title: RNA vEDS fibroblast P18_biological replicate

city: Brescia

first: Nicola

last: Chiarelli

state: Lombardy

address: Viale Europa 11

department: Molecular and Translational Medicine

country: Italy

institute: University of Brescia

data_processing:
Raw sequences were aligned and processed by Torrent Suite 5.12.3 according to standard analysis procedures. Output normalized counts (in read per milion) were generated (*.chp files ).  Differentially expressed genes (DEGs) were identified by using the Transcriptome Analysis Console (TAC) software (version 4.0.2) with the ampliSeqRNA plugin (Thermofisher Scientific) and applying an ANOVA test with a fold-change threshold of ±1.5 and an FDR-adjusted p-value ≤ 0.05.
Assembly: hg19_AmpliSeq_Transcriptome_21K_v1.transcript
Supplementary files format and content: Output normalized counts (in read per milion) were generated (*.chp files ).

source_name: dermis

growth_protocol: Control and vEDS fibroblasts were grown between 1st and 4th in vitro passage at 37 °C in a 5% CO2 atmosphere in Earle's Modified Eagle Medium supplemented with 2 mM L-glutamine, 10% FBS, and 100 µg/ml penicillin and streptomycin

extract_protocol:
Total RNA was purified from patient and control cells grown for 72 hours at 70-80% of confluency using the miRNeasy kit (Qiagen). Sample quality and quantity were evaluated with the QIAxcel (QIAGEN) and the Qubit fluorometer (Thermofisher Scientific), respectively.
To improve statistical power, RNA samples from all experimental groups were sequenced twice as biological replicates (BR) (at the 2nd and 4th in vitro passage, respectively), for a total of 108 sequenced libraries. Reverse transcription and library preparation were achieved following the standard protocol of the Ion Ampliseq Transcriptome Human Gene Expression Kit (Thermofisher Scientific), which targets by multiplexed PCR 18,574 protein-coding and 2,228 non-coding RNAs (mRNAs/ncRNAs). Libraries were quantified with the Ion qPCR Library Quantification Kit, diluted to 100 pM and pooled equally, with 8 samples per pool, followed by emulsion PCR and enrichment with the Ion OneTouch2 with ES system. Libraries were then loaded onto an Ion 540 chip and sequenced on the Ion S5 instrument.

value: dermis

value: Skin fibroblasts

value: vEDS

tag: disease state

value: affected


title: control24_rep3

status: Public on Oct 13 2023

type: SRA

city: Aachen

first: Mingbo

last: Cheng

state: North Rhine-Westphal

address: Pauwelsstr. 19

country: Germany

institute: Uniklinik RWTH Aachen

library_source: transcriptomic

data_processing:
sc/snRNA: The Cell Ranger Software Suite (version 3.1.0) was used for processing sequencing information and single cell barcodes.
sc/snRNA: Seurat (version 3.2.0) was used to integrate the scRNA-seq data and predict cell labels.
bulk RNA-seq: Demultiplexing is run using bcl2fastq,alignment peformed using STAR,counts matrix generated using Salmon. We utilized Nextflow nf-core/rnaseq(v3.0) to process the data
Genome_build: snRNA: mm10 premRNA
Genome_build: scRNA: mm10 RNA_tdTomato
Genome_build: bulk RNA-seq: GRCh38
Supplementary_files_format_and_content: barcodes: barcodes present in input data
Supplementary_files_format_and_content: matrix: a count matrix with Market Exchange Format (MEX). The first line contains the type code and the second line includes format and cell-ranger versions. Number of peaks, barcodes, and non-zero entries can be found in the third line.  The rest of the lines represent  nonzero entries with the first two numbers corresponding to peak and barcode indices and the last one corresponding to  the number of cut sites.
Supplementary_files_format_and_content: genes: a bed file contains genes corresponding to row indices. For each gene, its gene ID and name are stored in the first and second column of the gene.tsv file, respectively
Supplementary_files_format_and_content: bulkRNA_count_matrix: bulk RNA raw feature count matrix, each row is a gene, each column is a condition

molecule: total RNA

organism: Homo sapiens

source_name: PDGFR beta + cells of Human heart

extract_protocol:
All protocols to generate sc/snRNA-seq data on the 10x Chromium platform, including sample prep, library prep, instrument and sequencing settings, are available here: https://support.10xgenomics.com/single-cell-gene-expression
All protocols to generate bulk RNA-seq data on the Sanger / Illumina 1.9 platform

tag: tissue

value: Heart

tag: Stage

value: control


title: sc-RNA seq S1

city: Beijing

first: Xi-Yin

last: Zhang

address: College Road

department: Neuroscience Research Institute

country: China

institute: Peking University

description: New library for GSE198608

data_processing:
Illumina Casava1.7 software used for basecalling.
Extract Barcode and UMI: umi_tools extract.
Trim TSO and PolyA: trim_TSO_polyA.pl, seqtk trimfq.
Mapping With STAR, add feature using featureCounts of subread
Sort and index bam file using Samtools
Assembly: Rnor_6.0.102
Supplementary files format and content: Matrix table with raw gene counts for every gene and every cell-id

molecule: polyA RNA

organism: Rattus norvegicus

source_name: Dorsal root ganglia

growth_protocol: Specific pathogen-free (SPF) male Sprague-Dawley (SD) rats weighing 200 g were supplied by the Animal Center of Peking University Health Science Center. The animals were housed in an SPF laboratory in ventilated cages (IVC) (3-5 rats per cage) on a 12-hour light-dark cycle (lights on at 8:00 AM) with free access to food and water. All experiments were performed according to the guidelines of the Animal Care and Use Committee of Peking University, and efforts were made to minimize the discomfort of the animals.

extract_protocol:
A modified Smart-seq2 protocol developed by the Tang laboratory was used for single-cell RNA-seq. Briefly, acutely dissociated DRG neurons were plated on dishes as described previously. After 15 min, the DMEM was replaced with PBS, and a mouth pipette was then used to place each individual fluorescently labeled single DRG neuron into a volume of lysis buffer under a fluorescence microscope. Harvesting of the single DRG neurons from one rat was completed in 4 h, and the L4-5 DRG of at least 5 rats were harvested at each time point.
The reverse transcription reaction was performed with a TSO primer added at the 5’ end and with 25 nt oligo (dT) primer anchored with an 8 nt cell-specific barcode and 8 nt unique molecular identifiers (UMIs) added at the 3’ end. After reverse transcription, cDNAs were amplified by 15 cycles of PCR, during which the IS primer and the 3’P2 primer were added to the 5’ and 3’ ends of the cDNAs, respectively. The amplified cDNAs from single cells were pooled, purified using a DNA Clean & concentration-5 kit (Zymo Research, D4014) and AMPure XP beads (Beckman Coulter, A63882), and quantified using a Qubit dsDNA HS Assay Kit (Invitrogen, Q32854). To add biotin tags to the 3’ ends of the amplified cDNAs, biotinylated preindexed primers and IS primer were used to further amplify the PCR product through an additional 4 cycles of PCR. The biotinylated cDNAs were then sheared to approximately 300 bp using Covaris S2, and the 3’ termini of the cDNAs were captured using C1 beads (Dynabeads® Myone™ Streptavidin C1, Invitrogen, 65001). The RNA-seq library was constructed using a Kapa Hyper Prep Kit (KAPA Biosystems, KK8505), and short universal primers and QP2 primers were used in the last amplification. Finally, the genome and transcriptome libraries were subjected to 150 bp paired-end sequencing on an Illumina HiSeq 4000 platform (sequenced by Novogene).

treatment_protocol: During the surgery, the rats were anesthetized using isoflurane, and a 2 cm incision was made on the skin at the mid-thigh level of the left thigh to expose the left sciatic nerve. The three branches of the nerve were carefully separated using a glass dissecting needle, and the tibial and common peroneal nerves were crushed for 30 s using 3 clicks of an ultrafine microscope forceps (WA2030, JingZhong, Shanghai, China), leaving the remaining sural nerve intact. The muscle and skin were then closed in two layers, and penicillin was applied to the wound. For the sham surgery, the sciatic nerve was exposed but left uninjured. Animals with marked weight loss or severe immobility at 7 days postsurgery were excluded from the study. For the medial plantar sensory recovery (pinprick and light touch sensitivity) test and skin biopsy, we exposed the saphenous nerve above the knee region and ligated and transected it after SNC, making the medial plantar epidermis innervated only by regenerated sciatic nerve axons.

value: Dorsal root ganglia

tag: cell type

value: Neuron

tag: strain

value: SD

tag: post-surgical time_point

value: Sham-0 day


title: control24_rep2


title: control48_rep1


title: control24_rep1


title: RNA vEDS fibroblast P17

tag: genotype


title: US_rep2

description: bulkRNA_Hypertrophy_raw_feature_bc_counts.tsv

source_name: treatment uremic

value: treatment


title: US_rep3


title: RNA vEDS fibroblast P18


title: S_14_Ambs_214

city: Bethesda

first: Stefan

last: Ambs

state: MD

address: Building 37, Room 3050B

department: CCR

country: USA

institute: NCI

data_processing:
Reads of the samples were trimmed for adapters and low-quality bases using Cutadapt before alignment with the reference genome (hg38) and the annotated transcripts using STAR. The average mapping rate of all samples is 96%. Unique alignment is above 63%.
The gene expression quantification analysis was performed for all samples using STAR/RSEM tools.
Assembly: hg38
Supplementary files format and content: tab-delimited text files include raw count data for each Sample

source_name: frozen breast tumors

extract_protocol:
RNA isolation was done using TRIzol method . Integrity of isolated RNA was evaluated with the Agilent 2100 Bioanalyzer (Agilent Technologies) and  sequencing was performed on an Illumina NovaSeq S2 machine.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: accession

tag: type

value: normal

tag: age (at surgery)

tag: race

value: Kenyan

tag: er status

value: Positive

tag: pr

value: Negative


title: S_12_Ambs_212


title: GS_rep3

source_name: healthy serum


title: S_13_Ambs_213

value: tumor


title: RNA vEDS fibroblast P17_biological replicate


title: RNA vEDS fibroblast P16


title: S_15_Ambs_215


title: control48_rep2


title: RNA vEDS fibroblast P16_biological replicate


title: US_rep1


title: S_11_Ambs_211


title: GS_rep2


title: S_9_Ambs_209


title: GS_rep1


title: RNA vEDS fibroblast P15_biological replicate


title: S_10_Ambs_210


title: RNA vEDS fibroblast P15


title: RNA vEDS fibroblast P14_biological replicate


title: S_8_Ambs_208


title: RNA vEDS fibroblast P14


title: S_7_Ambs_207


title: RNA vEDS fibroblast P13_biological replicate


title: Naïve B cell rep 3

city: New York

first: Ashley

middle: Stephen

last: Doane

state: NY

address: 1300 York Ave

institute: Weill Cornell Medicine

library_source: genomic

data_processing:
complete processing pipeline avaialble at https://github.com/nf-core/atacseq
Read adapter trimming using ngtrim
Alignment to mm10 using bowtie2
PCR duplicate removeal by piccard tools
Peak calling using genrich
Assembly: GRCm38
Supplementary files format and content: Bigwig files contain ATAC-seq fragment density, scaled to library size.  Peak files are in narrowPeak format as output from MACS2.

molecule: genomic DNA

organism: Mus musculus

source_name: splenocytes

growth_protocol: Splenocytes from C57BL/6 mice were sorted for germinal center B cells (B220+, FAS+, GL7+) and naïve B cells (B220+, CD38+, FAS-, IgD+). B cells grown ex vivo were isolated from splenocytes using EasySep Mouse B Cell Isolation Kit (Stem Cell Technologies, catalog no. 19854).

extract_protocol:
Omni-ATAC-seq https://www.nature.com/articles/nmeth.4396
Illumina nextera per protocol https://www.nature.com/articles/nmeth.4396

treatment_protocol: For ex vivo experiments, B cells were enriched using EasySep Mouse B Cell Isolation Kit (Stem Cell Technologies, catalog no. 19854), grown in RPMI media with LPS, transduced with Pou2af1 expression + GFP vector or control empty +GFP vector, and sorted for GFP 3d after transduction.

