title: 10x multiome (RNA) on the mouse liver using the NST protocol

data_processing:
The generated fastq files were processed with cellranger-arc (v1.0.0) count function, with include introns =True option. Reads were aligned to Mus musculus reference genome (ata-cellranger-arc-mm10-2020-A-2.0.0).
10x snRNA-seq and 10x multiome (gene expression) runs were analyzed first independently using VSN-pipelines (v0.27.0). Briefly, cells with at least 350 genes expressed and a percentage of mitochondrial reads below 10% were kept. Scanpy (v1.8.2) was run with default parameters, using the number of principal components automatically selected by VSN-Pipelines and using Leiden clustering with resolutions 0.4, 0.6 and 0.8. Hepatocyte clusters with low gene expression and high percentage of mitochondrial reads were removed, as well as doublets called with Scrublet (v0.2.3) [REF]. The samples were merged, obtaining 29,798 high-quality cells, and reanalyzed with VSN-Pipelines. 10x scATAC-seq samples were processed with cisTopic,(v0.3.0) using the cells called by cellRanger (5,628 cells) and mm10 SCREEN regions (1,212,823 regions). For topic modelling, we used warpLDA with default parameters, using 500 iterations and inferring models with 2, 5, 10 to 30 (by a step of 1), 35, 40, 45 and 50. This resulted in a model with 19 topics. After correcting sample effects with harmony (v1.0, applied on the scaled topic distributions), we performed Leiden clustering with resolution 0.6, obtaining 11 clusters. The labelled 10x scATAC-seq and multiome cells (annotated based on the transcriptome labels) and the scATAC-seq fragments were used as input for pycisTopic (v1.0.1.dev75+g3d3b721). Briefly, we first created pseudobulks per cell type and performed peak calling using MACS2 (v2.2.7.1, with –format BEDPE –keep-dup all –shift 73 –ext_size 146 as parameters, as recommended for single-cell ATAC-seq data). To derive a set of consensus peaks, we used the iterative overlap peak merging procedure describe in Corces et al. (2018). This resulted in 486,888 regions. We further filtered the data set based on the scATAC-seq quality as well, keeping cells with at least 1,000 fragments, FRiP > 0.4 and TSS enrichment > 7, resulting in 22,600 high-quality cells.
Assembly: mm10
Supplementary files format and content: TEW__043783__523d9c__Multiome_Liver_10xprotocol_fragments.tsv: Tab-separated text file containing chromosome, start, end, cell barcode and counts for each read for the sample in which the 10x protocol was used.
Supplementary files format and content: TEW__ebb273__b33e6f__Multiome_Liver_CTRL_NSTprotocol_fragments.tsv: Tab-separated text file containing chromosome, start, end, cell barcode and counts for each read for the sample in which the NST protocol was used.
Supplementary files format and content: multiome_atac_counts.tsv: Tab-separated flat text file containing regions as rows, cells as columns and fragment counts in each region for each cell as values.
Supplementary files format and content: multiome_rna_counts.tsv: Tab-separated flat text file containing genes as rows, cells as columns and counts for each gene in each cell as values.

source_name: Liver (P56)

extract_protocol:
For the “Multiome-10x_Fresh_Mouse-4” sample we used a modified protocol from the Nuclei Isolation from Complex Tissues for Single Cell Multiome ATAC + Gene Expression Sequencing Protocol (CG000375) from 10x Genomics. Briefly, 100 mg of fresh mouse liver tissue was chopped and transferred to a Dounce homogenizer cylinder containing 1 mL of ice-cold homogenization buffer (10 mM NaCl, 10 mM Tris 7.4, 3 mM MgCl2, 0,1% IGEPAL CA-63, 1 mM DTT, 1 U/µL of Protector RNase inhibitor (Sigma)). The tissue was homogenized with 5 strokes of pestle A and 10 strokes of pestle B until a homogeneous nuclei suspension was achieved. The resulting homogenate was filtered through a 70-μm cell strainer (Corning). The homogenizer and the filter were rinsed with an additional 0.5 μL of homogenization buffer. The tissue material was spun down at 500 x g for 5 min and the supernatant was discarded. The tissue pellet was resuspended in wash buffer (1% BSA in PBS + 1 U/µlL of Protector RNase inhibitor (Sigma)). Nuclei were stained with 7AAD (Thermo Fisher Scientific) and viability sorted on a BD FACS Fusion into 5 mL low bind Eppendorf tube containing BSA with RNase inhibitor. The sorted nuclei were spun down at 500 x g for 5 min and the supernatant was discarded. Next, the nuclei were permeabilized by resuspending the pellet in 0.1x lysis buffer (10 mM NaCl, 10 mM Tris 7.4, 3 mM MgCl2, 0.1% IGEPAL CA-63, 0.01% Digitonin, 1% BSA, 1 mM DTT, 1 U/µL of Protector RNase inhibitor (Sigma)) and incubated on ice for 2 min. 1 mL Wash Buffer (10 mM NaCl, 10 mM Tris 7.4, 3 mM MgCl2, 0.1% Tween-20, 1% BSA, 1 mM DTT, 1 U/µL of Protector RNase inhibitor (Sigma)) was added. The nuclei were spun down at 500 x g for 5 min and the supernatant was discarded. The nuclei pellet was resuspended in diluted nuclei buffer (1x Nuclei buffer Multiome kit (10x Genomics)), 1 mM DTT, 1 U/µL of Protector RNase inhibitor (Sigma)). For the “Multiome-NST_Fresh_Mouse-5” sample nuclei isolation we used a modified protocol from the Slyper et al. (2020). Briefly, 100 mg of fresh mouse liver tissue was chopped and transferred to a Dounce homogenizer cylinder containing 1 mL of ice-cold homogenization buffer (Salt-tris solution - 146 mM NaCl, 10 mM Tris 7.5, 1 mM CaCl2, 21 mM MgCl2, 0.2% IGEPAL CA-63, 0.01% BSA, 0.2 U/µL of Protector RNase inhibitor (Sigma)). The tissue was homogenized with 5 strokes of pestle A and 10 strokes of pestle B until a homogeneous nuclei suspension was achieved. The resulting homogenate was filtered through a 70-μm cell strainer (Corning). The homogenizer and the filter were rinsed with an additional 1 mL of homogenization buffer and 3 mL salt-tris solution buffer (146 mM NaCl, 10 mM Tris 7.5, 1 mM CaCl2, 21 mM MgCl2). The tissue material was spun down at 500 x g for 5 min. The obtained pellet, after supernatant removal, was resuspended in 1.5 mL salt-tris solution buffer with 0.2 U/µL RNasin Plus RNase Inhibitor (Promega). The tissue material was spun down at 500 x g for 5 min. The obtained pellet, after supernatant removal, was resuspended in 1.5 mL wash buffer (1x PBS, 1% BSA and 0.2U/μL RNasin Plus RNase Inhibitor (Promega)). The wash step was repeated one more time. The final pellet was resuspended in 500 µL wash buffer, filtered, stained with DAPI (Thermo Fisher Scientific) and viability sorted on a BD FACS Fusion into 5 mL low bind Eppendorf tube containing BSA with RNase inhibitor. The sorted nuclei were spun down at 500 x g for 5 min and the supernatant was discarded. The nuclei pellet was resuspended in diluted nuclei buffer (1x Nuclei buffer Multiome kit (10x Genomics)), 1 mM DTT, 1 U/µL RNasin Plus RNase Inhibitor (Promega)). 9 μL of sample was mixed with 1 μL of arginine orange/propidium iodide (AO/PI) stain, loaded onto a LUNA-FL slide and visualized with the LUNA-FL Automated cell counter for nuclei yield, morphology and presence of clumps/debris.
Single-nuclei libraries were generated using the 10x Chromium Single-Cell Instrument and NextGEM Single Cell Multiome ATAC + Gene Expression kit (10x Genomics) according to the manufacturer’s protocol. In brief, the single mouse liver nuclei were incubated for 60 min at 37°C with a transposase that fragments the DNA in open regions of the chromatin and adds adapter sequences to the ends of the DNA fragments. After generation of nanolitre-scale gel bead-in-emulsions (GEMs), GEMs were incubated in a C1000 Touch Thermal Cycler (Bio-Rad) under the following program: 37°C for 45 min, 25°C for 30 min and hold at 4°C. Incubation of the GEMs produced 10x barcoded DNA from the transposed DNA (for ATAC) and 10x barcoded, full-length cDNA from poly-adenylated mRNA (for GEX). Next quenching reagent (Multiome 10x kit) was used to stop the reaction. After quenching, single-cell droplets were dissolved, and the transposed DNA and full-length cDNA were isolated using Cleanup Mix containing Silane Dynabeads. To fill gaps and generate sufficient mass for library construction, the transposed DNA and cDNA were amplified via PCR: 72°C for 5 min; 98°C for 3 min; 7 cycles of 98°C for 20 s, 63°C for 30 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. The pre-amplified product was used as input for both ATAC library construction and cDNA amplification for gene expression library construction. Illumina P7 sequence and a sample index were added to the single-strand DNA during ATAC library construction via PCR: 98°C for 45 s; 7-9 cycles of 98°C for 20 s, 67°C for 30 s, 72°C for 20 s; 72°C for 1 min; and hold at 4°C. The sequencing-ready ATAC-seq library was cleaned up with SPRIselect beads (Beckman Coulter). Barcoded, full-length pre-amplified cDNA was further amplified via PCR: 98°C for 3 min; 6-9 cycles of 98°C for 15 s, 63°C for 20 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. Subsequently, the amplified cDNA was fragmented, end- repaired, A-tailed and index adaptor ligated, with SPRIselect cleanup in between steps. The final gene expression library was amplified by PCR: 98°C for 45 s; 5-16 cycles of 98°C for 20 s, 54°C for 30 s, 72°C for 20 s. 72°C for 1 min; and hold at 4°C. The sequencing-ready GEX library was cleaned up with SPRIselect beads. Before sequencing, the fragment size of every library was analyzed using the Bioanalyzer high-sensitivity chip. All 10x Multiome ATAC libraries were sequenced on NovaSeq6000 instruments (Illumina) with the following sequencing parameters: 50 bp read 1 – 8 bp index 1 (i7) – 16 bp index 2 (i5) - 49 bp read 2. All 10x Multiome GEX libraries were sequenced on NovaSeq6000 instruments with the following sequencing parameters: 28 bp read 1 – 10 bp index 1 (i7) – 10 bp index 2 (i5) – 75 bp read 2.

value: Liver (P56)


title: Differentiated cells, CHIR+RA, Bio rep 1 [CR1]

status: Public on Sep 07 2023

type: SRA

city: Shanghai

first: Ting

last: Xu

address: 1239 Siping Road

country: China

institute: Tongji University

library_source: transcriptomic

data_processing:
The raw paired end reads were trimmed and quality controlled by SeqPrep and Sickle  with default parameters.
Then clean reads were separately aligned to reference genome with orientation mode using HISAT2.
The mapped reads of each sample were assembled by StringTie in a reference-based approach
Assembly: homo_sapiens GRCh38 V101
Supplementary files format and content: tab-delimited text files includeTPM values for each Sample

molecule: total RNA

organism: Homo sapiens

extract_protocol:
Total RNA was extracted from the cells using TRIzol® Reagent, about  1ug of total RNA was used for the construction of sequencing libraries.
RNA-seq transcriptome librariy was prepared following TruSeqTM RNA sample preparation Kit from Illumina (San Diego, CA).

tag: cell line

tag: cell type

value: Neural stem cells

tag: genotype

value: WT

tag: treatment

value: treat with CHIR and RA


title: 10x multiome (ATAC) on the mouse liver using the NST protocol

city: Leuven

first: Carmen

last: Bravo Gonzalez-Blas

address: Herestraat 49

country: Belgium

institute: VIB-KU Leuven Center for Brain & Disease Research

library_source: genomic single cell

molecule: genomic DNA

organism: Mus musculus

tag: tissue


title: 8 mg FA non-neoplastic cells [N1841]

city: Camden

first: Jaroslav

last: Jelinek

state: NJ

address: 403 Haddon Avenue

department: Research Labs

country: USA

institute: Coriell Institute for Medical Research

description:
N1841
2nd cohort

library_source: genomic

data_processing:
Illumina HiSeq 2500 paired ends sequencing.
Raw sequencing data were converted to fastq.gz files.
bowtie2 --sensitive alignment of the reads in fastq files to mm9 and generating SAM files.
Generating sorted bam files from the sam files with samtools.
Counting methylated CCGGG signatures and unmethylated GGG at the reads aligned to CCCGGG sites.
Adjusting methylation values based on the spiked in standards. At each SmaI/XmaI site, let Xma be the number of reads corresponding to methylated CpG (i.e., reads starting with CCGGG) and Sma be the number of reads corresponding to unmethylated CpG (i.e., reads starting with GGG), the methylation value was calculated as 100%*(c*Xma/(c*Xma+Sma)), where c was the correction factor calculated based on observed and expected methylation of spiked-in standards.
Genome_build: mm9 (MGSCv37)
Supplementary_files_format_and_content: The processed data are in tab-delimited text file. The columns of the file are: ID_SmaI_mm9 (CCCGGG site ID), chromosome (chromosome of the CCCGGG site), position (position of the CCCGGG site), meth_reads (observed number of reads corresponding to methylated CpG, Xma), unmeth_reads (observed number of reads corresponding to unmethylated CpG, Sma), total_reads, raw_methylation (unadjusted methylation value), adjusted_methylation (methylation value adjusted based on the spiked in standards).

