title: ChIP_501mel_set2_dCas9_ALK_sgint4_Cas9

description: Library name: Sample 8


title: ChIP_501mel_set2_dCas9_ALK_sgint19_Cas9

status: Public on May 01 2025

type: SRA

city: New York

first: Elissa

middle: WP

last: Wong

state: NY

address: 1275 York Avenue

department: HOPP

country: USA

institute: Memorial Sloan Kettering Cancer Center

description: Library name: Sample 10

library_source: genomic

data_processing:
ChIP-seq reads were aligned to hg19 using BWA (v0.7.17-r1188). Unmapped and low MAPQ reads (< 30) were filtered out using SAMtools (v1.9) and PCR duplicates were removed using Picard tools (v2.18.16). Depth normalized bigWig tracks were generated using deepTools (v3.1.1) bamCoverage with `bamCoverage -b ${input_bam} -o ${output_bigwig} --normalizeUsing RPGC --ignoreForNormalization chrX chrM -bl hg19-blacklist.v2.bed`.
Assembly: hg19
Supplementary files format and content: bigWig

molecule: genomic DNA

organism: Homo sapiens

source_name: skin

growth_protocol: All cell lines were cultured in media supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. All cells were cultured at 37 ºC in 5% CO2.

extract_protocol:
Cas9 ChIP was performed with 1% formaldehyde fixation for 15 min. Cells were washed once with PBS, resuspended in LB1 [50 mM HEPES (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100 and 1× complete EDTA-free protease inhibitor] and incubated with rotation at 4 °C for 10 min. Samples were pelleted, resuspended in LB2 [10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and 1× complete protease inhibitor] and incubated rotating at 4 °C for 10 min. Nuclei were pelleted, resuspended in LB3 with 0.2% SDS [0.2% SDS, 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100 and 1× complete protease inhibitor] and sonicated using Covaris E220 focused ultrasonicator (peak power = 150 W, duty factor = 2%, cycle per burst = 200) for 20 min. Soluble chromatin extracts were cleared by centrifugation and 1% was kept as input DNA. Sheared chromatin was diluted to contain 0.1% SDS for anti-Cas9 antibody (Active Motif, 61957) binding. 20 ul Pierce ChIP-grade magnetic beads were used for precipitation at 4 °C for 6 hr. Beads were washed 3x LiCl buffer [250 mM LiCl, 10 mM Tris-HCl (pH 8.0), 1% sodium deoxycholate, 1% NP-40 and 1 mM EDTA], 1x RIPA wash buffer [500 mM LiCl, 50 mM HEPES-KOH (pH 7.6), 0.7% sodium deoxycholate, 1% NP-40 and 1 mM EDTA] and 1x TE [10 mM Tris-HCl (pH 8.0) and 1 mM EDTA] + 50 mM NaCl. Chromatin was eluted in elution buffer [1% SDS, 50 mM Tris-HCl (pH 8.0) and 10 mM EDTA] and incubated at 65 °C for 30 min. Eluate was reverse cross-linked at 65 °C overnight, treated with RNaseA (Roche) at 37 °C for 1 hr and Proteinase K (Roche) at 55 °C for 2 hr before recovered using a miniElute PCR purification kit from Qiagen.
Library was prepared using KAPA HTP library kit (KAPA biosystems).
ChIP libraries were sequenced to obtain 30-40 M reads for each sample on NovaSeq 6000 with PE100.

treatment_protocol: dCas9-KRAB positive cells that were transduced with lentivirus carrying puromycin-resistant shRNA plasmids were treated with 2 µg/ml puromycin.

tag: tissue

value: skin

tag: cell line

tag: cell type

value: melanoma

tag: genotype

value: dCas9-KRAB, sgALK int19

tag: antibody

value: Cas9 antibody


title: ChIP_501mel_set2_dCas9_sgGFP_Cas9

description: Library name: Sample 6

value: dCas9-KRAB, sgGFP


title: ChIP_501mel_set2_dCas9_ALK_sgCtrl2_Cas9

description: Library name: Sample 4

value: dCas9-KRAB, sgALK Ctrl 2


title: ChIP_501mel_set1_dCas9_sgGFP_Cas9

description: Library name: Sample 5


title: ChIP_501mel_set1_dCas9_ALK_sgCtrl2_Cas9

description: Library name: Sample 3


title: ChIP_501mel_set2_dCas9_ALK_sgCtrl1_Cas9

description: Library name: Sample 2

value: dCas9-KRAB, sgALK Ctrl 1


title: ChIP_501mel_set1_dCas9_ALK_sgCtrl1_Cas9

description: Library name: Sample 1


title: CNR_501mel_set1_dCas9_ALK_sgCtrl2_H3K9me3

description: Library name: sample 36

data_processing:
CUTNRUN-seq reads were aligned to hg19 using BWA (v0.7.17-r1188). Unmapped and low MAPQ reads (< 30) were filtered out using SAMtools (v1.9) and PCR duplicates were removed using Picard tools (v2.18.16). Depth normalized bigWig tracks were generated using deepTools (v3.1.1) bamCoverage with `bamCoverage -b ${input_bam} -o ${output_bigwig} --normalizeUsing RPGC --ignoreForNormalization chrX chrM -bl hg19-blacklist.v2.bed`.
Assembly: hg19
Supplementary files format and content: bigWig
Library strategy: CUT&RUN

growth_protocol: All cells lines were cultured in media supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. All cells were cultured at 37 ºC in 5% CO2.