tag: facs markers

value: Naive B


title: S_6_Ambs_206


title: Naïve B cell rep 2


title: RNA vEDS fibroblast P13

value: dermis


title: S_4_Ambs_204


title: RNA vEDS fibroblast P12_biological replicate

value: Skin fibroblasts


title: S_5_Ambs_205


title: Naïve B cell rep 1


title: RNA vEDS fibroblast P12

value: vEDS


title: GC B cell rep 5

value: Germinal Center B cell


title: RNA vEDS fibroblast P11_biological replicate

value: affected


title: GC B cell rep 4


title: GC B cell rep 3


title: S_3_Ambs_203


title: RNA vEDS fibroblast P11


title: S_2_Ambs_202


title: GC B cell rep 2


title: RNA vEDS fibroblast P10_biological replicate


title: Naïve B cell rep 4


title: Naïve B cell rep 5


title: S_1_Ambs_201


title: RNA vEDS fibroblast P10


title: S_45_Ambs_245


title: S_46_Ambs_246


title: Embryoid_body_day_7_CD71+_R2-insertion_PolyAminus_RNA-seq

city: Oxford

first: Emily

last: Georgiades

address: John Radcliffe Hospital/Headley Way

country: United Kingdom

institute: Weatherall Institute of Molecular Medicine

description: EB, day 7, R2-insertion, PolyA-

data_processing:
All data was processed using the RNA-seq upstream pipeline (https://github.com/Hughes-Genome-Group/upstream_pipelines/tree/main/07_Bulk-RNA-Seq)
aligner_algorithm: STAR (v2.7.1a)
samtools_sort_extra: -m 4G (samtools v1.15.1)
bam_coverage: --binSize 1 --normalizeUsingRPKM --extendReads (deepTools v3.5.1)
Assembly: mm39
Supplementary files format and content: bigwig

source_name: Erythroid

extract_protocol:
Cells were grown, treated and processed as described in the Methods section of the paper.
Libraries prepared for Illumina sequencing using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs: E7490) and the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs: E7760) following the manufacturer’s instructions.

value: Erythroid

tag: origin cell_line

value: E14TG2a.IV mouse ES cells

tag: differentiation day

value: Day 7


title: Embryoid_body_day_7_CD71+_WT_PolyAminus_RNA-seq

description: EB, day 7, WT, PolyA-

growth_protocol: Cells were grown, treated and processed as described in the Methods section of the paper.


title: GC B cell rep 1


title: RNA vEDS fibroblast P8_biological replicate


title: Embryoid_body_day_7_CD71+_WT_PolyAplus_RNA-seq

description: EB, day 7, WT, PolyA+

treatment_protocol: Cells were grown, treated and processed as described in the Methods section of the paper.


title: S_42_Ambs_242


title: S_43_Ambs_243


title: S_41_Ambs_241


title: Embryoid_body_day_7_CD71+_ATAC-seq _rep3

description: EB, day 7, rep3

data_processing:
genome build/assembly: mm39
All data was processed using the CATCH-up pipeline (https://github.com/Hughes-Genome-Group/UpStreamPipeline/tree/main/genetics/catch-up)
aligner_algorithm: bowtie2 (bowtie2 v2.4.5)
samtools_sort_extra: -m 4G (samtools v1.15.1)
filtering_extra: -bShuF 4 -f 3 -q 30  (samtools v1.15.1)
bam_coverage: --binSize 1 --normalizeUsingRPKM --extendReads (deepTools v3.5.1)
Supplementary files format and content: bigwig

growth_protocol: mES E14 cells were differentiated into embryoid bodies and erythroid cells isolated using a CD71+ selection following the published protocol (Francis, 2022). Cells were then processed for ATAC-Seq as in (Buenrostro et al, 2013).

extract_protocol:
Assay for transposition of active chromatin sequencing (ATAC-Seq) was performed as previously published (Buenrostro, 2013). 75,000 cells were used per biological replicate.
Libraries were amplified and barcoded using the NEBNext 2xMastermix (NEB) and the custom primers as published in Buenrostro et al., 2013. ATAC-Seq libraries profiles were visualized using D1000 tape on the Tapestation (Agilent). The libraries were quantified using the universal library quantification kit (KAPA Biosystems). Samples were sequenced using 40 bp paired end reads (NextSeq platform).

tag: selection antibody

value: CD71 FITC, eBioscience #11-0711-82


title: S_40_Ambs_240


title: RNA vEDS fibroblast P8

tag: disease state


title: Embryoid_body_day_7_CD71+_R2-insertion_PolyAplus_RNA-seq

description: EB, day 7, R2-insertion, PolyA+


title: Embryoid_body_day_7_CD71+_ATAC-seq _rep2

description: EB, day 7, rep2


title: RNA vEDS fibroblast P7

tag: tissue


title: RNA vEDS fibroblast P7_biological replicate


title: S_38_Ambs_238_resent

tag: Stage


title: S_39_Ambs_239


title: mESC E14, WT, Capture-C

description:
mESC-E14_WT_CaptureC
mm39_LPx6_oligofile.bed

data_processing:
library strategy: Capture-C
All data was processed using the CapCruncher tool (https://github.com/sims-lab/CapCruncher), with viewpoints listed in mm39_LPx6_oligofile.bed.
restriction_enzyme: dpnii
reporter_exclusion_zone: 1000
aligner: bowtie2, index_flag: -x, options: -p 6 --very-sensitive
Normalisation scale_factor: 1000000
Assembly: mm39
Supplementary files format and content: bigwig

source_name: Embryonic Stem Cell

extract_protocol:
Cells were grown, treated and processed as described in the Methods section of the paper.
Capture-C library was generated as described in the Methods section of the paper and sequenced on the Illumina Nextseq platform using a 300-cycle paired-end kit (NextSeq 500/550 Mid Output Kit v2.5: 20024905)

tag: cell type

value: Embryonic Stem Cell


title: RNA vEDS fibroblast P9_biological replicate


title: S_37_Ambs_237


title: RNA vEDS fibroblast P6


title: EB, day 7, WT, Capture-C

description:
Embryoid_body_day_7_CD71+_WT_CaptureC
mm39_LPx6_oligofile.bed

tag: strain


title: EB, day 7, WT, RAD21

description: Embryoid_body_day_7_CD71+_WT_RAD21_ChIP-seq

extract_protocol:
Cells were grown, treated and processed as described in the Methods section of the paper.
The NEBNext Ultra II DNA Library Prep Kit (New England Biolabs: NEB: 7645S/L) was used to prepare indexed sequencing libraries following the manufacturer supplied protocol. PCR amplification was performed for 7-11 cycles (depending on input DNA concentration) using NEBNext Mulitplex Oligos (New England Biolabs: E7335S/L and E7500S/L). Indexed sample concentration was quantified using the KAPA Library Quantification Complete Kit (Universal)(Roche: KK4824). Samples were pooled as a 2 nM or 4 nM library and sequenced with a high-Output v2 75 cycle kits on the Illumina NextSeq 500 platform.

tag: antibody

value: abcam, ab992


title: S_36_Ambs_236

value: normal


title: S_44_Ambs_244

value: Kenyan


title: RNA vEDS fibroblast P9

tag: genotype


title: S_35_Ambs_235

tag: accession

value: Positive


title: RNA vEDS fibroblast P5


title: EB, day 7, WT, H3K4me3

description: Embryoid_body_day_7_CD71+_WT_H3K4me3_ChIP-seq

value: abcam, ab8580

tag: antibody lot_#


title: RNA vEDS fibroblast P4_biological replicate


title: EB, day 7, WT, H3K4me1

description: Embryoid_body_day_7_CD71+_WT_H3K4me1_ChIP-seq

value: abcam, ab8895


title: RNA vEDS fibroblast P4


title: Embryoid_body_day_7_CD71+_ATAC-seq _rep1

description: EB, day 7, rep1


title: RNA vEDS fibroblast P6_biological replicate


title: S_33_Ambs_233

tag: type

value: tumor

tag: age (at surgery)

value: Negative


title: RNA vEDS fibroblast P3_biological replicate

value: dermis

value: Skin fibroblasts


title: EB, day 7, WT, H3K27Ac

description: Embryoid_body_day_7_CD71+_WT_H3K27Ac_ChIP-seq

value: abcam, ab4729


title: EB, day 7, R2-only, RAD21

description: Embryoid_body_day_7_CD71+_R2-only_RAD21_ChIP-seq

value: CD34+ human erythroid

value: Day 13


title: S_31_Ambs_231

tag: race

tag: er status


title: S_30_Ambs_230

tag: pr


title: EB, day 7, WT, CTCF

description: Embryoid_body_day_7_CD71+_WT_CTCF_ChIP-seq

value: Erythroid

tag: origin cell_line

value: E14TG2a.IV mouse ES cells

value: Merck Millipore, 07-729


title: RNA vEDS fibroblast P2

value: vEDS

value: affected


title: mESC E14,  WT, CTCF

value: Embryonic Stem Cell


title: RNA vEDS fibroblast P1


title: RNA vEDS fibroblast P5_biological replicate


title: RNA vEDS fibroblast P1_biological replicate


title: S_153_Gati_615_redo

value: normal

value: African American

value: IIA


title: RNA vEDS fibroblast P3


title: S_155_Gati_617_redo

value: Intensity 3


title: S_34_Ambs_234

value: Kenyan


title: S_154_Gati_616_redo

value: tumor

value: Negative


title: EB, day 7, R2-insertion, RAD21

description: Embryoid_body_day_7_CD71+_R2-insertion_RAD21_ChIP-seq

tag: differentiation day

value: Day 7

value: Qiagen, 34850


title: S_32_Ambs_232

value: Positive


title: S_144_Gati_605_redo

value: Caucasian

value: Borderline


title: RNA Control fibroblast C36

value: dermis

value: Skin fibroblasts

value: healthy donors

value: unaffected


title: EB, day 7, R2-insertion, H3K4me1

description: Embryoid_body_day_7_CD71+_R2-insertion_H3K4me1_ChIP-seq

value: Erythroid

value: E14TG2a.IV mouse ES cells

value: abcam, ab8895


title: EB, day 7, DSE, RAD21

description: Embryoid_body_day_7_CD71+_DSE_RAD21_ChIP-seq

value: CD34+ human erythroid

value: Day 13


title: RNA vEDS fibroblast P2_biological replicate

value: vEDS

value: affected


title: RNA Control fibroblast C35


title: NFYa cKO

city: Dallas

first: Miao

last: Cui

state: Texas

address: UT Southwestern Medical Center Department of Molecular Biology 6000 Harry Hines Blvd.

institute: UTSW

description: Visium spatial transcriptome  RNAseq

data_processing:
The Space Ranger Single-Cell Software Suit was used to perform sample demultiplexing, reads alignment, and to generate feature-spot matrices.
The cDNA reads were aligned to the mm10/GRCm38 mRNA reference genome. The H&E staining image was supplemented to Spatial Ranger for tissue detection, fiducial detection, and alignment of the spatial position of sequenced data points.
Assembly: mm10
Supplementary files format and content: count matrix
Supplementary files format and content: H&E tissue staining image
Library strategy: Visium Spatial Transcriptome RNAseq

source_name: heart section

extract_protocol:
Hearts at E15.5 were dissected and immediately embedded in a tissue embedding mold filled with OCT and placed in a bath of isopentane and liquid nitrogen. OCT embedded heart tissue blocks were cryosectioned in a cryostat to generate appropriately sized sections for Visium Spatial slides. 10 um sections at the coronal view of the heart were collected and placed on the Visum Spatial slides capture areas.
Heart sections were permeabilized for 18 min followed by RNA reverse transcription. The optimized permeabilization time was determined using the Visum Spatial Tissue Optimization Kit (10xGenomics) according to the manufacturer’s protocol. Second strand cDNA was subsequently synthesized on tissue sections and was transferred to an Eppendorf tube after denaturation. The cDNA from the tissue slide that was spatially barcoded was next amplified via PCR and enzymatically fragmentated. P5, P7, i7 and i5 sample indexes are added via End Repair, A-tailing, Adaptor ligation and PCR to make the final sequencing library.

treatment_protocol: WT and NFYa cKO embryos at E15.5  were used for Visium analysis

tag: position

value: Four chamer section of the heart

value: NFYa cKO


title: EB, day 7, R2-insertion, H3K27Ac

description: Embryoid_body_day_7_CD71+_R2-insertion_H3K27Ac_ChIP-seq

value: Day 7

value: abcam, ab4729


title: RNA Control fibroblast C34

tag: disease state


title: RNA Control fibroblast C35_biological replicate

tag: tissue


title: RNA Control fibroblast C33_biological replicate


title: EB, day 7, R2-insertion, H3K4me3

description: Embryoid_body_day_7_CD71+_R2-insertion_H3K4me3_ChIP-seq

value: abcam, ab8580


title: RNA Control fibroblast C33


title: WT [atac]

description:
KO+WT_atac_fragments.tsv.gz
KO+WT_atac_fragments.tsv.gz.tbi

data_processing:
The Cell Ranger Single-Cell Software Suit was used to perform sample demultiplexing, barcode processing and single-cell 3′ gene counting.
The cDNA reads were aligned to the mm10/GRCm38 premRNA reference genome.
Further analyses for quality filtering was performed using the Seurat R package
Briefly, cellranger-ARC aggr was run for combined WT and NFYa KO samples and generated a single feature-barcode matrix containing all the data that were normalized between samples. “Gene Expression” and “Peaks” files from cellranger-ARC output were used to construct Seurat objects in R. A shared peak set derived from the integration of peaks identified for each sample was used to analyze the snATAC dataset. The combined Seurat object was QC’d using the same metrics: 100 <  nCount_ATAC < 100,000; 1000 < nCount_RNA < 100,000; and percent mitochondrial reads < 20
Assembly: mm10
Supplementary files format and content: count matrix

source_name: cadiac nuclei

extract_protocol:
Hearts at E15.5 were dissected and flash-frozen in liquid nitrogen. Genotyping was performed to ensure proper pooling of hearts with the same genotype. Four hearts with the same genotype were pooled for each sample. Cardiac nuclei isolation was performed as previously described.
Total cardiac nuclei were used to generate joint snRNA and snATAC libraries using Single Cell Multiome ATAC + Gene Expression (10×Genomics) according to the manufacturer’s protocol. Sequencing was performed on an Illumina Nextseq 500 system operated by the Next Generation Sequencing Core of Children’s Research Institute at UT Southwestern using the 150bp high output sequencing kit (Illumina).