source_name: non-neoplastic colon epithelium from colitic mice treated with 8 mg folic acid

growth_protocol: Mice were fed a diet containing folic acid 2 mg/kg chow, followed by carcinogen azoxymethane injection. Colitis was induced in all mice using 4 cycles of DSS. Immediately after the first DSS treatment, mice were randomized to receive folic acid 0, 2 or 8 mg/kg chow for 16 weeks. At the end of study, mice were euthanized and colons were collected and processed for gene expression analyses.

extract_protocol:
Cells were microdissected from paraffin-embedded tissues using Leica LMD6500 system. DNA was isolated using the RecoverAllTM total Nucleic Acid Isolation for FFPE kit.
Genomic DNA was spiked with 20 ng of methylation standards with defined methylation levels of 0, 25, 50, and 100% were digested with 100 units of SmaI endonuclease (NEB) for 3 hours at 25°C. Subsequently, 100 units of XmaI endonuclease (NEB) were added and the digestion was continued for an additional 16 hours at 37°C. Digested DNA was purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA) and eluted in TRIS-HCl 10 mM pH 8.5 (EB). Eluted DNA was supplemented with NEB buffer #2, dCTP, dGTP and dATP (0.4 mM final concentration of each), 15 units of Klenow Fragment (3'→5' exonuclease deficient) DNA polymerase (NEB) and incubated for 30 minutes at 37°C. This step filled in the recesses at 3' DNA ends created by XmaI digestion and added 3' dA tails to all fragments. Illumina paired end (Quail et al. 2008) or barcoded TruSeq (Illumina, San Diego, CA) sequencing adapters were then ligated at 10:1 adapter:fragment ratio using Rapid T4 DNA ligase (Enzymatics, Beverly, MA). The ligation mix was size selected by electrophoresis in 2% agarose. Two slices corresponding to 250-350 bp and 350-500 bp sizes based on a 100 bp DNA ladder (NEB) were cut out and DNA was extracted from agarose. DNA eluted from the slices was separately amplified with Illumina paired end PCR primers (Quail et al. 2008) using iProof high-fidelity DNA polymerase (Bio-Rad Laboratories, Hercules, CA) and 18 cycles of amplification. Resulting sequencing libraries were purified with AMPure magnetic beads (Agencourt, Beverly, MA).

treatment_protocol: AOM was injected at 7 weeks of age and DSS treatment was initiated at 9 weeks of age for 4 cycles (each cycle = 7 days of 2% DSS in drinking water plus 14 days of untreated water). At 11 weeks of age, mice were randomly assigned to receive folic acid at 0, 2 or 8 mg/kg chow for 16 weeks. Mice were euthanized at 27 weeks of age. Colons were fixed in 10% formalin and processed for embedding.

value: laser microdissected non-neoplastic colon epithelia cells

tag: strain

value: Swiss Webster

tag: age

value: 27 weeks

tag: diagnosis

value: Colitis induced by AOM and DSS

value: 8 mg folic acid


title: Differentiated cells, CHIR+RA, Bio rep 2 [CR2]


title: 8 mg FA non-neoplastic cells [N1830]

description:
N1830
2nd cohort


title: 8 mg FA non-neoplastic cells [N1848]

description:
N1848
2nd cohort


title: 8 mg FA non-neoplastic cells [N1800]

description:
N1800
2nd cohort


title: 8 mg FA polypoid tumor cells [1858P]

description:
1858P
2nd cohort

source_name: polypoid tumor cells from colitic mice treated with 8 mg folic acid

value: laser microdissected polypoid tumor cells


title: Differentiated cells, CHIR+PUR, Bio rep 3 [CP3]

value: treat with CHIR and Purmorphamine


title: 10x multiome (RNA) on the mouse liver using the 10x protocol

library_source: transcriptomic single cell


title: Patient LPDAC_15 Tumor CD45+CD3-CD19- sorted scRNAseq

city: Milano

first: Renato

last: Ostuni

address: Via Olgettina 58

country: Italy

institute: San Raffaele Telethon Institute for Gene Therapy

description: 10X Genomics

data_processing:
Fastq files were processed with Cell Ranger (v 4.0.0), using default parameters
Assembly: hg38
Supplementary files format and content: Tab-separated values files and matrix files (gzipped format)

molecule: polyA RNA

source_name: Primary tumor

extract_protocol:
Freshly resected human PDAC samples were minced in small pieces and digested with the Tumor Dissociation kit, human (Miltenyi Biotec). For the patient LiMeT PDAC15, cells were enriched in the myeloid fraction as CD45+CD3-CD19- by sorting into FACSAria instrument (BD Biosciences). Human PB samples were incubated with Red Blood Cell (RBC) lysis buffer (Biolegend) for 10 minutes on ice and washed with Phosphate Buffered Saline (PBS). Cells were centrifuged for 5 minutes at 450 x g and resuspended in the appropriate buffer for down-stream application.
scRNA-Seq libraries were generated using a microfluidics-based approach on Chromium Single-Cell Controller (10X Genomics) using the Chromium Single Cell 3′ Reagent Kit v3.1, according to the manufacturers’ instructions

value: Primary tumor


title: 8 mg FA polypoid tumor cells [1856P2]

description:
1856P2
2nd cohort


title: 8 mg FA polypoid tumor cells [1856P]

description:
1856P
2nd cohort


title: Differentiated cells, CHIR+PUR, Bio rep 2 [CP2]


title: 10x multiome (ATAC) on the mouse liver using the 10x protocol


title: 8 mg FA polypoid tumor cells [1848P]

description:
1848P
2nd cohort


title: Differentiated cells, CHIR+PUR, Bio rep 1 [CP1]


title: 10x snRNA-seq on the mouse liver (922e80)

description:
Mouse_3_Fresh
Single-cell RNA-seq (10X Genomics)

data_processing:
The generated fastq files were processed with cellranger (v1.0.0) count function. Reads were aligned to “pre-mRNA” Mus musculus reference genome, that included both exons and introns (10x Genomics, see https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/advanced/references#premrna).
10x snRNA-seq and 10x multiome (gene expression) runs were analyzed first independently using VSN-pipelines (v0.27.0). Briefly, cells with at least 350 genes expressed and a percentage of mitochondrial reads below 10% were kept. Scanpy (v1.8.2) was run with default parameters, using the number of principal components automatically selected by VSN-Pipelines and using Leiden clustering with resolutions 0.4, 0.6 and 0.8. Hepatocyte clusters with low gene expression and high percentage of mitochondrial reads were removed, as well as doublets called with Scrublet (v0.2.3) [REF]. The samples were merged, obtaining 29,798 high-quality cells, and reanalyzed with VSN-Pipelines.
Assembly: mm10
Supplementary files format and content: scrna_counts.tsv: Tab-separated flat text file containing genes as rows, cells as columns and counts for each gene in each cell as values.

extract_protocol:
The liver nuclei were isolated following the protocol described by Thrupp et al., 2020. For the fresh samples, 200 mg of fresh mouse liver tissue was minced and transferred to a Dounce homogenizer cylinder containing 1 mL of ice-cold homogenization buffer (320 mM Sucrose, 5 mM CaCl2, 3 mM Magnesium Acetate, 10 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 0.1% Igepal, 0.1 mM PMSF, 1 mM βME and 0.2 U/µL RNasin Plus RNase Inhibitor (Promega). For the frozen samples, a piece of 200 mg of liver tissue was sectioned on dry ice and transferred to a Dounce homogenizer cylinder containing 1 mL of ice-cold homogenization buffer and let to thaw for 5 minutes. From this step onward, both the fresh and frozen tissue were homogenized with 10 strokes of pestle A and 10 strokes of pestle B until a homogeneous nuclei suspension was achieved. The resulting homogenate was filtered through a 70-μm cell strainer (Corning). Next, we added 1.65 mL of homogenization buffer to top the homogenate up and mixed it with 2.65 mL of gradient medium (5 mM CaCl2, 50% Optiprep, 3 mM Magnesium Acetate, 10 mM Tris, 0.1mM PMSF, 1mM βME). 4 mL of 29% Iodoxanol cushion was prepared with a diluent medium (250 mM sucrose, 150 mM Potassium chloride, 30 mM Magnesium chloride, 60 mM Tris pH 8) and added into an ultracentrifuge tube. Following, 5.3 mL of sample (mixed with the homogenization buffer and the gradient medium) was gently layered on top of the 29% Iodoxanol cushion. Samples were centrifuged in a SW41Ti rotor at 7700 x g, 4°C for 30 min and the supernatant was gently removed without disturbing the nuclei pellet. Nuclei were resuspended in 200 μL of resuspension buffer (1x PBS, 1% BSA and 0.2 U/μL Rnasin Plus RNase Inhibitor (Promega)) and transferred to a 1.5 mL Eppendorf tube. 9 μL of sample was mixed with 1 μL of arginine orange/propidium iodide (AO/PI) stain, loaded onto a LUNA-FL slide and visualized with the LUNA-FL Automated cell counter for nuclei yield, morphology, and presence of clumps/debris.
Single-nuclei libraries were generated using the 10x Chromium Single-Cell Instrument and Chromium Single Cell 3’ Reagent v3 Kits (10x Genomics) according to the manufacturer’s protocol. In brief, the single mouse liver nuclei suspension was loaded in the Chromium chip B for partitioning into nanoliter-scale Gel Beads-in-emulsion (GEMs). After GEMs generation, the obtained emulsion was incubated in a C1000 Touch Thermal Cycler (Bio-Rad) under the following program: 53°C for 45 min, 85°C for 5 min and hold at 4°C. Incubation of the GEMs produced barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, single-cell droplets were dissolved, and full-length cDNA were isolated using Cleanup Mix containing Silane Dynabeads. To generate sufficient mass for library construction, the cDNA was amplified via PCR: 98°C for 3 min; 12 cycles of 98°C for 15 s, 63°C for 20 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. Subsequently, the amplified cDNA was fragmented, end- repaired, A-tailed and index adaptor ligated, with SPRIselect cleanup in between steps. The final gene expression library was amplified by PCR: 98°C for 45 s; 10-12 cycles of 98°C for 20 s, 54°C for 30 s, 72°C for 20 s. 72°C for 1 min; and hold at 4°C. The sequencing-ready libraries were cleaned up with SPRIselect beads. Before sequencing, the fragment size of every library was analyzed using the Bioanalyzer high-sensitivity chip and were sequenced on HiSeq2500 or NovaSeq6000 instruments with the following sequencing parameters: 28 bp read 1 – 10 bp index 1 (i7) – 10 bp index 2 (i5) – 75 bp read 2.

tag: sample state

value: Fresh


title: 8 mg FA non-neoplastic cells [1856N]

description:
1856N
2nd cohort


title: 8 mg FA non-neoplastic cells [N1858]

description:
N1858
2nd cohort


title: 10x snRNA-seq on the mouse liver (02ff09)

description:
Mouse_2_Frozen
Single-cell RNA-seq (10X Genomics)
NovaSeq6000/HiSeq4000

value: Frozen


title: 10x snRNA-seq on the mouse liver (7703fe)

description:
Mouse_2_Fresh
Single-cell RNA-seq (10X Genomics)
NovaSeq6000/HiSeq4000


title: 12K MPRA experiment on the mouse liver (5 prime library, enhancer-barcode assignments)

data_processing:
For the 5' enhancer-barcode assignments reads were first processed with fastqc (v0.11.8) to assess their quality, and then trimmed using cutadapt with options -g TGTCCCCAGTGCAAGTGCAG --discard-untrimmed -m 12 -l 12 for read 1 to extract the enhancer barcode and options -g AATTAATTCGGGCCCCGGTCC...GATCGGCGCGCCTGCTCG -j 10 --discard-untrimmed -m 17 -M 17 for read 2 to extract the plasmid barcode. For read 2, we then used seqkit (v0.10.2), with options seq -r -p, to get the reverse complement sequence. Reads were filtered to keep only those with quality > 30 using fastp (v0.20.0). This resulted in 8,835,050 enhancer-barcode assignments for the 5’ 12K library, with 78.3% barcodes assigned to a unique enhancer. For the 3' enhancer-barcode assignments, Raw signal fast5 files were re-basecalled using Guppy v6.0.7 using the super-accuracy model (dna_r10.4_e8.1_sup). To process the reads, we first generated a synthetic genome consisting of the plasmid sequence (with 100bp flanks) with all possible enhancers inserted. Reads were mapped using minimap2 (v2.22) with options -ax map-ont –secondary=no -N 1 -f 500 and a bam file was generated using SAMTools (v1.11). Next, we used the R package GenomicAlignments (v1.24.0) to extract the sequences overlapping the enhancer sequence and the enhancer barcodes from the reads and calculate the number of mismatches. This resulted in a library with 723,805 unique enhancer-barcode assignments, with 85.5% barcodes assigned to a unique enhancer. CHEQ-seq barcodes were extracted from the plasmid and cDNA samples (read 2) using cutadapt with parameters with options -g TTATCATGTCTGCTCGAAGC...GATCGGCGCGCCTGCTCG --discard-untrimmed -m 17 -M 17 and seqkit (v0.10.2), with options seq -r -p, was used to get the reverse complement sequence. Reads were filtered to keep only those with quality > 30 using fastp (v0.20.0). Reads were assigned to enhancers based on the corresponding enhancer-barcode assignments, resulting in a count matrix with number of reads per enhancer and sample.
Assembly: mm10
Supplementary files format and content: 12K_MPRA_counts.tsv: Tab-separated text file containing bulk MPRA samples as columns, tested enhancers as rows and assigned reads per sample as value.
Library strategy: CHEQ-seq

organism: synthetic construct

source_name: Plasmid library

growth_protocol: Culture of HepG2 cells: The HepG2 cell line was purchased from ATCC (HB-8065). HepG2 cells were cultured in Eagle’s Minimum Essential Medium (EMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 50 µg/mL penicillin/streptomycin (Thermo Fisher Scientific). Cell cultures were kept at 37°C, with 5% CO2.

