mouse ovary + UC-Exo 1X #2


title: Exp2 POI mouse ovary + UC-Exo 10X #3


title: MSX1FNV, long-term dTAG, rep2


title: MSX1FNV, long-term DMSO, rep2


title: MSX1FNV, long-term dTAG, rep1


title: Exp2 POI mouse ovary + UC-Exo 10X #2


title: Exp2 POI mouse ovary + UC-Exo 100X #2


title: MSX1FNV, long-term DMSO, rep1


title: Exp2 POI mouse ovary + UC-Exo 100X #3


title: Exp3 POI mouse ovary #2


title: PRRX1FV, long-term DMSO, rep2


title: PRRX1FV, long-term dTAG, rep2


title: PRRX1FV, long-term DMSO, rep3


title: PRRX1FV, long-term dTAG, rep1


title: Exp2 POI mouse ovary #1


title: ALX1FV, long-term dTAG, rep3


title: PRRX1FV, long-term dTAG, rep3


title: PRRX1FV, long-term DMSO, rep1


title: Exp2 POI mouse ovary #2


title: ALX1FV, long-term dTAG, rep2


title: Exp2 POI mouse ovary + UC-Exo 100X #1


title: MSX1FNV, long-term DMSO, rep3


title: ALX1FV, long-term DMSO, rep3


title: Exp2 Control mouse ovary #2


title: ALX1FV, long-term DMSO, rep2


title: Exp1 POI mouse ovary + MSC 1X #3

value: MSC


title: Exp1 POI mouse ovary + MSC 1X #2


title: ALX1FV, long-term dTAG, rep1


title: ALX1FV, long-term DMSO, rep1


title: Exp2 POI mouse ovary + UC-Exo 10X #1


title: Exp1 POI mouse ovary #3


title: Exp1 POI mouse ovary + MSC 1X #1


title: TWIST1FV, long-term dTAG, rep2


title: TWIST1FV, long-term dTAG, rep3


title: Exp1 POI mouse ovary #2


title: Exp2 Control mouse ovary #3


title: Exp1 Control mouse ovary #3


title: Exp1 POI mouse ovary #1


title: Exp2 POI mouse ovary #3


title: TWIST1FV, long-term DMSO, rep1


title: Exp1 Control mouse ovary #2


title: Exp1 Control mouse ovary #1


title: ALX1FV ALX4-, 24h dTAG, rep3


title: TWIST1FV, long-term dTAG, rep1


title: Exp2 Control mouse ovary #1


title: ALX1FV ALX4-, 3h dTAG, rep3


title: Stem-like Lewis cells, RT-MPs, 48 h, rep1

city: Wuhan, China

first: Xiao

last: Yang

state: 湖北省

address: 武汉市江汉区马场路109号协和医院肿瘤中心

institute: Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

data_processing:
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website (http:// ) directly. We selected Hisat2 as the mapping tool
The number of perfect clean tags for each gene was calculated and then normalized in fragments per kilo bases per million mapped Reads (FPKM) using the described method.
Assembly: mm10
Supplementary files format and content: FPKM

source_name: Lewis

growth_protocol: Lewis cells were maintained in DMEM supplemented with 2% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
Total RNA was extracted with Trizol (Invitrogen)
RNA sequencing libraries were generated using the KAPA Stranded mRNA-Seq Kit for illumina with multiplexing primers, according to the manufacturer’s protocol.

treatment_protocol: Stem-like Lewis cells were untreated or treated with RT-MPs for 48 h.