treatment_protocol: WT and NFYa cKO embryos at E15.5  were used for Multiome RNA + ATAC sequencing.

value: purified nuclei from heart

value: Wildtype


title: WT

description: Visium spatial transcriptome  RNAseq


title: RNA Control fibroblast C32_biological replicate

value: dermis

value: Skin fibroblasts


title: WT [gex]


title: RNA Control fibroblast C34_biological replicate

value: healthy donors

value: unaffected


title: S_150_Gati_612_redo

value: normal

value: Caucasian

value: Positive

value: Negative

tag: Stage

value: IIB


title: S_151_Gati_613_redo

value: tumor


title: NFYa cKO [atac]

tag: cell type


title: Male, Ad lib GEX

city: Birmingham

first: Kelly

middle: Anne

last: Hyndman

state: Al

address: 1918 University BLVD

department: Medicine

institute: University of Alabama at Birmingham

description:
single nuclues RNA and ATAC-seq, 10X Genomics Multiome
combined.rds
barcodes.xlsx

library_source: transcriptomic single cell

data_processing:
CellRanger arc-2.0.0 GEX and ATAC output files from filtered_peak_bc_matrix.  For each sample (630, 636, 641, 638) the metadata is in per_barcode_metrics.csv, ATAC fragments in ATAC_fragments.tsv.gz, GEX counts in  features.tsv.gz
These files were then further processed with Seurat v4.3.0 and Signacv1.9.  First low quality nuclei were filtered with this criteria sample_630 <- subset( x = sample_630, subset = nCount_RNA > 1000 & nCount_RNA < 25000 & nCount_ATAC > 1000 & nCount_ATAC < 100000 & nucleosome_signal < 2 &TSS.enrichment > 1)
The all peaks were recalled using the package MACS2, and then each sample went through Log Normalization and scale.factor = 10000
Each sample then underwent PCA, JAckStraw, Findneighbors,FindClusters, Run UMAP
All samples were then merged and underwent LogNormalization, ScaleData, RunPCA, JackStraw, FindNeighbors, Findcllusters, RunUMAP
Assembly: mm10
Supplementary files format and content: Seurat Object named: combined.rds
Supplementary files format and content: barcodes.xlsx

source_name: Kidney

extract_protocol:
Nuclei from that kidney were isolated with Nuclei EZ Lysis buffer (NUC-101; Sigma-Aldrich) supplemented with protease inhibitor (cOmplete™ ULTRA tab; Sigma-Aldrich) . The frozen kidney cubes were placed in a sterile petri dish with 1 ml of Nuclei EZ lysis buffer (plus protease and RNase inhibitors) and quickly minced.  The tissue was then homogenized with RNase-free, disposable pellet pestles and tubes (Fisher #12-141-368), and a hand held, Bel-Art™ Micro-Tube homogenizer (Wayne, NJ).  The homogenate was filtered through a 40 micron strainer (all strainers were purchase from pluriSelect Life Science).  The homogenate was moved to a sterile, RNase-free 15 ml tube with an additional 2 ml of Nuclei EZ lysis buffer (plus protease and RNase inhibitors).  After a 5 min incubation on ice, the homogenate was filtered through a 40 micron strainer.  The subsequent homogenate was centrifuged at 500 g for 5 min at 4°C.  The pellet was resuspending in Nuclei EZ lysis buffer (plus 1 ml/ml of SUPERaseIN and RNAsin), incubated on ice for 5 min and centrifuged again.  The pellet was resuspended in 0.5 ml of  10X Genomics 1X nuclei suspension buffer, filtered through a 5 micron strainer, and nuclei immediately counted with a hemocytometer.
5000 nuclei per sample were loaded in the Chromium Next GEM Chip J Single Cell Kit and the DNA libraries generated with the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle

treatment_protocol: mice were either left with ad lib food and water, or the water was removed for 9-11 h and euthanasia and kidney collection occurred at midnight.  1/2 kidney was cut into small 5 mm cubes and snap frozen

value: Kidney

tag: strain

tag: treatment

value: ad lib food, water deprived


title: Male, water deprived ATAC

value: ad lib food and water


title: S_146_Gati_608_redo

value: African American

value: IIA


title: S_148_Gati_610_redo

value: Borderline


title: S_134_Gati_594_redo

value: IA


title: S_149_Gati_611_redo


title: RNA Control fibroblast C30_biological replicate

value: dermis

tag: genotype


title: S_136_Gati_596_redo

tag: accession


title: NFYa cKO [gex]

value: purified nuclei from heart

value: NFYa cKO


title: RNA Control fibroblast C36_biological replicate

value: Skin fibroblasts

value: healthy donors

value: unaffected


title: Female, Ad lib GEX


title: RNA Control fibroblast C28_biological replicate


title: S_147_Gati_609_redo

value: Positive


title: S_152_Gati_614_redo

value: normal

value: Caucasian

value: Negative


title: Male, Ad lib ATAC

value: Kidney


title: RNA Control fibroblast C28


title: 5332-DKO-B-p53

first: Dolores

last: Hambardzumyan

address: 1425 Madison Ave.

department: Oncological Sciences

institute: Icahn School of Medicine

description: 10x Genomics

data_processing:
Raw fastq files were aligned to mouse genome reference mm10 customized to include the Rfp sequence, using CellRanger v5.0.0 (10X Genomics). Count matrices filtered by CellRanges algorithm were further filtered by discarding cells with either < 400 genes, < 1000 UMI (unique molecular identifier), or > 25% mitochondrial genes expressed. Data was processed and analyzed using R package Seurat v4.0.5. Normalization was performed using NormalizeData function with normalization.method = ‘LogNormalize’. Dimensionality reduction was computed on the top 2,000 variable features using FindVariableFeatures, ScaleData and RunPCA functions. UMAPs were generated using the top 15 PCs.
For subclustering the immune compartment, we used R package Harmony to mitigate for batch effects driven by technical variation between replicates. De novo clustering using the Louvain algorithm was applied at different resolutions (0.2; 0.8; 2; 5) on the KNN graph space. For high-level annotation, cell types were identified in an iterative and semi-supervised fashion by assigning de novo discovered clusters to cell types based on expression of known marker genes that define each cluster. Annotation of cell subtypes at a lower-level was performed in a similar manner as for the high-level and further aided by de novo marker discovery using the Seurat FindMarkers function and Wilcoxon Rank Sum test.
To identify doublet-enriched clusters we looked for clusters of cells displaying expression of canonical markers for two or more different cell types and higher number of genes/UMI; such clusters were removed from further analysis.
Cell-level proliferation analysis was carried out with Seurat function CellCycleScoring using a list of murine cell cycle genes. Cells were then assigned as ‘cycling’ or ‘non-cycling’ if either their S-phase score or G2M-score was above a threshold of 0.05 for the tumor compartment and 0.1 for the immune compartment.
Identification of modules of co-expressed genes was carried out using the R package scWGCNA (https://github.com/smorabit/scWGCNA)  by first computing metacells of 100 cells (k=100) using function construct_metacells. To identify modules, function blockwiseConsensusModules was called with following parameters: softPower=12, deepSplit=3, mergeCutHeight = 0.25. Only the top 2,000 variable genes were used.
Assembly: mm10
Supplementary files format and content: Cell Ranger output files (barcodes.tsv, features.tsv, matrix.mtx)

source_name: brain tumor

extract_protocol:
Tumors were dissected from the brain, minced into pieces < 1 mm3, and digested with an enzymatic mixture that includes papain (0.94 mg/ml, Worthington, LS003120), EDTA (0.18 mg/ml, Sigma, E6758), cysteine (0.18 mg/ml, Sigma, A8199), and DNase I (60 μg/ml, Roche, 11284932001) in 2 ml HBSS (Gibco, 14175-095). Tumor tissues were kept at 37°C for 15 minutes with occasional agitation. The digestion was terminated with the addition of 2 ml Ovomucoid (0.7 mg/ml, Worthington, LS003086). Following digestion, single cells were passed through a 70 μm cell strainer, centrifuged, and resuspended in 30% Percoll (GE Healthcare, 17-0891-01) solution containing 10% FBS (Hyclone SH30396.03). Cells were separated by centrifugation at 800g for 15 minutes at 4°C. The pellet was resuspended in RBC lysis buffer (BioLegend, 420301) for 1 min at 37°C before they were centrifuged at 450g for 5 min. The pellet was resuspend in PBS and immediately dilvered for GEM preparation.
Gel-Bead in Emulsions (GEMs) were generated on the sample chip in the Chromium controller. Barcoded cDNA was extracted from the GEMs by Post-GEM RT-cleanup and amplified for 12 cycles. Amplified cDNA was fragmented and subjected to end-repair, poly A-tailing, adapter ligation, and 10X-specific sample indexing following the manufacturer’s protocol. Libraries were quantified using Bioanalyzer (Agilent) and QuBit (ThermoFisher) analyses

value: brain tumor cells

value: brain tumor

tag: age

value: 8-16 weeks old


title: S_104_Gati_564_redo

tag: type

value: African American

value: IIA


title: S_145_Gati_607_redo

value: tumor

tag: age (at surgery)


title: RNA Control fibroblast C31_biological replicate

value: dermis


title: S_122_Gati_582_redo

tag: race


title: S_135_Gati_595_redo

tag: er status

value: IA


title: Male, water deprived GEX

value: ad lib food and water


title: Female, water deprived ATAC

value: ad lib food, water deprived


title: RNA Control fibroblast C31

value: Skin fibroblasts

value: healthy donors


title: RNA Control fibroblast C30

value: unaffected


title: S_140_Gati_601_redo

value: Positive

tag: pr


title: Female, water deprived GEX


title: RNA Control fibroblast C29_biological replicate


title: S_120_Gati_580_resent

value: normal

value: Caucasian


title: RNA Control fibroblast C26_biological replicate


title: S_139_Gati_600_redo

value: Negative


title: RNA Control fibroblast C32


title: Female, Ad lib ATAC

value: Kidney


title: S_105_Gati_565_redo


title: RNA Control fibroblast C29


title: S_123_Gati_583_redo


title: 5318-DKO-Bp53-ON

description: 10x Genomics

value: brain tumor cells

value: brain tumor


title: S_119_Gati_579_redo


title: RNA Control fibroblast C25


title: S_117_Gati_577_redo


title: S_116_Gati_576_resent


title: RNA Control fibroblast C27_biological replicate


title: 5726-TVA-B

description: 10x Genomics

value: WT;Ntv-a

value: 8-16 weeks old


title: 5304-DKO-B-53

description: 10x Genomics


title: S_121_Gati_581_redo

value: tumor

value: IIA


title: RNA Control fibroblast C27

value: dermis


title: S_115_Gati_575_redo


title: 5331-IL1b-B-p53

description: 10x Genomics


title: S_114_Gati_574_redo


title: Sham_rep

source_name: Mouse Heart of  Gli1+

value: Heart

value: Sham


title: IRI_rep

value: IRI


title: S_112_Gati_572_redo

value: African American

value: IA


title: 5855-IL1b-B-p53

description: 10x Genomics


title: RNA Control fibroblast C26

value: Skin fibroblasts

value: healthy donors

value: unaffected


title: RNA Control fibroblast C24_biological replicate


title: RNA Control fibroblast C25_biological replicate


title: RNA Control fibroblast C22


title: 5459-IL1b-B-p53

description: 10x Genomics


title: Exosc3-cKO-mESC-a-10-Label

first: Robert

last: Sebra

state: New York

address: 1 Gustave L. Levy Place

institute: Icahn School of Medicine at Mount Sinai

data_processing:
Illumina adapters were trimmed from reads using Trim Galore (v0.5.0). Reads were aligned to the GRCm38/mm10 reference genome using STAR (v2.7.5b) with the following custom parameters: --outFilterMultimapNmax 100 –winAnchorMultimapNmax 100. Gene and TE-level counts were calculated using TEcount (v2.2.1) using the Ensembl GRCm38 v102 transcript annotation and the GRCm38 RepeatMasker annotation downloaded from UCSC table browser.
Assembly: mm10
Supplementary files format and content: TSV file containing gene counts

source_name: mESC

extract_protocol:
For RNA extraction, cells were washed 3x with DPBS and lysed directly on the plate with 1mL of TRIzol reagent (Invitrogen, 15596026). Subsequently, RNA was purified using Direct-zol RNA Miniprep Plus Kits (Zymo, R2072) following manufacturer instructions. In brief, lysed cells in TRIzol were mixed 1:1 with 100% ethanol. Mixed lysates were added to the Zymo-spin columns, treated with DNase I, and washed with provided buffers. RNA was eluted with UltraPure DNase/RNase-Free Distilled Water (Invitrogen, 10977015). For DNA extraction, PureLink™ Genomic DNA Mini Kit (Invitrogen, K1820-00) was used followed manufacturer instructions. In brief, cells were washed 3x with DPBS and lysed directly on the plates using supplied Lysis/Binding buffer. Lysates were subsequently applied to the column and washed with buffers provided. DNA was eluted in PureLink Genomic Elution Buffer provided in the kit.
For complementary DNA synthesis, 100-200 µg of DNase I treated RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814). Random hexamers were used to capture total RNA.