extract_protocol:
Enhancer library cloning: The pSA293-CHEQseq plasmid (Addgene 174669), containing a SCP1 promoter, a chimeric intron and the Venus cDNA, was used as a reporter plasmid for MPRA. Two different versions of that plasmid were used for the cloning of the 12k library: pSA293-CHEQseq-5’BC contains a random 17 bp barcode (BC) upstream of the chimeric intron and pSA293-CHEQseq-3’BC-1 contains a random 17 bp BC between the Venus and the poly(A) tail. The oligonucleotide libraries were resuspended according to the manufacturer’s recommendation and amplified via PCR with the primers ‘CHEQ_liver_For’ and ‘CHEQ_liver_Rev’. To clone the amplified enhancer library upstream of the SCP1 promoter, the vectors were linearized via inverse PCR with primers ‘CHEQ_lin_For’ and ‘CHEQ_lin_Rev’. Amplified libraries and the corresponding linearized vector were combined in an NEBuilder reaction with a vector to insert ratio of 1:5. The NEBuilder reactions were dialyzed against water in a 6 cm Petri dish with a membrane filter MF-Millipore 0.05 µm (Merck, Kenilworth, NJ) for 1 hr. Reactions were recovered from the membrane, and 2.5 µL of the reaction was transformed into 25 µL of Lucigen Endura ElectroCompetent Cells (Biosearch Technologies, Hoddesdon, UK). Before culture for maxiprep, 1:100,000 of the transformed bacteria was plated on an LB-agar dish with carbenicillin to estimate the complexity of the cloned library. A volume of bacteria corresponding to a complexity of 500 BCs per enhancer was put in culture for maxiprep. Maxiprep was performed with the Nucleobond Xtra endotoxin-free maxiprep kit (Macherey-Nagel). Bulk MPRA in vitro: The MPRA libraries were transfected in HepG2 cells by use of the Lipofectamine 3000 reagent (Thermo Fisher Scientific). Briefly, 4 million HepG2 cells were seeded in a 10 cm cell culture dish. The next day, when cells reach 70-90% confluency, a tube A with 500 µL opti-MEM (Thermo Fisher Scientific) and 25 µL Lipofectamine 3000 reagent and a tube B with 500 µL opti-MEM and 15 µL of the liver enhancer library were prepared and incubated for 5 min at RT. Tube B was mixed carefully with tube A and incubated for 15 min at RT. The medium of the HepG2 cells was also changed to opti-MEM medium and finally the mixture was added dropwise to the cells. 48 hours post-transfection, cells were detached from the plate using trypsin (Thermo Fisher Scientific). One-fifth of the cells was used for plasmid DNA extraction (Qiagen). The remaining cells were used for RNA extraction using the innuPREP RNA Mini Kit 2.0 (Analytik Jena), followed by mRNA isolation using the Dynabeads mRNA purification kit (Ambion) and cDNA synthesis using the GoScript RT Kit and oligo dT primer (Promega). To amplify the random 5’ and 3’ BC from the plasmid DNA or cDNA sample, a PCR was performed for 16 cycles with ‘CHEQseq_barcode_5’_For’, ‘CHEQseq_barcode_5’_Rev’ and ‘CHEQseq_barcode_3’_For’, ‘CHEQseq_barcode_3’_Rev’, respectively. To add Illumina sequencing adaptors, all samples were finally amplified by PCR for 6 cycles with the primers ‘i5_Indexing_For’ and ‘i7_Indexing_Rev. Bulk MPRA in vivo: For intrahepatic delivery of the liver MPRA libraries, 8- to 10-week-old mice were secured and hydrodynamically injected with 20 µg of the libraries via the lateral tail vein. All libraries were diluted in sterile filtered 0.9% NaCl, and the total volume was adjusted to 10% (in mL) of the total body weight (in grams). 24 hours post-injection, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (Nembutal, 50 mg/kg) and whole livers were isolated. Liver tissues were homogenized by using M tubes (Miltenyi Biotec) and the GentleMACS Dissociator (Miltenyi Biotec). RNA and plasmid DNA extraction, mRNA purification and cDNA prep, and barcode amplification, were done as described for MPRA in vitro.
For the liver library cloned in the pSA293-CHEQseq-5’BC plasmid, a PCR amplification (12 cycles) of the enhancer, together with the random BC, was done with the primers ‘Enh_BC_5’_For’ and ‘Enh_BC_5’_Rev'. Illumina sequencing adaptors were added during a second round of PCR with the primers ‘i5_Indexing_For’ and ‘i7_Indexing_Rev’. Before sequencing, the fragment size of every library was analyzed using the Bioanalyzer high-sensitivity chip. All libraries were sequenced on a NextSeq2000 instrument (Illumina) with the following sequencing parameters: 51 bp read 1 – 8 bp index 1 – 8 bp index 2 – 51 bp read 2. For the liver libraries cloned in the pSA293-CHEQseq-3’BC plasmids, we performed Nanopore sequencing as follows. A total of 1.5 µg of the library was linearized by digestion with NcoI according to the manufacturer’s protocol. We next processed 200 ng of the cleaned up and linearized plasmid with an Oxford Nanopore Technologies Q20+ ligation sequencing kit early-access SQK-LSK112 following the manufacturer’s protocol (Genomic DNA by Ligation; revision GDE_9141_v112_revC_01Dec2021). A total amount of 10 fmol of the prepared library was loaded onto a MinION R10.4 flow cell and sequenced for at least 72 hr (MinKNOW ≥ v21.11.09).

value: Plasmid library


title: Differentiated cells, CHIR, Bio rep 2 [C2]

value: treat with CHIR


title: 8 mg FA polypoid tumor cells [1830P]

description:
1830P
2nd cohort


title: 8 mg FA non-neoplastic cells [LC1858]

description:
LC1858
1st cohort


title: 8 mg FA polypoid tumor cells [1800P]

description:
1800P
2nd cohort


title: Differentiated cells, CHIR, Bio rep 3 [C3]


title: Differentiated cells, CHIR, Bio rep 1 [C1]


title: 8 mg FA non-neoplastic cells [LC1856]

description:
LC1856
1st cohort


title: Mouse KC Heterotopic_2 Tumor CD45+Cd11b+ sorted scRNAseq

description: 10X Genomics

data_processing:
Fastq files were processed with Cell Ranger (v 4.0.0), using default parameters
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files (gzipped format)

source_name: Tumor

extract_protocol:
Murine healthy pancreas and tumors were manually minced in small pieces and dissociated with the Tumor Dissociation kit, mouse (Miltenyi Biotec). The obtained single cells suspensions were filtered on 70µm cell strainers, incubated with RBC lysis buffer for 10 minutes on ice and resuspended in the appropriate buffer for cell counting and down-stream application. For the heterotopic and orthotopic inoculation of KC cell line, cells were enriched in the myeloid fraction as CD45+Cd11b+ via FACS sorting. Mouse PB samples were incubated with Red Blood Cell (RBC) lysis buffer (Biolegend) for 10 minutes on ice and washed with Phosphate Buffered Saline (PBS). Cells were centrifuged for 5 minutes at 450 x g and resuspended in the appropriate buffer for down-stream application.
scRNA-Seq libraries were generated using a microfluidics-based approach on Chromium Single-Cell Controller (10X Genomics) using the Chromium Single Cell 3′ Reagent Kit v3.1, according to the manufacturers’ instructions. For the heterotopic and orthotopic inoculation of KC cell line, libraries generated using the Chromium Single Cell 3′ Reagent Kit v2.

value: Tumor

tag: Sex

value: M

tag: time

value: Day 33


title: 8 mg FA non-neoplastic cells [LC1848]

description:
LC1848
1st cohort


title: Mouse KC Heterotopic_1 Tumor CD45+Cd11b+ sorted scRNAseq

description: 10X Genomics

value: WT


title: SAD2, hpNPCs, treat rep2 (ATAC-seq)

city: London

first: Marco

last: Trizzino

state: UK

address: Imperial College Road

department: Life Sciences

country: United Kingdom

institute: Imperial College London

data_processing:
ATAC-seq data were aligned to hg19 using BWA-mem
RNA-seq data were aligned to hg19 using STARR-2pass
ATAC-seq peaks were called with MACS2.
RNA-seq gene expression was quantified using Kallisto
Assembly: Hg19
Supplementary files format and content: peak text files and Bigwig for ATAC-seq, Kallisto quantification (spreadsheet) for RNA-seq

growth_protocol:
Cells were passaged ~1:10 at 80% confluency using EDTA 0.5mM (STEMCELL Technologies) and small cell clusters (50?200 cells) were subsequently plated on tissue culture dishes coated overnight with Geltrex? LDEV-Free hESC-qualified Reduced Growth Factor Basement Membrane Matrix (Fisher-Scientific).
[hpNPCS differentiation protocol] iPSCs were treated with hpNPCs induction medium for five days: D-MEM F-12 medium (Invitrogen) supplemented with B-27 (A3582801, Gibco), N-2 (17502048, Gibco), DKK1 (778606, Biolegend), Cyclopamine (C-8700, LC Laboratories), Noggin (597004, Biolegend), and SB431542 (S1067, Selleck Chemicals LLC).
[hpNPCs proliferation protocol]  the hpNPCs are plated in a new geltrex coated well and cultured in proliferation medium, consisting in D-MEM F-12 medium (Invitrogen) supplemented with B-27 (A3582801, Gibco), N-2 (17502048, Gibco) and 20 ng/ml bFGF (713304, Biolegend)
[CA3 Neurons protocol]  iPSCs were treated with hpNPCs induction medium for 15 days. At day 16 the hpNPCs are plated in a new PLO-Laminin double- coated well in Neuron induction medium, consisting in DMEM/F12 medium (11320082, Gibco) supplemented with B-27 (A3582801, Gibco), N-2 (17502048, Gibco), BDNF (450-02, Prepotech), Dibutyryl-cAMP (11-415-0, Tocris) , laminin (23017015, Thermofisher Scientific), AA, Wnt3a. After 3 weeks the neurons are switched in the neuron medium, consisting in D-MEM F-12 medium (Invitrogen) supplemented with B-27 (A3582801, Gibco), N-2 (17502048, Gibco), BDNF (450-02, Prepotech), Dibutyryl-cAMP (11-415-0, Tocris), laminin (23017015, Thermofisher Scientific) and AA for one week.

extract_protocol:
[RNA extraction protocol] Cells were lysed in Tri-reagent (Zymo research) and total RNA was extracted using Quick-RNA Miniprep kit (Zymo research).
[RNA-seq]  RNA libraries were prepared using NEBNext® Poly(A) mRNA Magnetic Isolation Module, NEBNext® UltraTM II Directional RNA Library Prep Kit for Illumina® and NEBNext® UltraTM II DNA Library Prep Kit for Illumina® according to the manufacturer?s instructions (New England Biolabs). 1 ug of total RNA input was used for each library
[ATACseq]  50000 cells per condition were processed as described in the original ATAC-seq protocol paper (Buenrostro et al 2013)

tag: disease state

value: SAD

value: hpNPCs

value: cJun peptde +


title: 8 mg FA polypoid tumor cells [1841P]

description:
1841P
2nd cohort

value: Swiss Webster

value: 27 weeks


title: Mouse KC Orthotopic_2 Tumor CD45+Cd11b+ sorted scRNAseq

description: 10X Genomics


title: 8 mg FA non-neoplastic cells [LC1844]

description:
LC1844
1st cohort

value: laser microdissected non-neoplastic colon epithelia cells

value: Colitis induced by AOM and DSS

value: 8 mg folic acid


title: SAD2, hpNPCs, no treat rep2 (ATAC-seq)

value: cJun peptde -


title: BMDC, a-GalCer, 2

city: Gainesville

first: Alexandra

middle: E.