value: Lewis

value: Lung Carcinoma Cell Lines

value: RT-MPs


title: ALX1FV ALX4-, 0h dTAG, rep3

value: dTAGV-1 (500 nM)


title: ALX1FV ALX4-, 24h dTAG, rep2


title: ALX1FV ALX4-, 3h dTAG, rep2


title: Stem-like Lewis cells, RT-MPs, 48 h, rep3

address: 武汉市江汉区马场路109号协和医院肿瘤中心

value: WT


title: TWIST1FV, long-term DMSO, rep3


title: Stem-like Lewis cells, Control, 48 h, rep3

address: 武汉市江汉区马场路109号协和医院肿瘤中心

value: Control


title: ALX1FV ALX4-, 24h dTAG, rep1


title: ALX1FV ALX4-, 0h dTAG, rep2


title: TWIST1FV, long-term DMSO, rep2


title: Stem-like Lewis cells, Control, 48 h, rep2

address: 武汉市江汉区马场路109号协和医院肿瘤中心


title: ALX1FV ALX4-, 0h dTAG, rep1


title: mixed sample of medulloblastoma, CT-179-treated, POx-resiquimod-treated, untreated control

city: Atlanta

first: Timothy

last: Gershon

state: Georgia

address: 201 Dowman Dr

institute: Emory University

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation performed using the Parse proprietary software.
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: medulloblastoma

extract_protocol:
Brains were freshly harvested from treated and control mice, and then cut in half sagittally. One half was then drop-fixed in 4% paraformaldehyde for IHC, and tumor was dissected free from the other half. Dissected tumors were then processed for split-seq analysis using the Parse WT 1.0 kit. For processing, tumors were dissociated using the Papain Dissociation System (Worthington Biochemical) according to the manufacturer’s protocol, as in our prior studies, https://www.nature.com/articles/s41467-019-13657-6#Sec18. Cells were then counted and fixed using the Parse WT 1.0 Fixation Kit, per manufacturer’s instructions.
For library construction, fixed cells were plated out in the Parse WT 1.0 bar coding kit, according to manufacturer's protocol. A total of 8 sublibraries were generated and validated by Tapestation. Sequencing was then performed on an estimated 60,000 total cells across the three conditions.

value: heterogeneous

value: medulloblastoma

tag: age

value: CT-179 -or- POx-resiquimod -or- untreated


title: ALX1FV ALX4-, 3h dTAG, rep1


title: Stem-like Lewis cells, Control, 48 h, rep1

address: 武汉市江汉区马场路109号协和医院肿瘤中心


title: 8_M_PLAAT3_aff

city: Ghent

first: Bart

last: Dermaut

state: 10kb upstream, TSS, 10kb downstream

address: C. Heymanslaan 10

country: Belgium

institute: Ghent University

data_processing:
Adapter Trimming (Trimmomatic v0.38): $ java -jar trimmomatic-0.38.jar PE -phred33 -threads [threads] [R1 file] [R2 file] [R1 trim output] [R1 unpair output] [R2 trim output] [R2 unpair output] ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10:5:true LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
Alignment (HISAT2, v2.1.0): $ hisat2 -p [threads] --dta --rna-strandness RF -x [HISAT2 Index] -1 [R1 Processed Read] -2 [R2 Processed Read] -S [Output SAM]
Align Post-processing (SAMtools v1.9): $ samtools view -@ [threads] -bh [SAM file] > [BAM file], $ samtools sort -@ [threads] -o [POS-Sorted BAM] [BAM file]
Expression Profiling (stringtie 2.1.3b): $ stringtie [POS-Sorted BAM] -p [threads] --rf -e -l [Output prefix] -o [Output Dir] -G [annotation GTF] -A [gene abundance estimation output file]
Data filtering steps: gene : To exclude gene with at least one zero Counts, sample : No sample filtering.
Assembly: mm10
Supplementary files format and content: Excel file, contains gene, transcript and protein IDs, read counts/normalized read counts for each sample