value: mESC

tag: timepoint

tag: rna fraction

value: labeled


title: RNA Control fibroblast C21_biological replicate


title: S_128_Gati_588_redo

value: normal

value: Caucasian

value: Positive

value: Negative


title: S_118_Gati_578_redo


title: 5783-TVA-B

description: 10x Genomics

value: brain tumor cells

value: brain tumor

value: WT;Ntv-a

value: 8-16 weeks old


title: Sham_rep2

source_name: Mouse Heart of  Uremic

value: Heart

value: Sham


title: 5310-WT-B-p53

description: 10x Genomics


title: RNA Control fibroblast C24

value: dermis


title: RNA Control fibroblast C23


title: S_113_Gati_573_redo

value: tumor

value: African American

value: IA


title: RNA Control fibroblast C23_biological replicate

value: Skin fibroblasts

value: healthy donors

value: unaffected


title: IRI_rep2

value: IRI


title: RNA Control fibroblast C22_biological replicate


title: S_29_Ambs_229

value: Kenyan


title: S_129_Gati_589_redo

value: Caucasian

value: Positive


title: STNx_rep2

value: STNx


title: STNx_rep1


title: IRI_rep1


title: RNA Control fibroblast C21


title: S_127_Gati_587_redo

value: Negative

value: IIIA


title: PDAC, SN38, 4 hours, late S-phase, rep 2

city: Solna

first: Vladislav

last: Kuzin

state: Stockholm

address: Solnavägen 9

department: Cell and Molecular Biology

country: Sweden

institute: Karolinska Institute

description:
SN38_late.bedgraph
SN38_rep_timing.bedgraph

data_processing:
Basecalls were performed using bcl2fastq v2.19 for NextSeq 550 output.
Reads were trimmed using Cutadapt v2.10.
Reads were aligned to the UCSC hg38 genome using Bowtie version 2.4.1 with default settings and duplicates were marked with picard tool.
After alignment and deduplication, the established protocol for Repli-seq analysis (Marchal et al., 2018) was followed to determine read coverage in early and late S-phase and the replication timing resulting from the ratio between the two (E, L and E/L respectively). Afterwards, the replication timing was averaged for each condition (DMSO, SN38, JQ1, SN38+JQ1)
For determination of early- and late replicated regions the algorithm RepliScan was used (Zynda et al., 2017). The boundaries between those detected regions were then used to determine the distance from the TES of DoG genes detected in SN38+JQ1 and the respective list of non-DoG genes in early regions.
For visualisation of replication timing downstream of the TES or at early and late replicated regions of DoGs and non-Dogs, a custom-made python script was developed, before plotting the ensuing data in R with the default plotting function.
H3K36me peaks in late replicated regions were determined by taking the MACS2 called peaks and intersecting it with the determined late replicated regions via the intersect command from the BEDTools suite (Quinlan and Hall, 2010).
Assembly: hg38
Supplementary files format and content: tab-delimited text (bedgraph)files include RPKM values of early S-phase (E) coverage, late S-phase (L) coverage and replication timing (E/L) averaged for treatment duplicates
Library strategy: Repli-seq

source_name: Bo103, PDAC

growth_protocol: Cells were grown in a 1:1 mix of high glucose (4.5g/L) DMEM (Thermo, 61965059) and DMEM/F12 (Thermo, 11320033) containing 10% FBS (Thermo, 10270106), 1.6μg/ml amphotericin (Sigma, A2411), 10μM Y-27632 (Selleckchem, S1049), 6.2μg/ml ciprofloxacin (Sigma, 17850), 8.4ng/ml choleratoxin (Sigma, C8052), 500ng/mL insulin (Sigma, I9278) and 20nM 1-thioglycerol (Sigma, M6145), in a 37oC incubator supplied with 5% CO2.

extract_protocol:
Cells were trypsinized, resuspended in 2.5ml cold FACS buffer (PBS + 1% FBS), fixed with dropwise addition of 7.5ml ice-cold 100% ethanol while vortexing, and stored at -20°C. After washing twice in FACS buffer, 3x106 cells were stained for DNA content in 300μl FACS buffer + 50μg/ml propidium iodide + 250μg/ml RNase. 40-120K cells were sorted from the early S and late S populations for each sample according to the criteria outlined in Figure Sx. Cells were lysed in SDS-PK buffer (50mM Tris pH8, 10mM EDTA, 1M NaCl, 0.5% SDS, 200μg/ml Proteinase K) at 56°C for 2h, then DNA was extracted using the Zymo Quick-DNA Microprep Kit and eluted in 130μl H2O. DNA was sonicated to fragments of 200-300bps in microtubes with the Covaris ME220 sonicator using the following settings: Peak Incident Power – 70W, Duty Factor – 20%, Cycles/burst – 1000, Time – 130sec. Each sample was ligated with adapters using the NEBNext Ultra II DNA Library Kit and the adapter hairpins were processed with the USER enzyme and purified with the DNA Clean and Concentrator Kit. DNA was denatured to ssDNA by incubation at 95°C for 5m, then on ice for 2m. Each sample was diluted in IP buffer (10mM sodium phosphate pH7, 140mM NaCl, 0.05% Triton) and incubated with 0.5μg anti-BrdU for 20m at room temperature while rocking. Protein A/G Magnetic Beads were added and incubated for a further 30m, before washing the beads twice with cold IP buffer. The DNA was eluted by incubation in digestion buffer (50mM Tris pH8, 10mM EDTA, 0.5% SDS) with 50μg Proteinase K overnight in a 37°C air incubator, then by adding a further 25μg Proteinase K and incubating at 56°C shaking for 1h.
The eluted ssDNA was purified with the DNA Clean and Concentrator Kit. All samples were amplified and indexed using the NEBNext Multiplex Oligos for Illumina, primer-dimers were removed by Ampure XP enrichment, and sequenced on the Illumina NextSeq 500.

treatment_protocol:
This method was adapted from a published protocol (Marchal et al., 2018).
Bo103 cells were treated in duplicate with DMSO, 500 nM SN38, 1 µM JQ1, or both drugs in combination, then with 100μM BrdU, 4h and 2h before harvest, respectively.

tag: cell line

value: Bo103, PDAC

value: Pancreatic tumor patient-derived cells

value: 4 hours SN38, 2 hours BrdU


title: RNA Control fibroblast C20_biological replicate

source_name: dermis

value: dermis

tag: disease state


title: RNA Control fibroblast C18

tag: tissue


title: PDAC, DMSO, 4 hours, late S-phase, rep 2

description:
DMSO_late.bedgraph
DMSO_rep_timing.bedgraph

value: 4 hours DMSO, 2 hours BrdU


title: S_126_Gati_586_redo

value: normal

tag: Stage


title: RNA Control fibroblast C17_biological replicate

tag: cell type


title: PDAC, JQ1, 4 hours, late S-phase, rep 1

description:
JQ1_late.bedgraph
JQ1_rep_timing.bedgraph

value: 4 hours JQ1, 2 hours BrdU


title: RNA Control fibroblast C17

value: Skin fibroblasts


title: Sham_rep1

value: Heart

value: Sham


title: RNA Control fibroblast C20

value: healthy donors

value: unaffected


title: PDAC, SN38+JQ1, 4 hours, late S-phase, rep 2

description:
SN38+JQ1_late.bedgraph
SN38+JQ1_rep_timing.bedgraph

value: 4 hours SN38+JQ1, 2 hours BrdU


title: S_138_Gati_599_redo

value: tumor

value: African American

value: IIB


title: RNA Control fibroblast C19_biological replicate


title: S_137_Gati_598_redo

value: Negative


title: PDAC, JQ1, 4 hours, late S-phase, rep 2

value: Bo103, PDAC


title: PDAC, SN38, 4 hours, late S-phase, rep 1

value: Pancreatic tumor patient-derived cells


title: RNA Control fibroblast C19


title: S_125_Gati_585_redo

value: Caucasian

value: Positive

value: IIA


title: S_111_Gati_571_redo

value: IIIA


title: PDAC, SN38+JQ1, 4 hours, late S-phase, rep 1


title: S_132_Gati_592_redo

value: normal


title: RNA Control fibroblast C18_biological replicate

value: dermis

value: Skin fibroblasts


title: S_124_Gati_584_redo


title: S_101_Gati_561_redo

value: IA


title: S_103_Gati_563_redo

value: Weak Positive


title: S_110_Gati_570_redo


title: RNA Control fibroblast C16_biological replicate

value: healthy donors

value: unaffected


title: S_102_Gati_562_redo


title: PDAC, DMSO, 4 hours, late S-phase, rep 1

value: 4 hours DMSO, 2 hours BrdU


title: RNA Control fibroblast C16


title: PDAC, JQ1, 4 hours, early S-phase, rep 1

description:
JQ1_early.bedgraph
JQ1_rep_timing.bedgraph

value: 4 hours JQ1, 2 hours BrdU


title: PDAC, SN38+JQ1, 4 hours, early S-phase, rep 1

description:
SN38+JQ1_early.bedgraph
SN38+JQ1_rep_timing.bedgraph

value: 4 hours SN38+JQ1, 2 hours BrdU


title: RNA Control fibroblast C13


title: S_133_Gati_593_redo

value: tumor

value: African American

value: IIB


title: RNA Control fibroblast C15_biological replicate


title: S_58_Gati_511_redo

value: Caucasian

value: Negative


title: PDAC, SN38, 4 hours, early S-phase, rep 2

description:
SN38_early.bedgraph
SN38_rep_timing.bedgraph

value: Bo103, PDAC

value: Pancreatic tumor patient-derived cells

value: 4 hours SN38, 2 hours BrdU


title: RNA Control fibroblast C12_biological replicate

value: dermis


title: PDAC, JQ1, 4 hours, early S-phase, rep 2


title: S_100_Gati_560_redo


title: RNA Control fibroblast C14_biological replicate

value: Skin fibroblasts


title: PDAC, DMSO, 4 hours, early S-phase, rep 2

description:
DMSO_early.bedgraph
DMSO_rep_timing.bedgraph


title: S_61_Gati_514_redo

value: IIA


title: RNA Control fibroblast C14


title: S_131_Gati_591_redo

value: normal

value: IA


title: RNA Control fibroblast C11

value: healthy donors

value: unaffected


title: RNA Control fibroblast C13_biological replicate


title: PDAC, SN38+JQ1, 4 hours, early S-phase, rep 2


title: RNA Control fibroblast C15


title: S_70_Gati_524_redo


title: S_57_Gati_510_redo

value: Positive


title: PDAC, SN38, 4 hours, early S-phase, rep 1


title: S_130_Gati_590_redo


title: S_63_Gati_517_redo

value: IIIC


title: RNA Control fibroblast C10


title: RNA Control fibroblast C10_biological replicate


title: S_62_Gati_516_redo

value: tumor


title: S_69_Gati_523_redo

value: IIIA


title: S_71_Gati_525_redo

value: African American

value: IIB


title: RNA Control fibroblast C12


title: PDAC, DMSO, 4 hours, early S-phase, rep 1

value: 4 hours DMSO, 2 hours BrdU


title: PDAC, SN38 + JQ1, 4 hours, rep 2

data_processing:
Basecalls were performed using bcl2fastq v2.19 for NextSeq 550 output.
ChIP-seq reads were trimmed using Cutadapt v2.10 using --nextseq-trim 25 option, discarding reads left with < 30 bp, for mate 2 of a read pair, 12 bp were additionally cut-off to accord for the unique molecular identifier (UMI), additionally, stretches of polyA and polyT were cut-off to account for the polyA-tail of mature RNAs
RNA-seq reads were aligned to hg38  reference genome using the splice-aware aligner HISAT2 (Kim et al., 2019)
For deduplication umi_tools (Smith et al., 2017) was used which allows for differentiation between PCR duplicates and naturally occurring deduplication due to the unique molecular identifier (UMI) added through the library preparation.
Aligned reads were split into two files containing either exonic or non-exonic reads. This was done by first generating a .bed-file of the exon annotations from a .gtf file of Homo sapiens transcriptome assembly release 36 (respectively release 25 for Mus musculus) from GenCode (Frankish et al., 2019) using the grep and gtf2bed from the BEDOPS toolkit (Neph et al., 2012) commands in the shell. Using this .bed-file, the names of exonic reads were filtered from the respective .bam file using bam2bed and bedops also from BEDOPS, as well as awk in the shell. Finally, using Picard tools’ command FilterSamReads, the .bam file was filtered to either include or exclude the list of reads overlapping exons generating two separate .bam-files containing exonic or non-exonic reads.
For differential expression analysis the R package “Index” (Lee et al., 2020) which is based on the “edgeR” package was used to account for differential expression in both exonic and intronic reads. Exons and introns were counted separately using from the filtered .bam files (exonic, non-exonic).
Bigwig files for RNA-seq visualisation were generated using the bamCoverage command from the Deeptools suite (Ramírez et al., 2016) after .bam-files had been merged using samtools merge (Li et al., 2009)
Assembly: hg38
Supplementary files format and content: csv (differential expression analysis of human exonic reads), bigWig (bw, non-exonic reads)
Library strategy: SLAM-seq

growth_protocol: Cells were grown in a 1:1 mix of high glucose (4.5g/L) DMEM (Thermo, 61965059) and DMEM/F12 (Thermo, 11320033) containing 10% FBS (Thermo, 10270106), 1.6μg/ml amphotericin (Sigma, A2411), 10μM Y-27632 (Selleckchem, S1049), 6.2μg/ml ciprofloxacin (Sigma, 17850), 8.4ng/ml choleratoxin (Sigma, C8052), 500ng/mL insulin (Sigma, I9278) and 20nM 1-thioglycerol (Sigma, M6145), in a 37°C incubator supplied with 5% CO2.