last: Cuaycal

state: FL

address: 1275 Center Drive

department: Infectious Diseases and Immunology/Biomedical Engineering

institute: University of Florida

data_processing:
Raw data (raw reads) of FASTQ format were firstly processed through fastp to generate clean reads.
Paired-end clean reads were mapped to the reference genome Rnor_6.0 using HISAT2 software.
Novogene Co., Ltd Quantification FeatureCounts was used to count the read numbers mapped of each gene.
Assembly: Rnor_6.0
Supplementary files format and content: xlsx file with raw counts for all samples.

organism: Rattus norvegicus

source_name: Bone-marrow derived dendritic cell

growth_protocol: Rat BMDCs were generated from bone marrow progenitors by incubating in RPMI-1640 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine (as GlutaMAXTM supplement), 50 µM 2-mercaptoethanol, 100 units/mL of penicillin, 100 µg/mL streptomycin, 20 ng/mL of rat GM-CSF and 200 ng/mL of human FLT3-L (both from R&D Systems). Cells were incubated for 7 days at 37 °C and 5% CO2 with medium changes every 2-3 days.

extract_protocol:
Total RNA was isolated with the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. DNA was digested with the TURBO DNA-freeTM kit (Invitrogen). 1 ug of total RNA was used for the construction of sequencing libraries.
Sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer’s instructions.

treatment_protocol: Total lipids from Lactobacillus johnsonii N6.2 were resuspended in PBS pH 7.2 and added to BMDCs at 600 ng/mL. Synthetic alpha-galactosylceramide (a-GalCer), KRN700 was purchased from Adipogen, dissolved in DMSO following manufacturer's recommendations, diluted 1:10 in PBS pH 7.2 and added to the cells at 100 ng/mL. DMSO:PBS (1:10, v/v) added at equal volumes constituted the vehicle control.

value: Bone-marrow derived dendritic cell

value: alpha-galactosylceramide


title: BMDC, a-GalCer, 3


title: Mouse KC Orthotopic_1 Tumor CD45+Cd11b+ sorted scRNAseq

description: 10X Genomics


title: 8 mg FA non-neoplastic cells [LC1830]

description:
LC1830
1st cohort


title: BMDC, a-GalCer, 1


title: Mouse KPC GEMM_2 Tumor scRNAseq

description: 10X Genomics

value: F


title: SAD2, hpNPCs, treat rep1 (ATAC-seq)


title: αβ-TCR-stimulated αβ-TCR transduced γδ-T cells

type: RNA

city: Tsu

first: Mikiya

last: Ishihara

state: Mie

address: 2-174 Edobashi

country: Japan

institute: Mie University Hospital

hyb_protocol: 0.1 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a Agilent fragmentation buffer and was hybridized at 65°C for 17 hr using Gene Expression Hybridization Kit.

scan_protocol: Microarray slides were scanned at 3 μm resolution using an Agilent SureScan (G2600D).

data_processing: The scanned images were analyzed with Feature Extraction Software (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.

source_name: αβ-TCR transduced γδ-T cells, αβ-TCR-stimulated

label_protocol: For cRNA amplification and labeling, Agilent Low Input Quick Amp Labeling Kit, One-Color (Agilent Technologies) were used following the manufacter’s instructions.

extract_protocol: RNA extracts were prepared from γδ-T cells and αβ-TCR transduced γδ-T cells from a healthy donor using QIAamp RNA Blood Mini kit.

value: male

tag: disease

value: healthy donor

tag: agent

value: αβ-TCR-stimulated


title: 10x snRNA-seq on the mouse liver (7b2a3b)

description:
Mouse_1_Fresh
Single-cell RNA-seq (10X Genomics)
NovaSeq6000/HiSeq4000

value: Liver (P56)

value: Fresh


title: BMDC, Lj-N6.2_TL, 3

value: WT

value: Lactobacillus johnsonii N6.2 total lipids


title: unstimulated αβ-TCR transduced γδ-T cells

source_name: αβ-TCR transduced γδ-T cells, unstimulated

value: unstimulated


title: unstimulated γδ-T cells

source_name: γδ-T cells, unstimulated


title: γδ-TCR-stimulated γδ-T cells

source_name: γδ-T cells, γδ-TCR-stimulated

value: γδ-TCR-stimulated


title: 2 mg FA non-neoplastic cells [LC1832]

description:
LC1832
1st cohort

source_name: non-neoplastic colon epithelium from colitic mice treated with 2 mg folic acid

value: Swiss Webster

value: 27 weeks

value: 2 mg folic acid


title: SAD1, hpNPCs, treat rep2 (ATAC-seq)

value: SAD

value: hpNPCs

value: cJun peptde +


title: Mouse KPC GEMM_1 Tumor scRNAseq

description: 10X Genomics

value: Tumor

value: M


title: SAD2, hpNPCs, no treat rep1 (ATAC-seq)

value: cJun peptde -


title: BMDC, Lj-N6.2_TL, 2

value: Bone-marrow derived dendritic cell


title: BMDC, Lj-N6.2_TL, 1


title: SAD1, hpNPCs, no treat rep1 (ATAC-seq)


title: 2 mg FA non-neoplastic cells [LC1824]

description:
LC1824
1st cohort

value: laser microdissected non-neoplastic colon epithelia cells

value: Colitis induced by AOM and DSS


title: BMDC, Vehicle_Ctrl, 3

value: Vehicle control


title: SAD1, hpNPCs, no treat rep2 (ATAC-seq)


title: FAD2, hpNPCs, treat, rep2 (ATAC-seq)

value: FAD


title: 8 mg FA non-neoplastic cells [LC1841]

description:
LC1841
1st cohort

value: 8 mg folic acid


title: Mouse timecourse Healthy Blood scRNAseq pool of 3 mice

description: 10X Genomics

source_name: Blood

value: Blood

value: WT

value: F


title: 2 mg FA non-neoplastic cells [LC1835]

description:
LC1835
1st cohort

value: Swiss Webster

value: 27 weeks


title: Mouse KPC WT day7 Tumor scRNAseq pool of 3 mice

description: 10X Genomics

value: Day 7


title: 2 mg FA non-neoplastic cells [LC1822]

description:
LC1822
1st cohort

value: 2 mg folic acid


title: BMDC, Vehicle_Ctrl, 2


title: 2 mg FA non-neoplastic cells [LC1814]

description:
LC1814
1st cohort


title: Mouse timecourse KPC day30 Blood scRNAseq pool of 3 mice

description: 10X Genomics

value: Day 30


title: FAD2, hpNPCs, treat, rep1 (ATAC-seq)

value: hpNPCs

value: cJun peptde +


title: SAD1, hpNPCs, treat rep1 (ATAC-seq)

value: SAD


title: Mouse timecourse KPC day20 Blood scRNAseq pool of 3 mice

description: 10X Genomics

value: Day 20


title: 2 mg FA non-neoplastic cells [LC1829]

description:
LC1829
1st cohort

value: laser microdissected non-neoplastic colon epithelia cells


title: Mouse timecourse Healthy Pancreas scRNAseq pool of 3 mice

description: 10X Genomics

source_name: Pancreas

value: Pancreas


title: FAD2, hpNPCs, no treat rep2 (ATAC-seq)

value: cJun peptde -


title: 12K MPRA experiment on the mouse liver (3prime library, enhancer-barcode assignments)

description: 3p_Nanopore assignments

value: Plasmid library


title: BMDC, Vehicle_Ctrl, 1

value: Bone-marrow derived dendritic cell

value: Vehicle control


title: FAD2, hpNPCs, no treat rep1 (ATAC-seq)

value: FAD


title: Mouse timecourse KPC day10 Blood scRNAseq pool of 3 mice

description: 10X Genomics

value: Blood

value: WT

value: F

value: Day 10


title: FAD1, hpNPCs, treat rep2 (ATAC-seq)


title: 455 MPRA experiment on the mouse liver (3prime library, enhancer-barcode assignments)

data_processing:
For the 3' enhancer-barcode assignments, Raw signal fast5 files were re-basecalled using Guppy v6.0.7 using the super-accuracy model (dna_r10.4_e8.1_sup). To process the reads, we first generated a synthetic genome consisting of the plasmid sequence (with 100bp flanks) with all possible enhancers inserted. Reads were mapped using minimap2 (v2.22) with options -ax map-ont –secondary=no -N 1 -f 500 and a bam file was generated using SAMTools (v1.11). Next, we used the R package GenomicAlignments (v1.24.0) to extract the sequences overlapping the enhancer sequence and the enhancer barcodes from the reads and calculate the number of mismatches. This resulted in a library with 723,805 unique enhancer-barcode assignments, with 85.5% barcodes assigned to a unique enhancer. CHEQ-seq barcodes were extracted from the plasmid and cDNA samples (read 2) using cutadapt with parameters with options -g TTATCATGTCTGCTCGAAGC...GATCGGCGCGCCTGCTCG --discard-untrimmed -m 17 -M 17 and seqkit (v0.10.2), with options seq -r -p, was used to get the reverse complement sequence. Reads were filtered to keep only those with quality > 30 using fastp (v0.20.0). Reads were assigned to enhancers based on the corresponding enhancer-barcode assignments, resulting in a count matrix with number of reads per enhancer and sample.
Assembly: mm10
Supplementary files format and content: Bulk_455_MPRA_counts.tsv: Tab-separated text file containing bulk MPRA samples as columns, tested enhancers as rows and assigned reads per sample as value.
Supplementary files format and content: FACS_455_MPRA_counts.tsv: Tab-separated text file containing FACS bulk MPRA samples as columns, tested enhancers as rows and assigned reads per sample as value.
Library strategy: CHEQ-seq