source_name: inguinal white adipose tissue

extract_protocol:
Trizol Tissue RNA Extraction protocol - QIAzol® Lysis Reagent (Cat. No. 79306), RNeasy® Mini Kit (Cat. No. 74106), Tungsten Carbide Beads  (Cat. No. 69997)
Total RNA concentration was calculated by Quant-IT RiboGreen (Invitrogen, #R11490). To assess the integrity of the total RNA, samples are run on the TapeStation RNA screentape (Agilent, #5067-5576). Only high-quality RNA preparations, with RIN greater than 7.0, were used for RNA library construction.  A library was independently prepared with 1ug of total RNA for each sample by Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA, #RS-122-2101). The first step in the workflow involves purifying the poly‐A containing mRNA molecules using poly‐T‐attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using SuperScript II reverse transcriptase (Invitrogen, #18064014) and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I, RNase H and dUTP. These cDNA fragments then go through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. The products are then purified and enriched with PCR to create the final cDNA library.  The libraries were quantified using KAPA Library Quantificatoin kits for Illumina Sequecing platforms according to the qPCR Quantification Protocol Guide (KAPA BIOSYSTEMS, #KK4854) and qualified using the TapeStation D1000 ScreenTape (Agilent Technologies, # 5067-5582). Indexed libraries were then submitted to an Illumina NovaSeq (Illumina, Inc., San Diego, CA, USA), and the paired-end (2×100 bp) sequencing was performed by the Macrogen Incorporated.

value: inguinal white adipose tissue

tag: Sex

value: male


title: 9_M_PLAAT3_aff


title: 6_M_PLAAT3_cntr


title: MSX1FNV, 24h dTAG, rep2


title: 3_M_PLAAT3_cntr


title: MSX1FNV, 3h dTAG, rep2


title: 4_M_PLAAT3_cntr


title: Stem-like Lewis cells, RT-MPs, 48 h, rep2

address: 武汉市江汉区马场路109号协和医院肿瘤中心


title: 2_M_PLAAT3_cntr


title: 24_F_PLAAT3_aff

value: female


title: 23_F_PLAAT3_aff


title: 21_F_PLAAT3_aff


title: 19_F_PLAAT3_aff


title: PRRX1FV, 24h dTAG, rep2

value: Human cranial neural crest cells


title: MSX1FNV, 0h dTAG, rep1


title: PRRX1FV, 0h dTAG, rep2


title: PRRX1FV, 3h dTAG, rep2


title: PRRX1FV, 24h dTAG, rep1


title: PRRX1FV, 3h dTAG, rep1


title: 16_F_PLAAT3_cnt


title: MSX1FNV, 0h dTAG, rep2


title: PRRX1FV, 0h dTAG, rep1


title: 15_F_PLAAT3_cnt


title: MSX1FNV, 3h dTAG, rep1


title: ALX1FV, 3h dTAG, rep3


title: ALX1FV, 24h dTAG, rep3


title: 13_F_PLAAT3_cnt


title: ALX1FV, 0h dTAG, rep3


title: ALX1FV, 3h dTAG, rep2


title: ALX1FV, 24h dTAG, rep2


title: ALX1FV, 24h dTAG, rep1


title: ALX1FV, 3h dTAG, rep1


title: ALX1FV, 0h dTAG, rep2


title: 17_F_PLAAT3_cnt


title: ALX1FV, 0h dTAG, rep1


title: 11_M_PLAAT3_aff


title: MSX1FNV, 24h dTAG, rep1


title: WT, 24h dTAG, rep2


title: WT, 3h dTAG, rep2


title: WT, 24h dTAG, rep1


title: WT, 3h dTAG, rep1


title: WT, 0h dTAG, rep2


title: TWIST1FV, 24h dTAG, rep2


title: Sorted PDA MHCII-high maorphage, IFN gamma KO

city: Minneapolis

first: Jesse

middle: W

last: Williams

state: MN

address: 2101 6th St. SE

department: Dept. Integrative Biology & Physiology

institute: University of Minnesota

data_processing:
Alignment, mapping and counting were performed using the CHURP pipeline
Assembly: mm10
Supplementary files format and content: tab-delimited text files containing read count

source_name: pancrease primary tumor

extract_protocol:
Macrophages were sorted by CD45+CD11b+F4/80+. Cells were lysed using Trizol. RNA isolation was performed by the University of Minnesota Genomic Core using the Qiagen RNeasy kit.
Library was constructed by the University of Minnesota Genomic Core using the TruSeq RNA Library Prep Kit.