extract_protocol:
CUT&RUN was performed using CUT&RUN assay kit (Cell signaling technology). Briefly, 0.25 x 106 live cells were immobilized on activated Concanavalin A beads and permeabilized with digitonin. Cell:bead suspension was washed with 1x wash buffer supplemented with spermidine and protease inhibitor cocktail and incubated with 1 µg of antibody at 4 °C overnight. The following antibodies were used: anti-H3K27ac (Abcam, ab4729) and anti-H3K9me3 (Abcam, ab176916) antibodies. Antibody-targeted digestion of chromatin was performed by incubation of pAG-MNase at 4 °C for 1 hr and activation by calcium chloride at 4 °C for 30 min. MNase cleavage was stopped by addition of 1x stop buffer. Digested chromatin was allowed to diffuse out of cells at 37 °C for 20 min and DNA was purified by Nucleospin gel and PCR clean-up spin column (Takara bio).
Sequencing libraries were prepared using the KAPA HTP Library Preparation Kit (KAPA Biosystems).
Barcoded libraries were run on NovaSeq 6000 in a PE100 run. An average of 25 M reads were generated per sample.

value: H3K9me3 antibody


title: CNR_501mel_set2_dCas9_ALK_sgint4_H3K9me3

description: Library name: sample 39

value: dCas9-KRAB, sgALK int4


title: CNR_501mel_set1_dCas9_ALK_sgint19_H3K9me3

description: Library name: sample 40


title: CNR_501mel_set2_dCas9_ALK_sgint19_H3K9me3

description: Library name: sample 41


title: CNR_501mel_set2_dCas9_ALK_sgCtrl1_H3K9me3

description: Library name: sample 35


title: CNR_501mel_set2_dCas9_ULK4_sgint31_H3K9me3

description: Library name: sample 33

value: dCas9-KRAB, sgULK4 int31


title: CNR_501mel_set1_dCas9_ALK_sgCtrl1_H3K9me3

description: Library name: sample 34


title: CNR_501mel_set1_dCas9_ALK_sgint4_H3K9me3

description: Library name: sample 38


title: CNR_501mel_set1_dCas9_ULK4_sgint31_H3K9me3

description: Library name: sample 32


title: CNR_501mel_set1_dCas9_ULK4_sgint30_H3K9me3

description: Library name: sample 30

value: dCas9-KRAB, sgULK4 int30


title: CNR_501mel_set2_dCas9_ULK4_sgCtrl2_H3K9me3

description: Library name: sample 29

value: dCas9-KRAB, sgULK4 Ctrl 2


title: ChIP_501mel_set1_dCas9_NOsg_Cas9

description: Library name: Sample 11


title: CNR_501mel_set1_dCas9_ULK4_sgCtrl2_H3K9me3

description: Library name: sample 28


title: CNR_501mel_set1_dCas9_ULK4_sgCtrl1_H3K9me3

description: Library name: sample 26

value: dCas9-KRAB, sgULK4 Ctrl 1


title: CNR_501mel_set2_dCas9_ULK4_sgCtrl1_H3K9me3

description: Library name: sample 27


title: CNR_501mel_set2_dCas9_NOsg_H3K9me3

description: Library name: sample 23


title: CNR_501mel_set2_dCas9_sgGFP_H3K9me3

description: Library name: sample 25


title: CNR_501mel_set2_dCas9_ALK_sgint4_H3K27ac

description: Library name: sample 19

value: H3K27ac antibody


title: CNR_501mel_set1_dCas9_sgGFP_H3K9me3

description: Library name: sample 24


title: CNR_501mel_set1_dCas9_ALK_sgint19_H3K27ac

description: Library name: sample 20


title: CNR_501mel_set1_dCas9_NOsg_H3K9me3

description: Library name: sample 22


title: CNR_501mel_set2_dCas9_ALK_sgCtrl2_H3K9me3

description: Library name: sample 37


title: CNR_501mel_set1_dCas9_ALK_sgint4_H3K27ac