extract_protocol:
Enhancer library cloning: The pSA293-CHEQseq plasmid (Addgene 174669), containing a SCP1 promoter, a chimeric intron and the Venus cDNA, was used as a reporter plasmid for MPRA. The 455 library was cloned in a newly generated pSA293-CHEQseq-3'BC-2 containing an 18 bp BC optimized for ONT sequencing with the following pattern: NNNYRNNNYRNNNYRNNN. The oligonucleotide libraries were resuspended according to the manufacturer’s recommendation and amplified via PCR with the primers ‘CHEQ_liver_For’ and ‘CHEQ_liver_Rev’. To clone the amplified enhancer library upstream of the SCP1 promoter, the vectors were linearized via inverse PCR with primers ‘CHEQ_lin_For’ and ‘CHEQ_lin_Rev’. Amplified libraries and the corresponding linearized vector were combined in an NEBuilder reaction with a vector to insert ratio of 1:5. The NEBuilder reactions were dialyzed against water in a 6 cm Petri dish with a membrane filter MF-Millipore 0.05 µm (Merck, Kenilworth, NJ) for 1 hr. Reactions were recovered from the membrane, and 2.5 µL of the reaction was transformed into 25 µL of Lucigen Endura ElectroCompetent Cells (Biosearch Technologies, Hoddesdon, UK). Before culture for maxiprep, 1:100,000 of the transformed bacteria was plated on an LB-agar dish with carbenicillin to estimate the complexity of the cloned library. A volume of bacteria corresponding to a complexity of 500 BCs per enhancer was put in culture for maxiprep. Maxiprep was performed with the Nucleobond Xtra endotoxin-free maxiprep kit (Macherey-Nagel). Bulk MPRA in vitro: The MPRA libraries were transfected in HepG2 cells by use of the Lipofectamine 3000 reagent (Thermo Fisher Scientific). Briefly, 4 million HepG2 cells were seeded in a 10 cm cell culture dish. The next day, when cells reach 70-90% confluency, a tube A with 500 µL opti-MEM (Thermo Fisher Scientific) and 25 µL Lipofectamine 3000 reagent and a tube B with 500 µL opti-MEM and 15 µL of the liver enhancer library were prepared and incubated for 5 min at RT. Tube B was mixed carefully with tube A and incubated for 15 min at RT. The medium of the HepG2 cells was also changed to opti-MEM medium and finally the mixture was added dropwise to the cells. 48 hours post-transfection, cells were detached from the plate using trypsin (Thermo Fisher Scientific). One-fifth of the cells was used for plasmid DNA extraction (Qiagen). The remaining cells were used for RNA extraction using the innuPREP RNA Mini Kit 2.0 (Analytik Jena), followed by mRNA isolation using the Dynabeads mRNA purification kit (Ambion) and cDNA synthesis using the GoScript RT Kit and oligo dT primer (Promega). To amplify the random 5’ and 3’ BC from the plasmid DNA or cDNA sample, a PCR was performed for 16 cycles with ‘CHEQseq_barcode_5’_For’, ‘CHEQseq_barcode_5’_Rev’ and ‘CHEQseq_barcode_3’_For’, ‘CHEQseq_barcode_3’_Rev’, respectively. To add Illumina sequencing adaptors, all samples were finally amplified by PCR for 6 cycles with the primers ‘i5_Indexing_For’ and ‘i7_Indexing_Rev. Bulk MPRA in vivo: For intrahepatic delivery of the liver MPRA libraries, 8- to 10-week-old mice were secured and hydrodynamically injected with 20 µg of the libraries via the lateral tail vein. All libraries were diluted in sterile filtered 0.9% NaCl, and the total volume was adjusted to 10% (in mL) of the total body weight (in grams). 24 hours post-injection, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (Nembutal, 50 mg/kg) and whole livers were isolated. Liver tissues were homogenized by using M tubes (Miltenyi Biotec) and the GentleMACS Dissociator (Miltenyi Biotec). RNA and plasmid DNA extraction, mRNA purification and cDNA prep, and barcode amplification, were done as described for MPRA in vitro. FACS MPRA in vivo: For intrahepatic delivery of the liver MPRA libraries, 8- to 10-week-old mice were secured and hydrodynamically injected with 20 µg of the libraries via the lateral tail vein. All libraries were diluted in sterile filtered 0.9% NaCl, and the total volume was adjusted to 10% (in mL) of the total body weight (in grams). 48 hours post-injection, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (Nembutal, 50 mg/kg). Livers were perfused for 5 minutes with 40 mL of perfusion medium SC-1 (8 g/L Nacl, 400 mg/L KCl, 75.5 mg/L NaH2PO4, 120.5 mg/L Na2HPO4, 2,38 g/L HEPES, 350 mg/L NaHCO3, 190 mg/L EGTA, 900 mg/L D-(+)-Glucose, 1.2 mL Phenol Red solution) to remove the blood, followed by perfusion with 40 mL of SC-2 medium (8 g/L Nacl, 400 mg/L KCl, 75.5 mg/L NaH2PO4, 120.5 mg/L Na2HPO4, 2,38 g/L HEPES, 350 mg/L NaHCO3, 560 mg/L CaCl2.2H2O, 1.2 mL Phenol Red solution) containing 10 mg of collagenase P (Merck) for 5 minutes. Each lobe was dissected off and minced into small pieces in a beaker containing 39 mL SC-2 supplemented with 1 mL DNase I (Sigma-Aldrich) and 20 mg collagenase P, followed by rotating incubation for 15 min at 37°C. Hepatocytes were centrifuged for 2 min at 50 g, washed with PBS, centrifuged again for 2 min at 50 g, resuspended in 3 ml Hoechst buffer (DMEM + 10% FBS + 10 mM HEPES) and filtered through a 70 µm strainer. The protocol for hepatocyte staining was adapted from Ben-Moshe S. et al. After counting the cells on a LUNA cell counter, the concentration was adjusted to 5 million cells in 1 mL of Hoechst buffer. To determine the ploidy of hepatocytes, DNA was stained with Hoechst (Thermo Fisher Scientific) (15 µg/mL). Reserpine (5 µM) was also supplemented to prevent Hoechst expulsion from the cells. Cells were incubated for 30 min at 37°C. Hepatocytes were centrifuged for 5 min at 1,000 rpm at 4°C and the supernatant was discarded. Cells were resuspended in cold PBS in a concentration of 1 million cells in 100 µL. After spinning down (1,000 rpm for 5 min at 4°C), cells were resuspended in FACS buffer (2 mM EDTA, pH 8, and 0.5% BSA in 1x PBS) at a concentration of 1 million cells in 100 µL. Cells were stained with the following antibodies (BioLegend) at a dilution of 1:300: PE anti-mouse/human CD324 E-cadherin (catalogue no. 147304) and APC anti-mouse CD73 (catalogue no. 127210). FcX blocking solution (BioLegend catalogue no. 101319) was added at a dilution of 1:50. Cells were sorted by FACS-Aria-Fusion (BD Biosciences) using a 100 µm nozzle. FSC-A and SSC-A were used for hepatocytes size selection. Cells containing the library were selected based on GFP. Tetraploid hepatocytes were selected based on Hoechst stain. CD73 and Ecad were used to select hepatocytes bins along the porto-central axis, obtaining 100,000-200,000 cells per bin. RNA extraction, mRNA purification and cDNA prep were done as described for MPRA in vitro. To amplify the enhancer barcode on the cDNA small modifications were made. To amplify the random 3’ BC, a PCR with 24 cycles was performed. To add Illumina sequencing adaptors, a PCR with 10 cycles was done.
For the liver libraries cloned in the pSA293-CHEQseq-3’BC plasmids, we performed Nanopore sequencing as follows. A total of 1.5 µg of the library was linearized by digestion with NcoI according to the manufacturer’s protocol. We next processed 200 ng of the cleaned up and linearized plasmid with an Oxford Nanopore Technologies Q20+ ligation sequencing kit early-access SQK-LSK112 following the manufacturer’s protocol (Genomic DNA by Ligation; revision GDE_9141_v112_revC_01Dec2021). A total amount of 10 fmol of the prepared library was loaded onto a MinION R10.4 flow cell and sequenced for at least 72 hr (MinKNOW ≥ v21.11.09). All cDNA/Plasmid libraries were sequenced on a NextSeq2000 instrument (Illumina) with the following sequencing parameters: 51 bp read 1 – 8 bp index 1 – 8 bp index 2 – 51 bp read 2.


title: 0 mg FA non-neoplastic cells [N1820]

description:
N1820
2nd cohort

source_name: non-neoplastic colon epithelium from colitic mice treated with 0 mg folic acid

value: Swiss Webster

value: 27 weeks

value: Colitis induced by AOM and DSS

value: 0 mg folic acid


title: Mouse timecourse KPC day20 Tumor scRNAseq pool of 3 mice

description: 10X Genomics

value: Tumor


title: Mouse timecourse KPC day30 Tumor scRNAseq pool of 3 mice

description: 10X Genomics

value: Day 30


title: FAD1, hpNPCs, treat rep1 (ATAC-seq)

value: hpNPCs

value: cJun peptde +


title: Mouse timecourse KPC day10 Tumor scRNAseq pool of 3 mice

description: 10X Genomics


title: FAD1, hpNPCs, no treat rep1 (ATAC-seq)

value: cJun peptde -


title: C-K27

first: Yunhan

last: Liu

state: Shanghai

address: 639 Zhizaoju Road,

department: Department of Plastic and Reconstructive Surgery

institute: Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine

data_processing:
Basecalls performed using  Solexa pipeline v1.8
The trimmed reads (trimmed 5’,3’-adaptor bases using cutadapt) were aligned to reference genome using Hisat2 (2.0.5)software
The transcript abundances for each sample was estimated with StringTie（1.3.1c）
the FPKM value for gene and transcript level were calculated with R package Ballgown (v2.6.0)
For analyses of circRNA expression, after mapping reads to the genome using STAR, we used CIRCexplorer2 for back splice junction reads detection and reads count statistics, followed by differential expression calculation performed with the R software edge R.
Assembly: GRCh37
Supplementary files format and content: The Fastq format file contains the sequence of reads and the sequencing quality of base.

source_name: human skin

extract_protocol:
Total RNA was extracted using TRIzol Reagent (Invitrogen, USA), and RNA concentration was determined using NanoDrop (ND-1000). After removing the rRNAs, 2ug of total RNA was used for the preparation of the cDNA library with a KAPA-Stranded RNA-Seq Library Prep Kit (Illumina, USA).
The total RNA was processed by NEBNext® Poly(A) mRNA Magnetic Isolation Module (or rRNA removal by RiboZero Magnetic Gold Kit) to enrich mRNA, and the product RNA was used for construction Library, via KAPA Stranded RNA-Seq Library Prep Kit (Illumina). Sequencing libraries, denatured by 0.1 M NaOH to generate single-stranded DNA, as amplified in situ Illumina cBot (TruSeq SR Cluster Kit v3-cBot-HS (# GD-401-3001, Illumina)). The ends of the generated fragments was used to run 150 Cycles by the Illumina HiSeq 4000 Sequencer.

treatment_protocol: Tissue samples were cut into pieces and immersed in 0.3% dispase Ⅱ (0.3 g/ml; Gibco, USA) for 12 hours at 4℃. The dermal tissues were then obtained after the epidermis was separated.

value: skin

value: keloid, center


title: 0 mg FA non-neoplastic cells [N1808]

description:
N1808
2nd cohort

value: laser microdissected non-neoplastic colon epithelia cells


title: 0 mg FA non-neoplastic cells [N1809]

description:
N1809
2nd cohort


title: FAD1, hpNPCs, no treat rep2 (ATAC-seq)

value: FAD


title: 0 mg FA non-neoplastic cells [N1807]

description:
N1807
2nd cohort


title: C-K24


title: 0 mg FA polypoid tumor cells [1839P]

description:
1839P
2nd cohort

source_name: polypoid tumor cells from colitic mice treated with 0 mg folic acid

value: laser microdissected polypoid tumor cells


title: 0 mg FA non-neoplastic cells [N1805]

description:
N1805
2nd cohort


title: 2 mg FA non-neoplastic cells [LC1812]

description:
LC1812
1st cohort

value: 2 mg folic acid


title: SAD2, hpNPCs rep2 (ATAC-seq)

value: SAD


title: SAD2, hpNPCs rep1 (ATAC-seq)


title: Mouse timecourse KPC day30 Tumor spatial transcriptomics section B1

description: 10X Genomics

data_processing:
Fastq files were processed with Space Ranger (v.1.2.0)
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files (gzipped format)
Supplementary files format and content: Image outputs from Space Ranger (jpg and png format), scale factors (json) and tissue coordinates (csv format)

extract_protocol:
Tumor biopsies of day-30 orthotopic mouse PDAC were gently washed in PBS, snap frozen via bathing in liquid nitrogen-chilled isopentane and embedded with Optimal Cutting Temperature (OCT) compound.  From five to ten sections were collected to evaluate RNA quality at TapeStation system (Agilent). The tissue blocks were then processed to retrieve two non-sequential 10µm sections (100 µm apart), which were placed within the capture areas of a Visium slide equilibrated at cryostat temperature (-20°C). Sections were immediately fixed in chilled methanol at -20°C for 30 min and stained via immunofluorescence as stated above preserving RNA degradation using buffers supplemented with Recombinant RNase inhibitor (Takara 2313A, 2 U/µl). Whole-slide images were acquired using the MAVIG RS-G4 (Caliber I.D.) confocal microscope at 20x magnification. Barcoded libraries were generated by permeabilizing tissue at 37°C for 15 min and performing in situ reverse transcription at 53°C for 45 min, followed by second-strand synthesis at 65°C for 15 min. cDNA was denatured and transferred to tubes for PCR amplification and library construction, including fragmentation, adaptor ligation and sample indexing.
Spatial transcriptomics data were generated using the Visium Spatial Gene Expression Reagent Kits (10X Genomics) according to the manufacturer’s instruction.

value: WT

value: F


title: BMDM control RNA-seq replicate 3

data_processing:
Reads were aligned using STAR aligner (v STAR_2.5.3a). Read counts matrices were computed using the featureCounts function from Rsubread package (v 2.0.1).
Assembly: mm10
Supplementary files format and content: Tab-separated counts file (gzipped format)

source_name: Bone marrow

growth_protocol: For BMDMs, bone marrrow cells were counted and seeded in IMDM supplemented with 20% FBS, 20% L929-conditioned media containing M-CSF, antibiotics (penicillin G 100 U/ml and streptomycin sulfate 100 U/ml), 2 mM L-glutamine and 5 µM 2-mercaptoethanol. Four days after culture, fresh medium was added to the cells. At day 7 after plating, cells were stimulated as described below. KPC (K8484) cell lines were previously established from tumors arising in genetically engineered mouse model carrying the G12D oncogenic mutation in the Kras gene  and the missense point R720H mutation in the Tpr53 gene (KrasLSL-G12D/+;Tpr53LSL-R270H/+;Pdx1Cre/WT) and were cultured under standard conditions.

extract_protocol:
Total RNA was purified using the ReliaPrep RNA Cell Miniprep System.
RNA-Seq libraries were generated using the Smart-seq2 method with minor modification. Briefly, five ng of RNA were retrotranscribed, cDNA was PCR-amplified (15 cycles) and purified with AMPure XP beads. After purification, the concentration was determined using Qubit 3.0 and size distribution was assessed using Agilent 4200 TapeStation system. Then, the tagmentation reaction was performed starting from 0.5 ng of cDNA for 30 minutes at 55°C and the enrichment PCR was carried out using 12 cycles. Libraries were then purified with AMPure XP beads, quantified using Qubit 3.0, assessed for fragment size distribution on an Agilent 4200 TapeStation system.