extract_protocol:
Cells were scraped step-wise in 2x 0.5ml Trizol (Invitrogen, 15596018), snap frozen and stored at -80 °C. For RNA isolation we followed the manufacturer's instructions of SLAM-seq Kinetics Kit – Anabolic Kinetics Module (Lexogen, 061.24) except for the following modifications: frozen samples were thawed for 5 min at 65 °C and mixed with 200µl chloroform:isoamylalcohol mix (24:1) before further incubation for 15 min at 65 °C (strongly shaking and vortexing from time to time). RNA in the aqueous phase was separated by centrifugation, supplemented with reducing agent and again extracted with chloroform:isoamylalcohol mix (24:1) before precipitation overnight at -20°C (in the presence of reducing agent). RNA was washed  twice with 1 ml and 0.18 ml 75% EtOH (plus reducing agent), respectively and pelleted for 10 min at 7500g. Resuspension in H2O was facilitated by incubation for 15 min at 55 °C and 10 min on ice and repeated pipetting. RNA was quantified with the Qubit RNA nano Assay Kit (Thermo Fisher, Q33230). 4.5µg of the RNA (mixed with Spike In) was alkylated by addition of Iodoacetamide following the manufacturer's instructions.
Qubit RNA nano Assay Kit (Thermo Fisher, Q33230) was used to quantify the RNA and RNA integrity was controlled using the RNA 6000 nano kit (Agilent, 5067-1511) on a Bioanalyzer 2100 (Agilent). Ribosomal RNAs were depleted from 950ng of alkylated RNA using the RiboCop (HMR) kit (as part of the CORALL Total RNA-Seq library Prep Kit, Lexogen 146.24) following the manufacturer's instructions, except for increasing the volume for RNA elution to 16µl. Removal of rRNAs was confirmed using the RNA 6000 pico kit (Agilent, 5067-1413) on a Bioanalyzer 2100 (Agilent). To prepare cDNA libraries spanning the whole transcript length, the CORALL Total RNA-Seq library Prep Kit, Lexogen 146.24) was used, which includes reverse transcription with Displacement Stop primers. 10µl of the rRNA depleted RNA were used as input material and the final library was amplified by 14 PCR cycles. DNA concentration and molarity were determined by Qubit dsDNA HS Assay Kit (Thermo Fisher, Q33230) and Bioanalyzer High Sensitivity DNA Kit (5067-4626) on a Bioanalyzer 2100 (Agilent), respectively.

treatment_protocol:
A modified version of SLAM-seq (Herzog et al., 2017) was used to measure both, nascent RNAs and steady state RNAs, over the full transcript length.
1.5x10^6 Bo103 cells were treated with vehicle, 500nM SN38, 1µM JQ1 or SN38+JQ1 in combination for 4 hrs and in the last 2 hrs 100 μM 4 Thiouridine (S4U) was added to the medium in the dark (in triplicates).

value: Bo103, PDAC

value: 4 hours SN38+JQ1, 2 hours S4U


title: RNA Control fibroblast C11_biological replicate


title: RNA Control fibroblast C9_biological replicate


title: RNA Control fibroblast C9


title: PDAC, JQ1, 4 hours, rep 3

value: Pancreatic tumor patient-derived cells

value: 4 hours JQ1, 2 hours S4U


title: PDAC, SN38 + JQ1, 4 hours, rep 1


title: PDAC, SN38, 4 hours, rep 3

value: 4 hours SN38, 2 hours S4U


title: PDAC, JQ1, 4 hours, rep 1


title: S_68_Gati_522_redo

value: Caucasian

value: Negative


title: RNA Control fibroblast C8

value: dermis

value: Skin fibroblasts


title: PDAC, SN38, 4 hours, rep 2


title: RNA Control fibroblast C7_biological replicate

value: healthy donors

value: unaffected


title: S_65_Gati_519_resent

value: normal

value: Positive

value: IIA


title: RNA Control fibroblast C6_biological replicate


title: RNA Control fibroblast C8_biological replicate


title: S_67_Gati_521_redo

value: tumor


title: PDAC, SN38 + JQ1, 4 hours, rep 3


title: PDAC, JQ1, 4 hours, rep 2


title: PDAC, SN38, 4 hours, rep 1


title: PDAC, DMSO, 4 hours, rep 3

value: 4 hours DMSO, 2 hours S4U


title: PDAC, DMSO, 4 hours, rep 2


title: S_64_Gati_518_redo

value: African American


title: S_56_Gati_509_redo


title: S_66_Gati_520_redo


title: panNEC, PDX, vehicle, 5 days, 4 hours, rep 2

description: Table_S3.csv, column 9, "DMSO_2_RPKM"

data_processing:
Basecalls were performed using bcl2fastq v2.19 for NextSeq 550 output.
ChIP-seq reads were trimmed using Cutadapt v2.10 using --nextseq-trim 20 option, discarding reads left with < 30 bp. For mate 2 of a read pair, 12 bp were additionally cut-off to accord for the unique molecular identifier (UMI) (only for samples with treatment cycles C1, C2, C3), additionally, stretches of polyA and polyT were cut-off to account for the polyA-tail of mature RNAs
RNA-seq reads were aligned to hg38 or mm10 reference genome using the splice-aware aligner HISAT2 (Kim et al., 2019)
For deduplication umi_tools (Smith et al., 2017) was used which allows for differentiation between PCR duplicates and naturally occurring deduplication due to the unique molecular identifier (UMI) added through the library preparation. (only for samples with treatment cycles C1, C2, C3). Otherwise, deduplication, sorting and indexing were performed using samtools (Li and Durbin, 2009) and Picard (Picard, 2019).
Aligned reads were split into two files containing either exonic or non-exonic reads. This was done by first generating a .bed-file of the exon annotations from a .gtf file of Homo sapiens transcriptome assembly release 36 (respectively release 25 for Mus musculus) from GenCode (Frankish et al., 2019) using the grep and gtf2bed from the BEDOPS toolkit (Neph et al., 2012) commands in the shell. Using this .bed-file, the names of exonic reads were filtered from the respective .bam file using bam2bed and bedops also from BEDOPS, as well as awk in the shell. Finally, using Picard tools’ command FilterSamReads, the .bam file was filtered to either include or exclude the list of reads overlapping exons generating two separate .bam-files containing exonic or non-exonic reads.
Bigwig files for RNA-seq visualisation were generated using the bamCoverage command from the Deeptools suite (Ramírez et al., 2016).
For differential expression analysis the R package “Index” (Lee et al., 2020) which is based on the “edgeR” package was used to account for differential expression in both exonic and intronic reads. Exons and introns were counted separately using from the filtered .bam files (exonic, non-exonic).
Assembly: hg38, mm10
Supplementary files format and content: csv (differential expression analysis of human exonic reads), bigWig (bw, non-exonic reads)

source_name: Bo99, panNEC

growth_protocol: To establish treatment cohorts, tumor pieces (1-2 mm) from early passage PDXs (≤ F4 generation) were soaked in undiluted matrigel (Becton Dickinson) for 15 to 30 min. and subsequently implanted subcutaneously onto female mice (NMRI-Foxn1nu/Foxn1nu, Janvier, St Berthevin Cedex, France) at two sites (scapular region) using as many as 4 pieces per site. Tumors were allowed to grow to a size of approx. 100-200 mm3, at which time mice were randomized in the treatment and control groups with five to six mice in each group.

extract_protocol:
For RNA extraction, after tumor harvest and storage at -80°C, the tumors  were broken apart in liquid nitrogen using a mortar and pestle. The tumor fragments were then weighed in as to use at least 20 mg of tumor tissue and homogenized in either TRIzol reagent using a rotor stator instrument (C1, C2, C3 samples) or crushing in the mortar and pestle with buffer from the AllPrep DNA/RNA Mini Kit (Qiagen, 80204) (5 days samples). Both RNA and DNA were extracted either using the TRIzol (Invitrogen, 15596018) (C1, C2, C3 samples) or AllPrep kits (5 days samples). Afterwards 500 ng of RNA per sample was processed using the RiboCop kit (Lexogen, 037) for rRNA removal.
For the C1, C2, C3 samples, RNA extracted from xenografts was quantified with the Qubit RNA nano Assay Kit (Thermo Fisher, Q33230). Sequencing libraries were created according to the Corall kit protocol (Lexogen, 095) (C1, C2, C3 samples). Library preparation success was confirmed using the Agilent High Sensitivity DNA Kit (Agilent, 5067-4626) on the Agilent 2100 Bioanalyzer. For the  5 days treated samples, these steps were performed by Novogene. Libraries were then pooled and sequenced using the NextSeq 500/550 High Output Kit v2.5 (Illumina, 20024906). The sequencing runs were Paired end and Dual Index with 75 bp reads eachs.

treatment_protocol: Mice were treated daily with JQ1 (Hycultec) 50mg/kg by intraperitoneal (i.p.) injection and Irinotecan (Hycultec) i.p. three times per week (Mon., Wed., Fri.) at 15mg/kg, either followed by harvest (5 days treated samples) or followed by one week of treatment pause for up to two consecutive cycles (C1, C2, C3 samples).For the 5 days samples, mice were either treated with no, one or both drugs in combination. For C1, C2, C3 samples, the mice were subjected to zero, one or two cycles of treatment and subsequent tumor recovery and the tumors were harvested 0, 4, 12 or 24 hours after another treatment with JQ1 + Irinotecan.

value: Bo99, panNEC

value: Pancreatic tumor patient-derived xenografts (PDX)

value: 5 days, DMSO 4 hours


title: RNA Control fibroblast C6

extract_protocol:
Total RNA was purified from patient and control cells grown for 72 hours at 70-80% of confluency using the miRNeasy kit (Qiagen). Sample quality and quantity were evaluated with the QIAxcel (QIAGEN) and the Qubit fluorometer (Thermofisher Scientific), respectively.
To improve statistical power, RNA samples from all experimental groups were sequenced twice as biological replicates (BR) (at the 2nd and 4th in vitro passage, respectively), for a total of 108 sequenced libraries. Reverse transcription and library preparation were achieved following the standard protocol of the Ion Ampliseq Transcriptome Human Gene Expression Kit (Thermofisher Scientific), which targets by multiplexed PCR 18,574 protein-coding and 2,228 non-coding RNAs (mRNAs/ncRNAs). Libraries were quantified with the Ion qPCR Library Quantification Kit, diluted to 100 pM and pooled equally, with 8 samples per pool, followed by emulsion PCR and enrichment with the Ion OneTouch2 with ES system. Libraries were then loaded onto an Ion 540 chip and sequenced on the Ion S5 instrument.