value: pancrease primary tumor

value: macrophage

value: INFgKO


title: Sorted PDA MHCII-low maorphage, IFN gamma WT

value: INFgWT


title: PDA, no treatment, day7, AHTO

description: HTO

library_source: other

data_processing:
10x formated output files for GEX were generated using cellranger
10x formated output files for HTO and ADT were generated using CITE-seq-Count
Further analysis was performed using Seurat
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files
Supplementary files format and content: 10x Genomics output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz

molecule: protein

extract_protocol:
Single-cell dissociated tissue was CD45-sorted to only include immune cells. TdTomato+ and TdTomato- immune cells were labeled using Hashtag9 and Hashtag10. All cells were labeled by CITE-seq antibodies.
Library was generated according to Chromium Single Cell 3ʹ v3 protocol (10x Genomics).

tag: timepoint

tag: antibodies/tags

value: Anti-mouse Hashtag9/TotalSeq-A0309, Anti-mouse Hashtag10/TotalSeq-A0310


title: PDA, no treatment, day7, ADT

description: ADT

tag: treatment

value: Anti-CD274/TotalSeq-A0190, Anti-F4-80/TotalSeq-A0114, Anti-IA-IE/TotalSeq-A0117, Anti-CD11b/TotalSeq-A0014, Anti-FRb/TotalSeq-A0564, Anti-CD4/TotalSeq-A0001, Anti-CD8a/TotalSeq-A0002, Anti-Ly6C/TotalSeq-A0013, Anti-Ly6G/TotalSeq-A0015


title: Sorted PDA MHCII-low maorphage, IFN gamma KO

tag: cell type


title: PDA, antiCD4, day7, GEX

description: GEX

tag: tissue


title: PDA, no treatment, day3, GEX

description: GEX


title: Sorted PDA MHCII-high maorphage, IFN gamma WT


title: PDA, antiCD4, day7, ADT

description: ADT


title: PDA, antiCD4, day7, HTO

description: HTO


title: PDA, no treatment, day3, HTO

description: HTO


title: PDA, no treatment, day7, GEX

description: GEX


title: WT, 0h dTAG, rep1

tag: cell line


title: E10.5 HL, input

source_name: Mouse E10.5 hindlimb buds

value: Mouse E10.5 hindlimb buds

tag: reference genome_for_alignment

tag: reference genome_for_processed_files


title: E10.5 FL, input

source_name: Mouse E10.5 forelimb buds

value: Mouse E10.5 forelimb buds

tag: genotype

tag: clone


title: TWIST1FV, 3h dTAG, rep2

value: dTAGV-1 (500 nM)

tag: time


title: TWIST1FV, 0h dTAG, rep2


title: PDA, no treatment, day3, ADT

description: ADT


title: TWIST1FV, 24h dTAG, rep1


title: E10.5 Md, input

source_name: Mouse E10.5 mandibular prominences

value: Mouse E10.5 mandibular prominences

value: WT

tag: spike-in cell_type

tag: spike-in cell_line


title: E10.5 Mx, input

source_name: Mouse E10.5 maxillary prominences

value: Mouse E10.5 maxillary prominences

tag: antibody


title: E10.5 FNP, input

source_name: Mouse E10.5 frontonasal prominences

value: Mouse E10.5 frontonasal prominences


title: TWIST1FV, 0h dTAG, rep1


title: E10.5 HL, Twist1

value: TWIST1 (Abcam, #ab50887)


title: E10.5 FL, Twist1


title: E10.5 Md, Twist1