treatment_protocol: BMDMs were stimulated with reagents at the following concentrations: TNFa(10 ng/mL), PGE2 (1µM), alone or in combination for 1, 2 or 4 hours respectively. KPC cells were stimulated with IL-1b (10 ng/mL) for 24 hours. Controls were left untreated.

value: Bone marrow

value: Bone marrow derived macrophages

value: untreated


title: 0 mg FA non-neoplastic cells [N1839]

description:
N1839
2nd cohort

value: Swiss Webster

value: 27 weeks

value: Colitis induced by AOM and DSS

value: 0 mg folic acid


title: C-K23


title: BMDM control RNA-seq replicate 2


title: SAD1, hpNPCs rep3 (ATAC-seq)

value: hpNPCs


title: 0 mg FA polypoid tumor cells [1820P]

description:
1820P
2nd cohort


title: C-K20

value: skin


title: SAD1, hpNPCs rep2 (ATAC-seq)


title: BMDM control RNA-seq replicate 1


title: M-K24

value: keloid, margin


title: M-K23


title: 0 mg FA polypoid tumor cells [1808P]

description:
1808P
2nd cohort


title: BMDM TNFa+PGE2 stimulation 4 hour RNA-seq replicate 3

value: TNFa(10 ng/mL) + PGE2 (1uM)


title: M-K20


title: BMDM TNFa+PGE2 stimulation 4 hour RNA-seq replicate 2


title: BMDM TNFa+PGE2 stimulation 4 hour RNA-seq replicate 1


title: 0 mg FA polypoid tumor cells [1805P]

description:
1805P
2nd cohort


title: BMDM TNFa+PGE2 stimulation 2 hour RNA-seq replicate 3


title: N-K27

value: normal


title: N-K24


title: Mouse timecourse KPC day30 Tumor spatial transcriptomics section A1

description: 10X Genomics

value: Tumor

value: Day 30


title: 0 mg FA non-neoplastic cells [LC1820]

description:
LC1820
1st cohort

value: laser microdissected non-neoplastic colon epithelia cells


title: 0 mg FA polypoid tumor cells [1807P]

description:
1807P
2nd cohort

value: laser microdissected polypoid tumor cells


title: 0 mg FA non-neoplastic cells [LC1817]

description:
LC1817
1st cohort


title: BMDM TNFa+PGE2 stimulation 2 hour RNA-seq replicate 2

value: WT


title: N-K23


title: BMDM TNFa+PGE2 stimulation 2 hour RNA-seq replicate 1

value: Bone marrow


title: 0 mg FA polypoid tumor cells [1809P]

description:
1809P
2nd cohort


title: FAD2, hpNPCs rep2 (ATAC-seq)

value: FAD


title: FAD1, hpNPCs rep2 (ATAC-seq)


title: FAD2, hpNPCs rep1 (ATAC-seq)


title: Ctrl2, hpNPCs rep2 (ATAC-seq)

source_name: iPSM15 Sev4

value: Ctrl


title: FAD1, hpNPCs rep1 (ATAC-seq)


title: 0 mg FA non-neoplastic cells [LC1809]

description:
LC1809
1st cohort

value: Swiss Webster


title: 0 mg FA non-neoplastic cells [LC1810]

description:
LC1810
1st cohort

value: 27 weeks


title: N-K20


title: BMDM TNFa+PGE2 stimulation 1 hour RNA-seq replicate 3

value: Bone marrow derived macrophages


title: SAD1, hpNPCs rep1 (ATAC-seq)

value: SAD


title: Ctrl2, hpNPCs rep1 (ATAC-seq)

value: hpNPCs


title: RNAseq of Early treatment gastruloid at 96h replicate 3

city: LAUSANNE

first: ALEXANDRE

last: MAYRAN

state: Waadt

address: Station 19

country: Switzerland

institute: EPFL

data_processing:
Raw RNA-seq reads were trimmed to remove Nextera adapters or bad quality bases (Cutadapt v4.0 -a CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -A CTGTCTCTTATACACATCTGACGCTGCCGACGA -q 30 -m 15) (Martin 2011).
Mapping was done from filtered reads on the mouse genome mm10 with STAR version 2.7.8a (Dobin et al. 2013) with the gencode.vM24.primary assembly annotations (https://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M24/gencode.vM24.primary_assembly.annotation.gtf.gz) and ENCODE parameters.
FPKM values were obtained by Cufflinks version 2.2.1 (Trapnell et al. 2010; Roberts et al. 2011) with options --no-effective-length-correction -b 'mm10.fa' --multiread-correct --library-type fr-firststrand --mask-file 'chrM_mm10.gtf' --max-bundle-length 10000000 --max-bundle-frags 1000000 (where chrM_mm10.gtf contains a transcript on each strand of the whole chrM). FPKM of identical gene_ids were summed
Counts and FPKM values were aggregated with R scripts.
Assembly: mm10
Supplementary files format and content: output of cufflinks with FPKM values per gene for all samples
Supplementary files format and content: output of STAR with count values for all samples

source_name: Gastruloids

extract_protocol:
Gastruloids for each condition were collected and pooled in a 1.7 ml Eppendorf tube. After centrifugation and medium removal, pelleted stembryos were washed once in PBS, pelleted, snapfroze in liquid nitrogen and stored at -80°C until RNA extraction. RNeasy Plus mini kit (Qiagen) with on-column DNase digestion was used for RNA extraction following the manufacturer’s instructions.
We performed a "Illumina stranded mRNA ligation" (ISML) prep starting from 1000ng of RNA (see table below), according to Illumina protocol 1000000124518 v01 (internal exp code LPX362 and LPX364). We quantified the libraries by qubit DNA HS and performed profile analysis by TapeStation TS4200. All libraries look good and have sufficient yields.

value: Gastruloids

tag: stage (after_aggregation)

value: CHIR between 24h and 48h


title: BMDM TNFa+PGE2 stimulation 1 hour RNA-seq replicate 2

value: TNFa(10 ng/mL) + PGE2 (1uM)


title: BMDM TNFa+PGE2 stimulation 1 hour RNA-seq replicate 1


title: RNAseq of Early treatment gastruloid at 96h replicate 1


title: 0 mg FA non-neoplastic cells [LC1807]

description:
LC1807
1st cohort

value: Colitis induced by AOM and DSS

value: 0 mg folic acid


title: SAD2, CA3 neurons rep2 (RNA-seq)

value: CA3 neurons


title: Ctrl1, hpNPCs rep1 (ATAC-seq)

source_name: iPSM8 Sev3


title: BMDM TNFa stimulation 4 hour RNA-seq replicate 3

value: TNFa(10 ng/mL)


title: S068,Basilic vein,Healthy,NA

city: Miami

first: Anthony

last: Griswold

address: 1501 NW 10th Ave, BRB 318

institute: University of Miami

data_processing:
Raw sequence data was processed by the on-instrument Real Time Analysis software (v.2.4.11) to basecall files.
Basecall files were converted to de-multiplexed FASTQ files with the Illumina supplied scripts in the BCL2FASTQ software (v2.17).
The quality of reads was determined with the FASTQC software to evaluate per base sequence quality, duplication rates, and overrepresented k-mers.
Illumina adapters were trimmed from the ends of the reads using the Trim Galore! Package.
Reads were aligned to the human reference genome (hg38) with the STAR aligner (v2.5.0a).
Gene count quantification was performed using the GeneCounts function within STAR against the GENCODE v25 human transcript .gtf file.
Assembly: hg38
Supplementary files format and content: Processed file is a tab delimited matrix file with the first column representing the official gene symbol and subsequent columns raw gene counts generarted by --quantMode GeneCounts option in STAR. A read is counted if it overlaps (1bp or more) with an exon of only one gene on the appropriate strand.

source_name: Basilic vein

extract_protocol:
RNAlater-preserved samples were ground in liquid nitrogen and isolated with Trizol (Thermo Fisher Scientific) followed by column purification (EZNA Total RNA Kit I, Omega Bio-tek).
500 ng of total RNA was used as input for the Illumina TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero to generate ribosomal RNA-depleted sequencing libraries. Each sample had a unique barcode to allow multiplexing, and was sequenced to >40 million raw reads in a single end 75 bp sequencing run on the Illumina NextSeq500.

value: Basilic vein

tag: disease status

value: Healthy

tag: avf outcome


title: SAD2, CA3 neurons rep1 (RNA-seq)


title: BMDM TNFa stimulation 4 hour RNA-seq replicate 2

value: Bone marrow

tag: cell line

value: WT


title: SAD1, CA3 neurons rep1 (RNA-seq)

tag: cell type


title: BMDM TNFa stimulation 4 hour RNA-seq replicate 1

tag: genotype


title: RNAseq of control gastruloid at 96h replicate 3

tag: treatment

value: CHIR between 48h and 72h


title: S067,Basilic vein,Healthy,NA

tag: tissue


title: RNAseq of control gastruloid at 96h replicate 2

tag: strain


title: SAD1, CA3 neurons rep2 (RNA-seq)


title: BMDM TNFa stimulation 2 hour RNA-seq replicate 3


title: S062,Basilic vein,Healthy,NA


title: RNAseq of Early treatment gastruloid at 72h replicate 3


title: S059,Basilic vein,Healthy,NA


title: Ctrl1, hpNPCs rep2 (ATAC-seq)

value: Ctrl


title: RNAseq of Early treatment gastruloid at 96h replicate 2


title: RNAseq of Early treatment gastruloid at 72h replicate 2


title: FAD2, CA3 neurons rep2 (RNA-seq)

value: FAD


title: FAD2, CA3 neurons rep1 (RNA-seq)


title: BMDM TNFa stimulation 2 hour RNA-seq replicate 1

value: Bone marrow derived macrophages


title: S056,Basilic vein,Healthy,NA


title: RNAseq of Early treatment gastruloid at 72h replicate 1


title: FAD1, CA3 neurons rep2 (RNA-seq)


title: FAD1, CA3 neurons rep1 (RNA-seq)


title: S053,Basilic vein,Healthy,NA


title: BMDM TNFa stimulation 1 hour RNA-seq replicate 3


title: S051,Cephalic vein,Healthy,NA

source_name: Cephalic vein

value: Cephalic vein


title: RNAseq of control gastruloid at 72h replicate 2


title: S061,Basilic vein,Healthy,NA


title: RNAseq of control gastruloid at 72h replicate 1


title: S049,Basilic vein,Healthy,NA


title: RNAseq of control gastruloid at 96h replicate 1


title: Ctrl1, CA3 neurons rep2 (RNA-seq)


title: RNAseq of Early treatment gastruloid at 48h replicate 3


title: BMDM TNFa stimulation 1 hour RNA-seq replicate 1


title: BMDM TNFa stimulation 2 hour RNA-seq replicate 2


title: BMDM PGE2 stimulation 4 hour RNA-seq replicate 3

value: PGE2 (1uM)


title: RNAseq of Early treatment gastruloid at 48h replicate 2

value: Gastruloids


title: S045,Basilic vein,Healthy,NA


title: Ctrl1, CA3 neurons rep1 (RNA-seq)


title: BMDM PGE2 stimulation 4 hour RNA-seq replicate 2


title: SAD2, hpNPCs, treat rep1 (RNA-seq)

value: SAD

value: hpNPCs

value: cJun peptde +


title: BMDM PGE2 stimulation 4 hour RNA-seq replicate 1


title: RNAseq of Early treatment gastruloid at 48h replicate 1

value: CHIR between 24h and 48h


title: RNAseq of control gastruloid at 72h replicate 3


title: S044,Basilic vein,Healthy,NA


title: BMDM PGE2 stimulation 2 hour RNA-seq replicate 3


title: BMDM TNFa stimulation 1 hour RNA-seq replicate 2


title: S050,Basilic vein,Healthy,NA


title: Ctrl2, CA3 neurons rep2 (RNA-seq)

value: CA3 neurons


title: RNAseq of control gastruloid at 48h replicate 3

value: not treated


title: Ctrl2, CA3 neurons rep1 (RNA-seq)


title: S043,Basilic vein,Healthy,NA

value: Basilic vein


title: SAD2, hpNPCs, no treat rep1 (RNA-seq)

value: cJun peptde -


title: SAD1, hpNPCs, treat rep1 (RNA-seq)


title: BMDM PGE2 stimulation 2 hour RNA-seq replicate 1

value: Bone marrow


title: RNAseq of control gastruloid at 48h replicate 2

value: WT


title: BMDM PGE2 stimulation 2 hour RNA-seq replicate 2


title: S042,Basilic vein,Healthy,NA

value: Healthy


title: BMDM PGE2 stimulation 1 hour RNA-seq replicate 3


title: RNAseq of control gastruloid at 48h replicate 1


title: S040,Basilic vein,Healthy,NA


title: S041,Basilic vein,Healthy,NA


title: S047,Basilic vein,Healthy,NA


title: FAD2, hpNPCs, no treat rep1 (RNA-seq)


title: BMDM PGE2 stimulation 1 hour RNA-seq replicate 2


title: S039,Basilic vein,Healthy,NA


title: BMDM PGE2 stimulation 1 hour RNA-seq replicate 1


title: SAD1, hpNPCs, no treat rep1 (RNA-seq)


title: S037,Basilic vein,Healthy,NA


title: FAD1, hpNPCs, treat rep1 (RNA-seq)


title: FAD2, hpNPCs, treat, rep1 (RNA-seq)


title: shHEXB_3

city: shenyang

first: Xin

last: Chen

address: No. 36, Sanhao Street

institute: Shengjing Hospital Affiliated to China Medical University

data_processing:
CLC Genomics Workbench v 11.0.1
Sequence reads were trimmed for adaptor sequence/low-quality sequence using CLC genomic benchwork (parameter- Quality limit: 0.05)
Trimmed sequence reads were mapped to GrCM38/mm10 using CLC genomic benchwork (parameters- mismath cost: 2 insertion cost: 3 deletion cost: 3 length fraction: 0.8 similarity fraction: 0.8)
Read count extraction and normalization were performed using CLC genomic benchwork
Supplementary files format and content: mm10
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

source_name: Brain

growth_protocol: GSC1 cells were cultured in DMEM/F-12 medium (10565018, Gibco) containing 2% B27 supplement (17504044, Gibco), epidermal growth factor (EGF, 20 ng/ml), basic fibroblast growth factor (bFGF, 20 ng/ml) and heparin (2.5 μg/ml).