title: RNA Control fibroblast C5_biological replicate


title: PDAC, DMSO, 4 hours, rep 1

value: Bo103, PDAC

value: Pancreatic tumor patient-derived cells


title: panNEC, PDX, vehicle, 5 days, 4 hours, rep 1

description: Table_S3.csv, column 8, "DMSO_1_RPKM"


title: S_91_Gati_551_redo

value: IIB


title: RNA Control fibroblast C5


title: panNEC, PDX, JQ1, 5 days, 4 hours, rep 2

description: Table_S3.csv, column 15, "JQ1_2_RPKM"

value: 5 days, JQ1 4 hours


title: panNEC, PDX, JQ1, 5 days, 4 hours, rep 1

description: Table_S3.csv, column 14, "JQ1_1_RPKM"


title: S_143_Gati_604_resent

value: Caucasian

value: Negative

value: IA


title: RNA Control fibroblast C7

value: dermis

value: Skin fibroblasts

value: healthy donors

value: unaffected


title: S_90_Gati_550_redo

value: tumor

value: Positive


title: RNA Control fibroblast C4_biological replicate


title: S_141_Gati_602_redo


title: RNA Control fibroblast C3_biological replicate


title: panNEC, PDX, Irinotecan, 5 days, 4 hours, rep 2

description: Table_S3.csv, column 12, "Irinotecan_2_RPKM"

value: 5 days, Irinotecan 4 hours


title: S_93_Gati_553_redo

value: normal

value: African American

value: IIIA


title: panNEC, PDX, Irinotecan, 5 days, 4 hours, rep 1

description: Table_S3.csv, column 11, "Irinotecan_1_RPKM"

value: Bo99, panNEC

value: Pancreatic tumor patient-derived xenografts (PDX)


title: RNA Control fibroblast C3


title: panNEC, PDX, JQ1 + Irinotecan, 5 days, 4 hours, rep 2

description: Table_S3.csv, column 18, "Irinotecan+JQ1_2_RPKM"

value: 5 days, JQ1+Irinotecan 4 hours


title: panNEC, PDX, JQ1 + Irinotecan, 5 days, 4 hours, rep 1

description: Table_S3.csv, column 17, "Irinotecan+JQ1_1_RPKM"


title: RNA Control fibroblast C1


title: S_89_Gati_547_redo

value: IIA


title: S_88_Gati_546_redo


title: PDAC, PDX, C1, 24 hours, rep 2

description: Table_S5.csv and Table S7.csv, column 24, "C1_24h_PDX_22_FPKM"

source_name: B103, PDAC

value: B103, PDAC

value: C1, JQ1+Irinotecan 24 hours


title: S_92_Gati_552_redo

value: Negative


title: RNA Control fibroblast C2_biological replicate

value: dermis


title: S_55_Gati_508_redo


title: RNA Control fibroblast C1_biological replicate

value: Skin fibroblasts


title: PDAC, PDX, C2, 12 hours, rep 2

description: Table_S5.csv and Table S7.csv, column 21, "C2_12h_PDX_23_FPKM"

value: C2, JQ1+Irinotecan 12 hours


title: S_109_Gati_569_redo


title: S_108_Gati_568_redo


title: PDAC, PDX, C2, 0 hours, rep 2

description: Table_S5.csv and Table S7.csv, column 10, "C2_0h_PDX_19_FPKM"

value: C2, JQ1+Irinotecan 0 hours


title: PDAC, PDX, C1, 12 hours, rep 2

description: Table_S5.csv and Table S7.csv, column 19, "C1_12h_PDX_21_FPKM"

value: C1, JQ1+Irinotecan 12 hours


title: RNA Control fibroblast C4

value: healthy donors

value: unaffected


title: S_97_Gati_557_redo

value: tumor

value: IIB


title: PDAC, PDX, C3, 4 hours, rep 1

description: Table_S5.csv and Table S7.csv, column 16, "C3_4h_PDX_14_FPKM"

value: C3, JQ1+Irinotecan 4 hours


title: S_107_Gati_567_redo

value: normal

value: African American


title: miRNA vEDS fibroblast P17_biological replicate

data_processing:
After read trimming using cutadapt v1.18 (adapter 3' AACTGTAGGCACCATCAAT; adapter 5' GTTCAGAGTTCTACAGTCCGACGATC), quantification was performed with mirdeep2 pipeline (Friedländer et al., 2012) using as reference miRNAs and hairpins as defined in miRBase 22.1. Reads shorter than 17 nucleotides were discarded. MiRNAs with average expression across samples lower than 20 count were excluded from further analysis. Differential expression analysis was performed with DESeq2 R package v1.38.3 (Love et al., 2014). Differentially expressed miRNAs (DE-miRNAs) were selected by using an adjusted p-value ≤ 0.05 (p-value correction with Benjamini & Hochberg).
Quantification was performed with mirdeep2 pipeline (Friedländer et al., 2012) using as reference miRNAs and hairpins as defined in miRBase 22.1. Reads shorter than 17 nucleotides were discarded.  MiRNAs with average expression across samples lower than 20 count were excluded from further analysis.
Assembly: Reference miRNAs and hairpins as defined in miRBase 22.1.
Supplementary files format and content: Differential expression analysis was performed with DESeq2 R package v1.38.3 (Love et al., 2014).  Differentially expressed miRNAs (DE-miRNAs) were selected by using an adjusted p-value ≤ 0.05 (p-value correction with Benjamini & Hochberg).

growth_protocol: Control and patient fibroblasts were grown between 1st and 4th in vitro passage at 37 °C in a 5% CO2 atmosphere in Earle's Modified Eagle Medium supplemented with 2 mM L-glutamine, 10% FBS, and 100 µg/ml penicillin and streptomycin

extract_protocol:
Total RNA was purified from patient and control cells grown for 72 hours at 70-80% of confluency using the miRNeasy kit (Qiagen). Sample quality and quantity were evaluated with the QIAxcel (QIAGEN) and the Qubit fluorometer (Thermofisher Scientific), respectively.
To ensure the accuracy and reproducibility of the data, a biological replicate (BR) was prepared for each patient and control sample, resulting in a total of 72 miRNAs libraries. Reverse transcription and library preparation were achieved following the standard protocol of the QIAseq miRNA Library kit (QIAGEN). All procedures were carried out in accordance with the manufacturer's instructions. This method incorporates unique molecular indexes during cDNA synthesis, which uniquely tag each strand before amplification, resulting in a more accurate reflection of endogenous miRNA levels by controlling for library amplification bias (Wong et al., 2019). Libraries were quantified with the QIAxcel (QIAGEN) diluted to 100 pM and pooled equally, with 6 samples per pool, followed by emulsion PCR and enrichment with the Ion OneTouch2 with ES system. Libraries were then loaded onto an Ion 540 chip and sequenced on the Ion S5 instrument.

value: vEDS

value: affected


title: PDAC, PDX, C1, 4 hours, rep 2

description: Table_S5.csv and Table S7.csv, column 14, "C1_4h_PDX_20_FPKM"

value: B103, PDAC

value: Pancreatic tumor patient-derived xenografts (PDX)

value: C1, JQ1+Irinotecan 4 hours


title: miRNA vEDS fibroblast P18_biological replicate

value: dermis


title: S_96_Gati_556_redo

value: Negative


title: RNA Control fibroblast C2

data_processing:
Raw sequences were aligned and processed by Torrent Suite 5.12.3 according to standard analysis procedures. Output normalized counts (in read per milion) were generated (*.chp files ).  Differentially expressed genes (DEGs) were identified by using the Transcriptome Analysis Console (TAC) software (version 4.0.2) with the ampliSeqRNA plugin (Thermofisher Scientific) and applying an ANOVA test with a fold-change threshold of ±1.5 and an FDR-adjusted p-value ≤ 0.05.
Assembly: hg19_AmpliSeq_Transcriptome_21K_v1.transcript
Supplementary files format and content: Output normalized counts (in read per milion) were generated (*.chp files ).

value: Skin fibroblasts


title: S_106_Gati_566_redo

value: IIA


title: PDAC, PDX, C3, 0 hours, rep 1

description: Table_S5.csv and Table S7.csv, column 11, "C3_0h_PDX_13_FPKM"

value: C3, JQ1+Irinotecan 0 hours


title: miRNA vEDS fibroblast P17


title: miRNA vEDS fibroblast P16_biological replicate


title: S_99_Gati_559_redo

value: Positive

value: IIIC


title: PDAC, PDX, C2, 24 hours, rep 1

description: Table_S5.csv and Table S7.csv, column 25, "C2_24h_PDX_12_FPKM"

value: C2, JQ1+Irinotecan 24 hours


title: miRNA vEDS fibroblast P18


title: miRNA vEDS fibroblast P16


title: miRNA vEDS fibroblast P15_biological replicate


title: S_95_Gati_555_redo

value: Caucasian


title: S_98_Gati_558_redo


title: PDAC, PDX, C2, 12 hours, rep 1

description: Table_S5.csv and Table S7.csv, column 20, "C2_12h_PDX_11_FPKM"

value: C2, JQ1+Irinotecan 12 hours


title: PDAC, PDX, C2, 4 hours, rep 1

description: Table_S5.csv and Table S7.csv, column 15, "C2_4h_PDX_10_FPKM"

value: C2, JQ1+Irinotecan 4 hours


title: miRNA vEDS fibroblast P15


title: PDAC, PDX, C2, 0 hours, rep 1

description: Table_S5.csv and Table S7.csv, column 9, "C2_0h_PDX_9_FPKM"

value: C2, JQ1+Irinotecan 0 hours


title: CSPα KO2

city: New Haven

first: Sreeganga

last: Chandra

state: CT

address: 295 Congress Avenue, BCMM 154D

department: Depts. of Neuroscience and Neurology

institute: Yale University

data_processing:
snRNA-seq data were aligned to Mouse mm10 (2020-A) reference to map both unspliced pre-mRNA and mature mRNA using CellRanger version 6.0.1
Assembly: mm10
Supplementary files format and content: matrix files

source_name: cortex

extract_protocol:
Freshly dissected mouse cortex from P24 littermate mice were gently Dounce homogenized in 2 ml of ice-cold Nuclei EZ Prep buffer (Sigma, Cat.NUC101-1KT) with a loose pestle, and then with a tight pestle each for 25 times. The resulting homogenate was incubated on ice for five minutes with an additional 2 mL of cold EZ Prep buffer. Nuclei were centrifuged at 500xg for five minutes at 4°C.
SnRNA-seq libraries were prepared by the Chromium Single Cell 3′ Reagent Kit v3 chemistry according to the manufacturer’s instructions (10x Genomics). The generated snRNA-seq libraries were sequenced using Illumina NovaSeq6000 S4 at a sequencing depth of 300 million reads per sample.

value: cortex

value: cortical cells

value: CSPalpha KO


title: S_94_Gati_554_redo

source_name: frozen breast tumors

extract_protocol:
RNA isolation was done using TRIzol method . Integrity of isolated RNA was evaluated with the Agilent 2100 Bioanalyzer (Agilent Technologies) and  sequencing was performed on an Illumina NovaSeq S2 machine.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: tumor


title: CSPα KO1


title: miRNA vEDS fibroblast P14_biological replicate

value: vEDS

value: affected


title: S_84_Gati_541_redo

value: Asian

value: Negative

value: Weak Positive


title: PDAC, PDX, C1, 0 hours, rep 1

description: Table_S5.csv and Table S7.csv, column 8, "C1_0h_PDX_8_FPKM"

value: B103, PDAC

value: Pancreatic tumor patient-derived xenografts (PDX)

value: C1, JQ1+Irinotecan 0 hours


title: S_83_Gati_540_redo

value: normal

value: IIA


title: WT3

value: WT


title: miRNA vEDS fibroblast P14

value: dermis

value: Skin fibroblasts


title: PDAC, PDX, C1, 24 hours, rep 1

description: Table_S5.csv and Table S7.csv, column 23, "C1_24h_PDX_7_FPKM"

value: C1, JQ1+Irinotecan 24 hours


title: WT2

value: cortex

value: cortical cells


title: miRNA vEDS fibroblast P13_biological replicate


title: S_80_Gati_536_redo

value: tumor

value: Caucasian

value: Positive

value: IA


title: miRNA vEDS fibroblast P13

value: vEDS

value: affected


title: CSPα KO3

value: CSPalpha KO


title: PDAC, PDX, C1, 12 hours, rep 1

description: Table_S5.csv and Table S7.csv, column 18, "C1_12h_PDX_6_FPKM"

value: B103, PDAC

value: Pancreatic tumor patient-derived xenografts (PDX)

value: C1, JQ1+Irinotecan 12 hours


title: WT1


title: S_82_Gati_539_redo

value: normal

value: African American

value: Negative

value: IIIA


title: miRNA vEDS fibroblast P12_biological replicate

value: dermis

value: Skin fibroblasts


title: panNEC, PDX, C3, 0 hours, rep 2

description: Table_S4.csv and Table S6.csv, column 12, "C3_0h_PDX_27_FPKM"

value: Bo99, panNEC

value: C3, JQ1+Irinotecan 0 hours


title: miRNA vEDS fibroblast P12


title: PDAC, PDX, C1, 4 hours, rep 1

description: Table_S5.csv and Table S7.csv, column 13, "C1_4h_PDX_5_FPKM"

value: C1, JQ1+Irinotecan 4 hours


title: S_81_Gati_538_redo

value: tumor

value: Positive


title: 1 ng mESC genomic DNA treated with ultrafast BS replicate 2

city: Chicago

first: Chang

last: Ye

state: Illinois

institute: University of Chicago

data_processing:
After trimming the TruSeq sequencing adapters from the 3′ ends of read 1 and read 2 using the cutadapt tool, low-quality and short reads were filtered out by applying ‘-q 20 –nextseq-trim 20 –max-n 0 -m 20’ arguments. The clean reads were then aligned to the spike-in sequences (found in Extended Data Table 1) and reference genomes (mouse reference GRCm39 for mESC samples and human reference GRCh38 for cfDNA samples) using the hisat-3n tool. The ‘--base-change C,T --no-splice-alignment –bowtie2-dp 1 –score-min L,0,1’ argument was used for all DNA libraries to ensure accurate alignment. To make use of the strand-specific property of the library, the ‘--directional-mapping’ argument was applied for 10 ng / 3.3 ng mES gDNA and cfDNA libraries, while ‘--directional-mapping-reverse’ arguments were used for single cell mESC libraries. The sorted mapped reads were then processed to remove PCR duplicates using the MarkDuplicateSpark command in the GATK tool. Finally, the number of converted and unconverted reads at all C sites were counted using the hisat-3n-table command, and the methylation ratio was calculated as the number of unconverted reads divided by total coverage.
Assembly: hg38, mm10
Supplementary files format and content: For bw files, number of conversion and unconverted are counted from mapped bam files using samtools mplieup and cpup tool. Methylation level is calculated by converstion count over sequence coverage and bw files were created by bedGraphToBigWig tools.
Supplementary files format and content: For tsv files, unconversion ratio on RNA sites are detected by the hisat3n-table tools. m5C sites are filter by statatistical cutoff and annoated based of ensembl release 108.