description: Library name: sample 18


title: CNR_501mel_set2_dCas9_ALK_sgCtrl2_H3K27ac

description: Library name: sample 17


title: CNR_501mel_set2_dCas9_ALK_sgCtrl1_H3K27ac

description: Library name: sample 15


title: CNR_501mel_set1_dCas9_ALK_sgCtrl2_H3K27ac

description: Library name: sample 16


title: CNR_501mel_set2_dCas9_ULK4_sgint31_H3K27ac

description: Library name: sample 13


title: CNR_501mel_set1_dCas9_ALK_sgCtrl1_H3K27ac

description: Library name: sample 14


title: CNR_501mel_set2_dCas9_ULK4_sgint30_H3K9me3

description: Library name: sample 31


title: CNR_501mel_set2_dCas9_ULK4_sgint30_H3K27ac

description: Library name: sample 11


title: CNR_501mel_set1_dCas9_ULK4_sgCtrl2_H3K27ac

description: Library name: sample 8


title: CNR_501mel_set2_dCas9_ULK4_sgCtrl1_H3K27ac

description: Library name: sample 7


title: CNR_501mel_set2_dCas9_sgGFP_H3K27ac

description: Library name: sample 5


title: CNR_501mel_set1_dCas9_ULK4_sgCtrl1_H3K27ac

description: Library name: sample 6


title: CNR_501mel_set2_dCas9_ULK4_sgCtrl2_H3K27ac

description: Library name: sample 9


title: CNR_501mel_set1_dCas9_ULK4_sgint30_H3K27ac

description: Library name: sample 10


title: CNR_501mel_set1_dCas9_ULK4_sgint31_H3K27ac

description: Library name: sample 12


title: CNR_501mel_set2_dCas9_NOsg_H3K27ac

description: Library name: sample 3


title: CNR_501mel_set1_dCas9_NOsg_H3K27ac

description: Library name: sample 2


title: CNR_501mel_set2_dCas9_ALK_sgint19_H3K27ac

description: Library name: sample 21


title: CAGEseq_501mel shNIPBL_#5 6D rep1

description: Library name: CAGEseq_501mel shNIPBL_#5 6D rep1

library_source: transcriptomic

data_processing:
CAGE-seq reads were analyzed based on the Nextflow CAGE-seq analysis pipeline.
Index sequences, EcoP15I recognition site and the first G following the EcoP15I site at the 5’-end from reads were removed using Cutadapt (v2.6).
rRNA sequences were filtered out using SortMeRNA (v4.2.0) and filtered reads were aligned to the hg19 genome using STAR (v2.7.1a).
At this step, two sets of BAM files were created for each sample: one with uniquely mapped reads (adding --outFilterMultimapNmax 1 to STAR command) and one with multi-mapped reads (adding --outFilterMultimapNmax 10 to STAR command). Unmapped reads from BAM files were removed.
BAM files were converted to BED files with the summed up 1 bp unclustered CAGE tags using SAMtools (v1.9) and bamtobed (v.2.26.0). We pooled CAGE tags across samples, filtered them by TPM threshold of 0.5 and then clustered them with paraclu to generate a BED file with the clustered CAGE transcription start sites (CTSSs). CAGE tags were intersected with the clusters identified by paraclu and a raw count table with columns for each sample and rows for each tag cluster was generated. DESeq2 (v1.30.1) was used to perform differential CAGE expression using the raw count table. CTSS clusters were annotated using R/Bioconductor packages ChIPseeker (v1.26.2) and AnnotationDbi (v1.52) using GENCODE (v19) gene annotations.
Assembly: hg19
Supplementary files format and content: Differential expression analysis by DESeq2
Library strategy: CAGE-seq