extract_protocol:
Total RNA from GSC1 cells were isolated by Trizol 1mL.
RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols.

value: Brain

value: Glioblastoma

value: shHEXB


title: shHEXB_1


title: shHEXB_2


title: BMDC(cDC1) cells, Zeb1 knockout [KO2]

city: Xiamen

first: Nengming

last: Xiao

state: 福建

address: Xiang an Campus Xiang an south Road Xiamen University, fengting 2 -2044

institute: Xiamen University

data_processing:
CLC Genomics Workbench v 11.0.1
Sequence reads were trimmed for adaptor sequence/low-quality sequence using trim_galore (parameter- Quality limit: 20)
Trimmed sequence reads were mapped to mm10 using hisat2 (parameters- hisat2 -x faindex -p 4 --dta -t --phred33 -1 _1.clean.fq.gz -2 *1.unmap.fq.gz --un-conc-gz .unmap.fq.gz)
Read count extraction and normalization were performed using featureCounts benchwork
Assembly: Ensemble version 94
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

extract_protocol:
Total RNA was isolated by Qiagen RNeasy Mini Kits (Qiagen) following the manufacturer’s protocol. 1-2ug of total RNA was used for the construction of sequencing libraries
RNA libraries for RNA-seq were prepared using NEBNextR UltraTM RNA Library Prep Kit following manufacturer's protocols.

value: bone marrow cells

value: Konckout

value: Unstimulation


title: shNC_3

value: shNC


title: FAD1, hpNPCs, no treat rep1 (RNA-seq)

value: FAD

value: hpNPCs


title: shNC_2


title: BMDC(cDC1) cells, Zeb1 knockout [KO1]


title: SAD2, hpNPCs rep2 (RNA-seq)

value: SAD


title: S018,Cephalic vein,Healthy,NA

value: Cephalic vein


title: BMDC(cDC1) cells, Control [WT2]


title: V578,Basilic vein,ESKD,Failed

value: ESKD

value: Failed


title: shNC_1


title: BMDC(cDC1) cells, Control [WT1]


title: SAD2, hpNPCs rep1 (RNA-seq)


title: V533,Basilic vein,ESKD,Failed

value: Basilic vein


title: SAD1, hpNPCs rep2 (RNA-seq)


title: Tumor_nonEC, scRNAseq

city: Chicago

first: Jalees

last: Rehman

state: IL

address: 909 S. Ashland Ave. MBRB 2072, Mailcode 669

department: Biochemistry and Molecular Genetics

institute: UIC

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v6.0.2 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Skeletal muscle

extract_protocol:
ECtdTomato mice with or without tumor for 3 weeks were used to dissect skeletal muscles (gastrocnemius, tibialis anterior, or quadriceps) from hindlimbs. The muscle tissues were minced and enzymatically digested with digestion buffer (2 mg/mL collagenase A, 1 mg/mL dispase II, 0.1 mg/mL DNase I in 1x PBS) at 37 °C for 30 min with gentle shaking.
Library was performed according to the manufacter’s instructions (single cell 3’ v3 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.

value: Non-Endothelial cells

value: Skeletal muscle

tag: age

value: 8-10 weeks old


title: V466,Basilic vein,ESKD,Failed


title: Normal fetal CD140a+ CTX_5

city: Rochester

first: Steven

last: Goldman

state: NY

address: 601 Elmwood Ave

department: Center for Translational Neuromedicine

institute: University of Rochester Medical Center

data_processing:
Basecalling was performed with Illumina CASAVA.
Raw FASTQ files were aligned to GRCh38 using Ensembl 95 gene annotations with STAR 2.5.4b in 2-pass mode across all samples.
Reads were quantified with RSEM 1.3.1.
Genome_build: GRCh38
Supplementary_files_format_and_content: bulkRNA_Expected_Counts.csv: Expected gene count matrix - output from RSEM.

source_name: Dissociated cells from normal fetal CTX, sorted on CD140a

extract_protocol:
mRNA was extracted and purified with the Qiagen RNeasy kit, according to the manufacturer's instructions.
Libraries were created by first generating cDNA with the SMARTer Ultra Low RNA kit (Clontech) and then taking 1ng of the resulting cDNA for the NexteraXT DNA kit (Illumina), according to the manufacturers’ instructions.

treatment_protocol: Tissue samples were dissociated with papain, cultured in suspension plates in serum-free media, and sorted via MACS and FACS on A2B5 (adult normal samples) or CD140a (fetal normal samples).

tag: tissue source

value: cortex

tag: developmental stage

value: fetal

tag: cell sort marker

value: CD140a+ (PDGFRA)


title: SAD1, hpNPCs rep1 (RNA-seq)


title: V459,Brachial vein,ESKD,Failed

source_name: Brachial vein

value: Brachial vein


title: FAD2, hpNPCs rep2 (RNA-seq)


title: Normal fetal CD140a+ CTX_4


title: Tumor_EC, scRNAseq

description: 10x Genomics

value: Endothelial cells


title: V388,Basilic vein,ESKD,Matured

value: Matured


title: Normal fetal CD140a+ CTX_1


title: Control_nonEC, scRNAseq

description: 10x Genomics


title: DC-WTA_FKDL192546079-1a

description: BD Rhapsody

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the BD Rhapsody bioinformatics pipeline (https://docs.sevenbridges.com/docs/command-line-interface)
sudo docker pull bdgenomics/rhapsody:1.7.1
sudo docker images
Choose the scripts version corresponding to your pipeline image version, for example, if you use the Version 1.7.1 pipeline
Choose rhapsody_targeted_1.7.1.cwl as the script to run the pipeline and template_targeted_1.7.1.yml to set up the input file.
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: spleen

extract_protocol:
We sorted cDC from spleen by FACS.
Library was performed according to the manufacte's instructions (BD Rhapsody Single-Cell Analysis System Instrument User Guid).

value: cDCs cells

value: spleen

value: spleen primary cell


title: V082,Basilic vein,ESKD,Matured


title: Control_EC, scRNAseq

description: 10x Genomics


title: FAD1, hpNPCs rep2 (RNA-seq)

value: FAD

value: hpNPCs


title: Normal adult A2B5+ WM_414

source_name: Dissociated cells from normal adult WM (epileptic resection), sorted on A2B5

value: white matter

value: adult


title: FAD2, hpNPCs rep1 (RNA-seq)


title: Normal adult A2B5+ WM_412


title: FAD1, hpNPCs rep1 (RNA-seq)


title: DC-SMK_FKDL192546080-1a

description: BD Rhapsody


title: V026,Basilic vein,ESKD,Failed

value: Basilic vein

value: ESKD

value: Failed


title: V077,Basilic vein,ESKD,Matured


title: cKO_3

city: Guangzhou

first: Meixiang

last: Yang

state: Guangdong

address: No.601, West Huangpu Avenue

institute: Jinan University

data_processing:
The libraries were sequenced on an llumina Novaseq 6000 platform and 150 bp paired-end reads were generated. About 50M raw reads for each sample were generated. Raw reads of fastq format were firstly processed using fastp and the low quality reads were removed to obtain the clean reads.
Then about 50M clean reads  for each sample were retained for subsequent analyses. The clean reads were mapped to the reference genome  using HISAT2.
FPKM of each gene was calculated and the read counts of each gene were obtained by HTSeq-count. PCA analysis were performed using R (v 3.2.0) to evaluate the biological duplication of samples.
Differential expression analysis was performed using the DESeq2. P value < 0.05 and foldchange > 1.5 or foldchange < 2/3 was set as the threshold for significantly differential expression gene (DEGs) .
Based on the hypergeometric distribution, GO, KEGG pathway, Reactome and WikiPathways enrichment analysis of DEGs were performed to screen the significant enriched term using R (v 3.2.0), respectively. R (v 3.2.0) was used to draw the column diagram, the chord diagram and bubble diagram of the significant enrichment term. Gene Set Enrichment Analysis (GSEA) was performed using R package (clusterProfiler).
Assembly: GRCm38.p6
Supplementary files format and content: xls file include FPKM values for each sample

source_name: uterus

extract_protocol:
Total RNA was extracted using the TRIzol reagent  (Invitrogen, CA, USA) according to the manufacturer’s protocol. RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Then the libraries were constructed using VAHTS Universal V6 RNA-seq Library Prep Kit according to the manufacturer’s instructions.

value: uterus

value: Mettl5 cKO


title: Normal adult A2B5+ WM_357


title: Ctrl2, hpNPCs rep2 (RNA-seq)

value: Ctrl


title: Normal adult A2B5+ WM_411


title: cKO_2

value: Mettl4 cKO


title: Ctrl1, hpNPCs rep2 (RNA-seq)


title: V121,Basilic vein,ESKD,Matured


title: MaplKO_Female5

city: Montreal

first: Aurèle

last: Besse-Patin

address: 3801 University Street

country: Canada

institute: McGill University

data_processing:
Filtering with Trimmomatic to remove adapter sequences, contamination, and low quality reads
Mapping using HISAT2
Counting using featureCounts
Differential expression analysis was done using DeSeq2
Assembly: mm10
Supplementary files format and content: excel sheet, differential expression analysis and data used in manscript graphs

source_name: Liver

growth_protocol: Mice were maintained in a vivarium with controlled temperature and light cycles, and allowed access to food and water ad libitum.

extract_protocol:
RNA was extracted using Trizol-chloroform, followed by purification on RNA-binding column.
Library was prepared using PolyA selection followed by KAPA RNA Hyperprep protocol, using xGen Dual Index UMI Adapters (IDT).

treatment_protocol: MAPL-/- and MAPLwt/wt mice were fasted overnight and refed for 2 hours. Pieces of liver were snap frozen in liquid nitrogen.

value: Liver

value: MaplKO


title: Normal fetal CD140a+ CTX_2

value: cortex

value: fetal

value: CD140a+ (PDGFRA)


title: cKO_1

value: Mettl3 cKO


title: Ctrl2, hpNPCs rep1 (RNA-seq)


title: MaplKO_Female4


title: Hey2 enhancer-nls-LacZ Tg, scRNAseq

city: Bunkyo-ku

first: Akiyasu

last: Iwase

state: Tokyo

department: Graduate School of Medicine

institute: The University of Tokyo

description: 10x Genomics 3' scRNA-seq

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v6.0.2 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Supplementary files format and content: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: embryonic tissue

extract_protocol:
E7.5 embryos were collected in DMEM + 25mM HEPES, and kept on ice during genotyping PCR.  Four Hey2 enhancer-LacZ Tg embryos were selected, and distal part of extraembryonic tissue and posterior half of the embryo were removed.  The sample was treated in 0.25% Trypsin-EDTA with DNase I (50U/ml) (260913-10MU, Millipore) for 10 min at room temperature, and then the reaction was stopped by adding 1/10 volume of fetal bovine serum (FBS).  The dissociated cells were centrifuged at 500g for 5 minutes, and resuspended in PBS + 10% FBS.  The cells were filtered through the cell strainer (70 micrometer, Falcon #352350), and centrifuged at 300g for 5 minutes.  The cells were resuspended in PBS + 10% FBS, and measured cell count and viability.
Library was performed according to the manufacter’s instructions (single cell 3’ v3 protocol, 10x Genomics).