growth_protocol: Cells were purchased from the American Type Culture Collection (ATCC). Cells were cultured at 37 °C with 5.0% CO2 in a Heracell VIOS 160i incubator (Thermo Scientific). All cell lines were grown in DMEM medium (GIBCO, catalog no. 11995) supplemented with 10% v/v FBS and 1% penicillin/streptomycin (Gibco).

extract_protocol: Library preparation starting from mES genomic DNA: The mixture of mES gDNA (100-200 ng/µl) and spike-in lambda DNA (0.1%) were fragmented by sonication in 100 µl tube for 11 cycles of 30 seconds on and 30 seconds off using Bioruptor® Pico (Diagenode). The fragmented genomic DNA was size selected by Agencourt AMPure XP (Beckman Coulter) to 300-500 bp and eluted with 17 µl water. 3′-Dephosphorylation was conducted by adding 2 µl 10x T4PNK buffer (NEB) and 1 µl of T4 PNK (NEB) and the mixture was incubated at 37 °C for 30 min followed by 65 °C for 20 min. After purification by DNA Clean & Concentrate kit (Zymo Research), 164 bp dsDNA oligos (0.001%) was added as spike-in.  After BS treatment, all samples were desulphonated on Zymo-Spin IC columns following manufacturer’s protocol except that final washing step was conducted for four times instead of twice. Then the samples were incubated at 95 °C for 5 min to denature DNA, and cold-shocked on ice for 2 min followed by libraries construction using xGen™ Methyl-Seq Lib Prep kit for 96rxn (IDT, catalog no: 10009824).  Library preparation starting from small number of mES cells (100-cell, 10-cell and single-cell): The DNA libraries starting from small number of mES cells were constructed following the previous method with slight modifications40,41. In brief, mES cells were sorted by fluorescence-activated cell sorting (FACS) into lysis buffer containing 20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 20 mM KCl, 0.3% Triton-X 100, 1 mg/mL QIAGEN protease and 0.6 pg lambda DNA (dam-, dcm-). Genomic DNA was released under the program of 50 °C for 3 h, followed by 75 °C for 30 min. Bisulfite treatment was performed using UBS-1 and Zymo DNA Methylation-Gold kit side by side. The first-strand DNA was synthesized by random priming using 50 U Klenow exo-, 400 μM P5-N6-oligo1 (CTACACGACGCTCTTCCGATCTNNNNNN, IDT) at 37 °C for 30 min. After 4 rounds of random priming, samples were purified with 0.8x AMPure XP beads. The second strand was synthesized using P7-N6-oligo2 (AGACGTGTGCTCTTCCGATCTNNNNNN, IDT). After purification with 0.8x AMPure XP beads, the sequencing index was introduced by PCR using Multiplex Oligos for Illumina (NEB) and KAPA HiFi HotStart ReadyMix. Library preparation for human plasma cfDNA: 41 ng of plasma cfDNA either from healthy people or from patient’s sample was spiked-in with 0.1% of the 164 bp dsDNA containing four 5mC sites and water was added to give each sample total volume 25 µl. Then to each sample, End Repair & A-Tailing Buffer (3.5 µl) and End Repair & A-Tailing Enzyme Mix from KAPA HyperPlus kit (Roche, catalog no: 07962428001) were added and the mixture was incubated at 65 °C for 30 min. Then a mixture of 15 µl ligation buffer, 5 µl DNA ligase, 2.5 µl water and 2.5 µl 15 µM methylated NEBNext adaptor was added, and the mixture was incubated at 20 °C for 1 h. Then 3 µl USER enzyme (NEB, catalog no: M5505S) was added and incubated at 37 °C for 15 min. Then the ligated DNA was purified by 1.0x Ampure beads and eluted with 41 µl water and divided into 5 parts. One part equivalent to 1 ng cfDNA will be directly amplified as input library. Two parts containing 10 ng cfDNA were treated with UBS condition followed by desulphonation the same procedure as described above for mES gDNA, and the other two parts equivalent to 10 ng cfDNA were treated with the conventional BS condition following manufacture’s protocol. The purified DNA was then amplified using KAPA HiFi Uracil+ DNA polymerase (Roche, catalog no: 07959079001) and the libraries was purified by 0.7x Ampure beads.

value: mESC

value: stem cell

value: WT

tag: method

value: ultrafast BS


title: panNEC, PDX, C2, 24 hours, rep 2

description: Table_S4.csv and Table S6.csv, column 21, "C2_24h_PDX_26_FPKM"

value: Pancreatic tumor patient-derived xenografts (PDX)

tag: treatment

value: C2, JQ1+Irinotecan 24 hours


title: S_87_Gati_545_redo

data_processing:
Reads of the samples were trimmed for adapters and low-quality bases using Cutadapt before alignment with the reference genome (hg38) and the annotated transcripts using STAR. The average mapping rate of all samples is 96%. Unique alignment is above 63%.
The gene expression quantification analysis was performed for all samples using STAR/RSEM tools.
Assembly: hg38
Supplementary files format and content: tab-delimited text files include raw count data for each Sample


title: 10 ng mESC genomic DNA treated with ultrafast BS replicate 2

tag: genotype


title: 1 ng mESC genomic DNA treated with ultrafast BS replicate 1


title: S_86_Gati_544_resent

tag: accession

value: normal


title: miRNA vEDS fibroblast P11_biological replicate

value: vEDS

value: affected


title: panNEC, PDX, C2, 0 hours, rep 2

description: Table_S4.csv and Table S6.csv, column 10, "C2_0h_PDX_25_FPKM"

value: C2, JQ1+Irinotecan 0 hours


title: panNEC, PDX, C1, 4 hours, rep 2

description: Table_S4.csv and Table S6.csv, column 15, "C1_4h_PDX_24_FPKM"


title: S_77_Gati_533_redo

value: Caucasian

value: Negative

value: IA


title: S_76_Gati_532_resent


title: 10 ng mESC genomic DNA treated with ultrafast BS replicate 1


title: miRNA vEDS fibroblast P10

value: dermis

value: Skin fibroblasts


title: 1 ng mESC genomic DNA treated with conventional BS replicate 2

value: conventional BS


title: panNEC, PDX, C3, 0 hours, rep 1

description: Table_S4.csv and Table S6.csv, column 11, "C3_0h_PDX_18_FPKM"

value: Bo99, panNEC

value: C3, JQ1+Irinotecan 0 hours


title: 1 ng mESC genomic DNA treated with conventional BS replicate 1

value: mESC

value: stem cell


title: panNEC, PDX, C2, 24 hours, rep 1

description: Table_S4.csv and Table S6.csv, column 20, "C2_24h_PDX_17_FPKM"


title: miRNA vEDS fibroblast P9_biological replicate


title: S_79_Gati_535_redo

value: tumor

value: African American

value: Positive


title: 10 ng mESC genomic DNA treated with conventional BS replicate 2

value: WT


title: panNEC, PDX, C2, 4 hours, rep 1

description: Table_S4.csv and Table S6.csv, column 16, "C2_4h_PDX_16_FPKM"

value: Pancreatic tumor patient-derived xenografts (PDX)

value: C2, JQ1+Irinotecan 4 hours


title: 10 ng mESC genomic DNA treated with conventional BS replicate 1


title: miRNA vEDS fibroblast P9

value: vEDS

value: affected


title: S_78_Gati_534_redo

value: normal

value: Negative


title: miRNA vEDS fibroblast P11

value: dermis


title: miRNA vEDS fibroblast P8_biological replicate

value: Skin fibroblasts


title: panNEC, PDX, C1, 24 hours, rep 1

description: Table_S4.csv and Table S6.csv, column 19, "C1_24h_PDX_4_FPKM"

value: C1, JQ1+Irinotecan 24 hours


title: miRNA vEDS fibroblast P8


title: MAIT_naive

city: Hamburg

first: Jan-Eric

last: Turner

address: Martinistraße 52

institute: University Medical Center Hamburg-Eppendorf

description: 10X Genomics

data_processing:
Demultiplexing and mapping reads to the reference genome was performed using CellRanger (version 4.0.0)
Data was analysed using single Seurat object (v.4.0.0).
After excluding low quality cells, low expressed genes and cells with high mitochondrial gene number, unsupervised clustering was performed using standard workflow by Seurat
Assembly: Mus_musculus.(mm10-Ensemble 98)
Supplementary files format and content: Tab-separated values files
Supplementary files format and content: Matrix files

source_name: kidney

extract_protocol:
Leukocytes were isolated from kidneys by digestion and percoll gradient. The MAIT cells were sorted as CD11c-CD19-B220-TCR-γδ-CD3+TCR-β+Mr1 tet (5-OP-RU)+ ; CD45-non-MAIT cells where sorted as CD45+ that were not MAIT cells
Single-cell libraries of sorted cells were generated with the 10X Genomics Chromium Single Cell 5’v1.1 reagents kit according to the manufacturer’s protocol.
The libraries were sequenced on Illumina NovaSeq 6000 with 150 base pairs and paired-end configurations

value: kidney

value: MAIT

value: CAST


title: S_52_Gati_505_redo

value: Caucasian

value: IA


title: S_51_Gati_504_redo

value: tumor


title: miRNA vEDS fibroblast P7_biological replicate


title: panNEC, PDX, C1, 12 hours, rep 1

description: Table_S4.csv and Table S6.csv, column 18, "C1_12h_PDX_3_FPKM"

value: Bo99, panNEC

value: C1, JQ1+Irinotecan 12 hours


title: miRNA vEDS fibroblast P10_biological replicate


title: S_50_Gati_503_resent

value: African American

value: Positive


title: panNEC, PDX, C1, 4 hours, rep 1

description: Table_S4.csv and Table S6.csv, column 14, "C1_4h_PDX_2_FPKM"

value: Pancreatic tumor patient-derived xenografts (PDX)

value: C1, JQ1+Irinotecan 4 hours


title: MAIT_d9

description: 10X Genomics

value: cGN


title: S_85_Gati_542_redo

value: Negative

value: IIA


title: miRNA vEDS fibroblast P7

value: dermis

value: Skin fibroblasts

value: vEDS

value: affected


title: miRNA vEDS fibroblast P6_biological replicate


title: S_49_Gati_502_resent

value: normal


title: CD45_nonMAIT_d9

description: 10X Genomics

value: kidney

value: CAST


title: miRNA vEDS fibroblast P6


title: S_60_Gati_513_redo

value: tumor

value: IIB


title: panNEC, PDX, C2, 0 hours, rep 1

description: Table_S4.csv and Table S6.csv, column 9, "C2_0h_PDX_15_FPKM"

value: Bo99, panNEC

value: C2, JQ1+Irinotecan 0 hours


title: S_75_Gati_531_redo

value: Asian

value: Positive


title: miRNA vEDS fibroblast P5


title: miRNA vEDS fibroblast P5_biological replicate


title: S_74_Gati_530_redo


title: B Cells Ocab 2

data_processing:
complete processing pipeline avaialble at https://github.com/nf-core/atacseq
Read adapter trimming using ngtrim
Alignment to mm10 using bowtie2
PCR duplicate removeal by piccard tools
Peak calling using genrich
Assembly: GRCm38
Supplementary files format and content: Bigwig files contain ATAC-seq fragment density, scaled to library size.  Peak files are in narrowPeak format as output from MACS2.

source_name: B cells ex vivo from splenocytes

extract_protocol:
Omni-ATAC-seq https://www.nature.com/articles/nmeth.4396
Illumina nextera per protocol https://www.nature.com/articles/nmeth.4396

tag: strain

tag: facs markers

value: GFP+

value: Total B cells


title: S_48_Gati_501_redo

tag: type

tag: age (at surgery)

value: African American

value: Negative


title: miRNA vEDS fibroblast P4_biological replicate

value: dermis


title: miRNA vEDS fibroblast P4

value: Skin fibroblasts


title: B Cells Ocab 1


title: S_47_Gati_500_redo

tag: race

tag: er status


title: B Cells Control 5


title: MAIT_d1

description: 10X Genomics

value: MAIT

value: cGN


title: B Cells Control 4


title: S_59_Gati_512_redo

value: normal

tag: pr


title: S_72_Gati_526_redo

value: Caucasian

value: IA


title: miRNA vEDS fibroblast P2_biological replicate

value: vEDS

value: affected


title: B Cells Control 2


title: S_53_Gati_506_redo

value: IIIC


title: S_73_Gati_529_redo

value: tumor

value: IIB


title: miRNA vEDS fibroblast P2


title: miRNA vEDS fibroblast P3_biological replicate


title: miRNA vEDS fibroblast P1_biological replicate


title: B Cells Control 3


title: Cstf64 combo rep 1

data_processing:
Basecalls were performed using bcl2fastq v2.20 for NextSeq 550 output.
ChIP-seq reads were trimmed using Cutadapt v4.0 using --nextseq-trim 25 option, discarding reads left with < 30 bp
ChIP-seq reads were aligned to the UCSC hg38 genome (or mm10 genome to determine spike-in factors) using Bowtie version 2.3.5.1 with default settings.
To create ChIP-seq average profiles, coverage matrices were generated from markduped bam files using ngs.plot.r v2.61. Spike-in normalisation of average profiles was performed by multiplying the total number of reads by a factor inversely proportional to Mouse spike-in unique deduplicated reads and an average of two normalised replicas was used for average profile visualisation with R default plot functions and ggplot2.
BigWig files were generated with bamCoverage from Deeptools v3.3.2 package with --scaleFactor option for spike-in normalization.
Assembly: hg38
Supplementary files format and content: spike-in normalized bigWig

extract_protocol:
Cells were crosslinked with 1% formaldehyde (Thermo Fisher, 28906) for 5 min. Cross-linking was stopped by the addition of glycine (125 mM) and cells were washed twice with cold PBS. After harvesting cells by scraping, the pellet was washed once with PBS plus 0.5% BSA and resuspended in RIPA buffer to a final concentration of 1x10^7 cells/ml. Samples were sonicated with a Bandelin probe sonicator to produce chromatin fragments of 400 bps on average.
DNA from ChIP was quantified with the Qubit dsDNA HS Assay Kit (Thermo Fisher, Q33230). Sequencing libraries were created according to the ThruPLEX DNA-seq kit protocol (Takara, R400676). Size selection was performed in the range of 200-700bp with AMPure XP beads (Beckman, A63880) and confirmed using the Agilent High Sensitivity DNA Kit (Agilent, 5067-4626) on the Agilent 2100 Bioanalyzer. Libraries were pooled and sequenced using the NextSeq 500/550 High Output Kit v2.5 (Illumina, 20024906). The sequencing run was Single End and Dual Index with 75 bp reads.

treatment_protocol: Cells were treated (in biological duplicates for the sequencing assays) with DMSO (vehicle), SN38, JQ1 or their combination (SN38+JQ1) for 1 hour.

value: Bo103, PDAC

value: Pancreatic tumor patient-derived cells

tag: chip antibody

value: Cstf64 (Bethyl Laboratories, #A301092A)


title: Cstf64 vehicle rep 1

tag: cell line

value: DMSO


title: RNAPII-ser2P combo rep 2

value: RNAPII-ser2P (Abcam, #ab5095)


title: Input before IP, reference for peak calling

description: Input.RPM.bw


title: Cstf64 vehicle rep 2


title: Cstf64 SN38 rep 2


title: Cstf64 SN38 rep 1


title: Cstf64 combo rep 2


title: Cstf64 JQ1 rep 2


title: panNEC, PDX, C1, 0 hours, rep 1

description: Table_S4.csv and Table S6.csv, column 8, "C1_0h_PDX_1_FPKM"

value: Bo99, panNEC

value: Pancreatic tumor patient-derived xenografts (PDX)

value: C1, JQ1+Irinotecan 0 hours


title: Cstf64 JQ1 rep 1


title: S_142_Gati_603_redo

value: African American

value: Positive

value: Negative


title: single mES cell treated with ultrafast BS, replicate 3

data_processing:
After trimming the TruSeq sequencing adapters from the 3′ ends of read 1 and read 2 using the cutadapt tool, low-quality and short reads were filtered out by applying ‘-q 20 –nextseq-trim 20 –max-n 0 -m 20’ arguments. The clean reads were then aligned to the spike-in sequences (found in Extended Data Table 1) and reference genomes (mouse reference GRCm39 for mESC samples and human reference GRCh38 for cfDNA samples) using the hisat-3n tool. The ‘--base-change C,T --no-splice-alignment –bowtie2-dp 1 –score-min L,0,1’ argument was used for all DNA libraries to ensure accurate alignment. To make use of the strand-specific property of the library, the ‘--directional-mapping’ argument was applied for 10 ng / 3.3 ng mES gDNA and cfDNA libraries, while ‘--directional-mapping-reverse’ arguments were used for single cell mESC libraries. The sorted mapped reads were then processed to remove PCR duplicates using the MarkDuplicateSpark command in the GATK tool. Finally, the number of converted and unconverted reads at all C sites were counted using the hisat-3n-table command, and the methylation ratio was calculated as the number of unconverted reads divided by total coverage.
Assembly: hg38, mm10
Supplementary files format and content: For bw files, number of conversion and unconverted are counted from mapped bam files using samtools mplieup and cpup tool. Methylation level is calculated by converstion count over sequence coverage and bw files were created by bedGraphToBigWig tools.
Supplementary files format and content: For tsv files, unconversion ratio on RNA sites are detected by the hisat3n-table tools. m5C sites are filter by statatistical cutoff and annotated based of ensembl release 108.
Library strategy: ultrafast bisulfite-seq

extract_protocol: Library preparation starting from mES genomic DNA: The mixture of mES gDNA (100-200 ng/µl) and spike-in lambda DNA (0.1%) were fragmented by sonication in 100 µl tube for 11 cycles of 30 seconds on and 30 seconds off using Bioruptor® Pico (Diagenode). The fragmented genomic DNA was size selected by Agencourt AMPure XP (Beckman Coulter) to 300-500 bp and eluted with 17 µl water. 3′-Dephosphorylation was conducted by adding 2 µl 10x T4PNK buffer (NEB) and 1 µl of T4 PNK (NEB) and the mixture was incubated at 37 °C for 30 min followed by 65 °C for 20 min. After purification by DNA Clean & Concentrate kit (Zymo Research), 164 bp dsDNA oligos (0.001%) was added as spike-in.  After BS treatment, all samples were desulphonated on Zymo-Spin IC columns following manufacturer’s protocol except that final washing step was conducted for four times instead of twice. Then the samples were incubated at 95 °C for 5 min to denature DNA, and cold-shocked on ice for 2 min followed by libraries construction using xGen™ Methyl-Seq Lib Prep kit for 96rxn (IDT, catalog no: 10009824).   Library preparation starting from small number of mES cells (100-cell, 10-cell and single-cell): The DNA libraries starting from small number of mES cells were constructed following the previous method with slight modifications40,41. In brief, mES cells were sorted by fluorescence-activated cell sorting (FACS) into lysis buffer containing 20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 20 mM KCl, 0.3% Triton-X 100, 1 mg/mL QIAGEN protease and 0.6 pg lambda DNA (dam-, dcm-). Genomic DNA was released under the program of 50 °C for 3 h, followed by 75 °C for 30 min. Bisulfite treatment was performed using UBS-1 and Zymo DNA Methylation-Gold kit side by side. The first-strand DNA was synthesized by random priming using 50 U Klenow exo-, 400 μM P5-N6-oligo1 (CTACACGACGCTCTTCCGATCTNNNNNN, IDT) at 37 °C for 30 min. After 4 rounds of random priming, samples were purified with 0.8x AMPure XP beads. The second strand was synthesized using P7-N6-oligo2 (AGACGTGTGCTCTTCCGATCTNNNNNN, IDT). After purification with 0.8x AMPure XP beads, the sequencing index was introduced by PCR using Multiplex Oligos for Illumina (NEB) and KAPA HiFi HotStart ReadyMix. Library preparation for human plasma cfDNA: 41 ng of plasma cfDNA either from healthy people or from patient’s sample was spiked-in with 0.1% of the 164 bp dsDNA containing four 5mC sites and water was added to give each sample total volume 25 µl. Then to each sample, End Repair & A-Tailing Buffer (3.5 µl) and End Repair & A-Tailing Enzyme Mix from KAPA HyperPlus kit (Roche, catalog no: 07962428001) were added and the mixture was incubated at 65 °C for 30 min. Then a mixture of 15 µl ligation buffer, 5 µl DNA ligase, 2.5 µl water and 2.5 µl 15 µM methylated NEBNext adaptor was added, and the mixture was incubated at 20 °C for 1 h. Then 3 µl USER enzyme (NEB, catalog no: M5505S) was added and incubated at 37 °C for 15 min. Then the ligated DNA was purified by 1.0x Ampure beads and eluted with 41 µl water and divided into 5 parts. One part equivalent to 1 ng cfDNA will be directly amplified as input library. Two parts containing 10 ng cfDNA were treated with UBS condition followed by desulphonation the same procedure as described above for mES gDNA, and the other two parts equivalent to 10 ng cfDNA were treated with the conventional BS condition following manufacture’s protocol. The purified DNA was then amplified using KAPA HiFi Uracil+ DNA polymerase (Roche, catalog no: 07959079001) and the libraries was purified by 0.7x Ampure beads.

value: mESC

tag: sample amount

value: single mES cell

value: WT

value: ultrafast BS


title: miRNA vEDS fibroblast P1

tag: tissue

value: dermis

value: Skin fibroblasts

value: vEDS

tag: disease state

value: affected


title: miRNA vEDS fibroblast P3

tag: cell type


title: RNAPII-ser2P JQ1 rep 2

value: Bo103, PDAC

value: Pancreatic tumor patient-derived cells


title: siNSUN6 HeLa polyA+ RNA treated with ultrafast BS, replicate 2

description:
RNA-siNSUN6-UBS2
HeLa-siNSUN6_sites.tsv

source_name: HeLa

value: HeLa

value: epithelial cell

tag: method


title: S_54_Gati_507_resent

value: tumor

value: Caucasian

tag: Stage

value: IIIC


title: single mES cell treated with ultrafast BS, replicate 2

tag: genotype


title: B Cells Ocab 4

value: GFP+

value: Total B cells


title: B Cells Ocab 5


title: B Cells Ocab 3


title: miRNA Control fibroblast C18_biological replicate

value: healthy donors

value: unaffected


title: B Cells Control 1


title: miRNA Control fibroblast C18


title: RNAPII-ser2P SN38 rep 2

tag: treatment

value: RNAPII-ser2P (Abcam, #ab5095)


title: RNAPII-ser2P JQ1 rep 1


title: RNAPII-ser2P vehicle rep 2

value: DMSO


title: siControl HeLa polyA+ RNA treated with ultrafast BS, replicate 2

description:
RNA-siControl-UBS2
HeLa-siControl_sites.tsv

value: siControl

value: ultrafast BS


title: RNAPII-ser2P SN38 rep 1


title: 10 mES cells treated with ultrafast BS, replicate 3

value: mESC

value: 10 mES cells

value: WT


title: siControl HeLa polyA+ RNA treated with ultrafast BS, replicate 1

description:
RNA-siControl-UBS1
HeLa-siControl_sites.tsv


title: miRNA Control fibroblast C16_biological replicate

value: dermis

value: Skin fibroblasts


title: 100 mES cells treated with ultrafast BS, replicate 3

value: 100 mES cells


title: RNAPII-ser2P combo rep 1

value: Bo103, PDAC

value: Pancreatic tumor patient-derived cells


title: single mES cell treated with ultrafast BS, replicate 1

value: single mES cell


title: siNSUN6 HeLa polyA+ RNA treated with ultrafast BS, replicate 1

description:
RNA-siNSUN6-UBS1
HeLa-siNSUN6_sites.tsv

value: HeLa

value: epithelial cell


title: miRNA Control fibroblast C17_biological replicate

value: healthy donors


title: miRNA Control fibroblast C17

value: unaffected


title: 10 mES cells treated with ultrafast BS, replicate 1


title: shNSUN2 HeLa polyA+ RNA treated with ultrafast BS, replicate 2

description:
RNA-shNSUN2-UBS2
HeLa-shNSUN2_sites.tsv


title: 10 mES cells treated with ultrafast BS, replicate 2


title: miRNA Control fibroblast C16


title: shControl HeLa polyA+ RNA treated with ultrafast BS, replicate 2

description:
RNA-shControl-UBS2
HeLa-shControl_sites.tsv

value: shControl


title: shNSUN2 HeLa polyA+ RNA treated with ultrafast BS, replicate 1

description:
RNA-shNSUN2-UBS1
HeLa-shNSUN2_sites.tsv


title: shControl HeLa polyA+ RNA treated with ultrafast BS, replicate 1

description:
RNA-shControl-UBS1
HeLa-shControl_sites.tsv


title: WT HEK293T polyA+ RNA treated with ultrafast BS, replicate 3

description:
RNA-HEK293T-UBS3
HEK293T-WT_sites.tsv

value: embryonic kidney


title: RNAPII-ser2P vehicle rep 1

value: DMSO

value: RNAPII-ser2P (Abcam, #ab5095)


title: BRD4 combo rep 2

value: BRD4 (Bethyl Laboratories, #A301985A100)


title: 100 mES cells treated with ultrafast BS, replicate 2

value: mESC

value: ultrafast BS


title: BRD4 JQ1 rep 2


title: miRNA Control fibroblast C15_biological replicate

value: dermis


title: miRNA Control fibroblast C14_biological replicate

value: Skin fibroblasts


title: BRD4 combo rep 1