value: embryonic tissue

value: Hey2 enhancer-nls-LacZ


title: MaplKO_Female3


title: Ctrl1, hpNPCs rep1 (RNA-seq)

data_processing:
ATAC-seq data were aligned to hg19 using BWA-mem
RNA-seq data were aligned to hg19 using STARR-2pass
ATAC-seq peaks were called with MACS2.
RNA-seq gene expression was quantified using Kallisto
Assembly: Hg19
Supplementary files format and content: peak text files and Bigwig for ATAC-seq, Kallisto quantification (spreadsheet) for RNA-seq

extract_protocol:
[RNA extraction protocol] Cells were lysed in Tri-reagent (Zymo research) and total RNA was extracted using Quick-RNA Miniprep kit (Zymo research).
[RNA-seq]  RNA libraries were prepared using NEBNext® Poly(A) mRNA Magnetic Isolation Module, NEBNext® UltraTM II Directional RNA Library Prep Kit for Illumina® and NEBNext® UltraTM II DNA Library Prep Kit for Illumina® according to the manufacturer?s instructions (New England Biolabs). 1 ug of total RNA input was used for each library
[ATACseq]  50000 cells per condition were processed as described in the original ATAC-seq protocol paper (Buenrostro et al 2013)

value: hpNPCs


title: Control_3

value: WT


title: Control_2


title: Control_1


title: Input

city: west Haven

first: Sarah

middle: A.

last: Slavoff

state: CT

address: 600 west campus drive

department: Chemistry

institute: Yale university

description: HEK 293T stably expressing EMBOW-FH, Input for FLAG

data_processing:
All reads were mapped to the human genome (hg38) using Bowtie2 (version 2.4.5)
The BigWig normalized signal files were generated using bamCoverage from deepTools (version 3.5.1)
WDR5- or EMBOW-enriched regions were predicted with MACS2 (version 2.2.7.1)
To detect changes in WDR5 and H3K4me3 levels in the wild-type and EMBOW KO HEK 293T cells, we calculated normalized WDR5 and H3K4me3 ChIP-seq tag densities according to the EMBOW-dependent WDR5 target genes using computeMatrix from deepTools, and plotted the tag densities using plotProfile from deepTools.
Assembly: hg38
Supplementary files format and content: bigWig, narrowPeak (except for Input sample)

growth_protocol: DMEM, 10% fetal bovine serum, 1x penicillin/streptomycin. Cells were cultured at 37C at 5% CO2.

extract_protocol:
Lysates were clarified from Mnase digested nuclei and histone-DNA complexes were isolated with antibody.
Libraries were prepared by Yale Center for Genomic Analysis.

value: Human embryonic kidney

tag: chip antibody


title: MaplKO_Female2


title: FLAG-IP

description: HEK 293T stably expressing EMBOW-FH, anti-FLAG beads ChIP

value: anti-FLAG beads (Sigma, A2220)


title: MaplKO_Female1


title: KO-Input-2

description: EMBOW KO HEK 293T, Input for H3K4me3


title: BMDC(cDC1) cells, Zeb1 knockout, Hours4, 2

data_processing:
microRNA Workbench
Sequence reads were trimmed for adaptor sequence/low-quality sequence using fastx_toolkit (parameter- Quality limit: 20)
Trimmed mapped against mature miRNAs in the mirBase using BWA tool
Trimmed sequence reads were mapped to mature.fa and hairpin.fa using bowtie2
miRNAs expression counts were obtained with miRBase
Assembly: mature.mmu.fa hairpin.mmu.fa
Supplementary files format and content: tab-delimited text files include TPM values for each Sample

extract_protocol:
Total RNA was isolated by Qiagen RNeasy Mini Kits (Qiagen) following the manufacturer’s protocol. 1-2ug of total RNA was used for the construction of sequencing libraries.
The small RNA libraries were prepared from a total of 2 ug total RNA isolated from each sample using NEBNextR Multiplex Small RNA Library Prep set for IlluminaR (NEB, USA.) according to the manufacturer's instruction.

value: bone marrow cells

value: Konckout

value: HKLM-OVA

tag: time


title: Control_Female5

value: wt/wt


title: BMDC(cDC1) cells, Zeb1 knockout, Hours4, 1


title: WT-Input-2

description: Wild-type HEK 293T, Input for H3K4me3


title: KO-H3K4me3

description: EMBOW KO HEK 293T, anti-H3K4me3 ChIP

value: anti-H3K4me3 antibody (Abclonal, A2357)


title: BMDC(cDC1) cells, Control, Hours4, 2


title: Control_Female3

value: Liver


title: Control_Female2


title: BMDC(cDC1) cells, Control, Hours4, 1


title: Control_Female1


title: Mouse KPC COX2-KO day7 Tumor scRNAseq pool of 3 mice

description: 10X Genomics

data_processing:
Fastq files were processed with Cell Ranger (v 4.0.0), using default parameters
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files (gzipped format)

source_name: Tumor

extract_protocol:
Murine healthy pancreas and tumors were manually minced in small pieces and dissociated with the Tumor Dissociation kit, mouse (Miltenyi Biotec). The obtained single cells suspensions were filtered on 70µm cell strainers, incubated with RBC lysis buffer for 10 minutes on ice and resuspended in the appropriate buffer for cell counting and down-stream application. For the heterotopic and orthotopic inoculation of KC cell line, cells were enriched in the myeloid fraction as CD45+Cd11b+ via FACS sorting. Mouse PB samples were incubated with Red Blood Cell (RBC) lysis buffer (Biolegend) for 10 minutes on ice and washed with Phosphate Buffered Saline (PBS). Cells were centrifuged for 5 minutes at 450 x g and resuspended in the appropriate buffer for down-stream application.
scRNA-Seq libraries were generated using a microfluidics-based approach on Chromium Single-Cell Controller (10X Genomics) using the Chromium Single Cell 3′ Reagent Kit v3.1, according to the manufacturers’ instructions. For the heterotopic and orthotopic inoculation of KC cell line, libraries generated using the Chromium Single Cell 3′ Reagent Kit v2.

value: Tumor

tag: Sex

value: F

value: Day 7


title: KO-Input-1

description: EMBOW KO HEK 293T, Input for WDR5


title: WT-Input-1

description: Wild-type HEK 293T, Input for WDR5


title: BMDC(cDC1), Zeb1, proteintech 2 [CUT&Tag-Seq]

data_processing:
library strategy: CUT&Tag-Seq
Basecalls were performed using bcl2fastq v2.17 for Novaseq output.
CUT&Tag-Seq reads were trimmed from 3' end until the final base had a quality score > 30, using Trimmomatic v0.33, discarding reads left with < 20 bp
CUT&Tag-seq reads were aligned to the UCSC mm10 genome using Bowtie version 1.1.2. Only uniquely mapping reads with at most two mismatches were retained.
Peaks were called uing MACS v2.1.0 with the significance cut-off q-value <=0.01
bigWig files were generated using the bedGraphToBigWig. Score represents the normalized coverage of DNA fragments at a given genomic coordinate. narrowPeak files were generated using MACS v2 with default settings.
Assembly: mm10
Supplementary files format and content: bigWig, narrowPeak

extract_protocol:
cDC1 sorted from BMDC and stimulated with HKLM-OVA for 4 hours.
CUT&Tag libraries were prepared using Hyperactive In-Situ ChIP Library Prep Kit for Illumina (pG-Tn5)

value: Bone Marrow

tag: antibody

value: Zeb1 (proteintech, 21544-1-AP)


title: MaplKO_Male5

value: MaplKO


title: KO-WDR5

description: EMBOW KO HEK 293T, anti-WDR5 ChIP

value: anti-WDR5 antibody (Invitrogen, 9H25L13)


title: Control_Female4


title: MaplKO_Male4


title: BVC5050

city: Leicester

first: Nicolas

last: Sylvius

address: University Road

department: CBS-Genetics

institute: University of Leicester

hyb_protocol: Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C .  After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.

scan_protocol: Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides

data_processing: The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent)

source_name: Vehicle

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA).  Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.

extract_protocol: RNA was extracted on a Promega Maxwell 16 robot using the Maxwell 16 LEV simplyRNA Cells Kit (Reference AS1270). RNA quality and quantity was assessed using the 2100 Bioanalyzer Instrument (Agilent)

treatment_protocol: Thirteen-week-old female VillinCreER/0/BrafV600E-LSL/+ mice received either 10 mg/kg atorvastatin by oral gavage or 5% DMSO/PBS vehicle control daily for 12 days.  A 20 mg/mL atorvastatin (Generon) stock in DMSO was freshly diluted to a 1 mg/mL working solution in PBS. Days 1-7 of the study were a daily pre-treatment period of atorvastatin/vehicle. Days 8-10 consisted of both atorvastatin/vehicle gavage and intraperitoneal tamoxifen injection to induce BrafV600E expression. On the final day of the study at 3-days post- BrafV600E induction, mice received intraperitoneal injections of 200 μL of 20 mM BrdU solution (Sigma) 3 hrs before tissue harvesting.

value: Intestinal tissue

value: Treated with vehicle


title: BMDC(cDC1), Zeb1, proteintech 1 [CUT&Tag-Seq]


title: BVC5063


title: WT-WDR5

description: Wild-type HEK 293T, anti-WDR5 ChIP

value: Human embryonic kidney


title: MaplKO_Male3


title: WT-H3K4me3

description: Wild-type HEK 293T, anti-H3K4me3 ChIP


title: BVC5043

source_name: MEKi

value: treated with MEK inhibitor PD184352


title: BVC5018


title: BVC5045


title: BMDC(cDC1) cells, Zeb1 knockout, Hours4 [HKLM_KO2]

data_processing:
CLC Genomics Workbench v 11.0.1
Sequence reads were trimmed for adaptor sequence/low-quality sequence using trim_galore (parameter- Quality limit: 20)
Trimmed sequence reads were mapped to mm10 using hisat2 (parameters- hisat2 -x faindex -p 4 --dta -t --phred33 -1 _1.clean.fq.gz -2 *1.unmap.fq.gz --un-conc-gz .unmap.fq.gz)
Read count extraction and normalization were performed using featureCounts benchwork
Assembly: mm10
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

value: bone marrow cells

value: 4 hours


title: BVC 5009 (5)


title: BVC5039


title: BVC5058


title: PBMC-scATAC

city: Shenzhen

first: Jiacheng

last: Zhong

address: Longmen north rd

institute: shenzhen people's hospital

description: 10x Genomics

data_processing:
PBMC-scRNA/scVDJ: Sequencing data were processed using the Cell Ranger 6.0.1 pipeline and aligned to GRCh38-1.1.0 reference genome to generate cell-by-gene count matrix (GEX) or filtered contig annotations (scVDJ).
BALF-scRNA: Sequencing data were processed using the Cell Ranger 6.0.1 pipeline and aligned to GRCh38-1.1.0 reference genome to generate cell-by-gene count matrix (scRNA).
PBMC-scATAC: Sequencing reads were processed through the Cell Ranger ATAC 1.1.0 pipeline and aligned to GRCh38-1.1.0 reference genome for mapping, de-duplication, and identification of Tn5 cut sites.
Assembly: GRCh38-1.1.0
Supplementary files format and content: filtered_contig_annotations.csv, consensus_annotations.csv
Supplementary files format and content: Tar archives include barcodes.tsv, features.tsv, matrix.mtx files.
Supplementary files format and content: fragments.tsv

source_name: Peripheral blood

extract_protocol:
Nuclei were isolated from the patient's PBMC and counted by Trypan Blue.
scATAC-seq libraries were prepared following the 10X Genomics Chromium Single Cell ATAC Reagent Kits User Guide.

value: Peripheral blood

value: Peripheral blood mononuclear cells

tag: diagnosis

value: Hyper-IgE syndrome (HIES)

value: Male

value: 18 years


title: BVC5017


title: MaplKO_Male2

value: Liver


title: BMDC(cDC1) cells, Zeb1 knockout, Hours4 [HKLM_KO1]

value: Konckout

value: HKLM-OVA


title: BMDC(cDC1),Rabbit IgG Isotype Control, proteintech [CUT&Tag-Seq]

value: Bone Marrow

value: Rabbit IgG Isotype control(CST,3900)


title: VCB 5477 (5)


title: BMDC(cDC1) cells, Control, Hours4 [HKLM_WT2]

value: WT


title: MaplKO_Male1

value: MaplKO


title: BALF-scRNA

description: 10x Genomics

source_name: Bronchoalveolar lavage fluid

extract_protocol:
BALF sample was collected from the patient and subjected to scRNA-seq according to the manufacturer’s protocol.
For BALF sample, Single Cell 3' v3 Reagent Kits (10X Genomics, Pleasanton, CA, USA) were used during library preparation.

value: Bronchoalveolar lavage fluid

value: Bronchoalveolar lavage cells


title: PBMC-scRNA

description: 10x Genomics

extract_protocol:
PBMC sample was collected from the patient and subjected to GEX/V(D)J-seq according to the manufacturer’s protocol.
For PBMC, single cell suspension was loaded into 10x Genomics Chromium system using Chromium Next GEM Single Cell 5' v1.1 to generate TCR/BCR libraries and 5' GEX libraries.


title: Control_Male5

value: wt/wt


title: VCB 5489 (5)

value: Intestinal tissue


title: BMDC(cDC1) cells, Control, Hours4 [HKLM_WT1]

value: bone marrow cells

value: 4 hours