molecule: total RNA

extract_protocol:
Cells were lyzed directly on culture dish with TRIzol RNA isolation reagent (Thermo fisher scientific). Total RNA was extracted by thorough mixing with chloroform and isopropanol precipitation of upper aqueous phase. 5 µg total RNA was reverse transcripted using a primer that carried a 15-nt random region and EcoP15I recognition sequence and SuperScriptIII RNase H- reverse transcriptase (Thermo fisher scientific) in the presence of trehalose/sorbitol to increase efficiency of reverse transcription. Diol groups on RNA/cDNA duplex were oxidated and labelled with biotin hydrazide (Vector laboratories). RNase I (Promega) treatment was performed to cleave single-stranded RNA regions that were not hybridized with first-strand cDNA. tRNA-coated streptavidin beads (Thermo fisher scientific) were used to capture biotinylated RNA/cDNA duplex. Sample was washed three times with wash buffer A [4.5 M NaCl, 50 mM EDTA (pH 8.0) and 0.1 % Tween20], once with wash buffer B [10 mM Tris–HCl (pH 8.5), 1 mM EDTA (pH 8.0), 0.5 M NaOAc (pH 6.1) and 0.1 % Tween20] and once with wash buffer C [0.3 M NaCl, 1 mM EDTA (pH 8.0) and 0.1 % Tween20]. Single-stranded cDNAs were released from streptavidin beads by heating at 95 °C for 5 min. Released cDNA was treated with RNase H (Thermo fisher scientific) and RNase I (Promega) at 37 °C for 45 min and proceed for library construction.
Library was constructed with modified primers for compatible sequencing on Illumina Novaseq 6000 sequencer. Ligation of bar-coded 5’ linker was performed. Second-strand cDNA was synthesized using Deep Vent (exo-) DNA polymerase (NEB) and biotinylated second-strand primer. cDNA was treated with shrimp alkaline phosphatase (Thermo fisher scientific) to remove phosphate group from 5’ linker. EcoP15I (NEB) was used to cleave 27 nt downstream of the recognition site at the 5’ linker. 3’ linker was ligated to the 27-nt-long tags, purified with tRNA-coated streptavidin beads and amplified with 2x Phusion high-fidelity PCR mix (NEB). CAGE libraries were treated with Exonuclease I (NEB) to degrade any single-stranded DNA before sequencing.
Libraries were sequenced on NovaSeq 6000 using SR50 to obtain around 40 M reads for each library.

treatment_protocol: Cells that were transduced with lentivirus carrying puromycin-resistant shRNA plasmids were treated with 2 µg/ml puromycin.

tag: treatment


title: CNR_501mel_dCas9_input

description: Library name: sample 1

value: input


title: CAGEseq_501mel shNIPBL_#4 6D rep2

description: Library name: CAGEseq_501mel shNIPBL_#4 6D rep2


title: CAGEseq_501mel shNIPBL_#5 6D rep2

description: Library name: CAGEseq_501mel shNIPBL_#5 6D rep2


title: CAGEseq_501mel shNIPBL_#4 6D rep1

description: Library name: CAGEseq_501mel shNIPBL_#4 6D rep1


title: CAGEseq_501mel shLuc 6D rep3

description: Library name: CAGEseq_501mel shLuc 6D rep3

value: shLuc


title: Colonic mucosa, UC3

city: Hangzhou

first: Shaopeng

last: Sun

state: Zhejiang

address: No. 54, Youdian Road, Shangcheng District

department: Department of Gastroenterology

country: China

institute: The First Affiliated Hospital of Zhejiang Chinese Medical University

description:
Library name: Sample6
UC.3

data_processing:
Use FastP software to perform quality filtering on the raw sequencing data of each sample. We used StringTie for the assembly of eukaryotic transcriptomes, extracted transcriptome sequences, and annotated species using the ensembl database. Salmon algorithm for gene expression quantification
Assembly: GRCh38
Supplementary files format and content: Excel file includes FPKM values for each Sample

source_name: Colonic mucosa

extract_protocol:
Perform preliminary quantification of library concentration using Qubit 3.0 and dilute the library to 1 ng/ul.
RNA libraries for RNA-seg were prepared using SMARTER mRNA-Seq LibraryPrep Kit following manufacturer's protocols.

value: Colonic mucosa


title: Colonic mucosa, UC1

description:
Library name: Sample4
UC.1


title: Colonic mucosa, UC2

description:
Library name: Sample5
UC.2


title: Colonic mucosa, Control2

description:
Library name: Sample2
Cont.2


title: Colonic mucosa, Control3

description:
Library name: Sample3
Cont.3


title: LDH

city: Nanjing

first: YONG

middle: XIN

last: CHEN

address: 167 Hushan Road

department: Department of Spine Surgery

institute: The Affiliated Jiangning Hospital of Nanjing Medical University

description:
Library name: Sample2
10x Genomics

library_source: transcriptomic single cell

data_processing:
Raw FASTQ sequencing data were processed using CellRanger (V6.1.1), a software officially provided by 10x Genomics for 10x single-cell transcriptome data analysis. Normalize gene expression matrices using Seurat's “LogNormalize” method.
Assembly: GRCh38
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Ligamentum flavum

extract_protocol:
The LF specimens were sliced into pieces measuring 0.5 mm^² and washed repeatedly with 1× PBS to remove surface impurities. The treated LF tissues were added to the dissociation solution, and the process was executed at 37°C in a water-bath shaker for a duration of 20 minutes. After filtering the cell suspension through a stacked cell strainer, it was centrifuged at 300 g for 5 minutes at 4°C. Then, the cell precipitate was collected and resuspended with PBS solution. Lysis buffer was added on ice to remove red blood cells, followed by centrifugation at 300 g for 5 minutes, and the cell pellet was collected. Dead cells were removed using the Miltenyi® Dead Cell Removal Kit (MACS 130-090-101). Following this, the suspension was resuspended in 0.04% BSA and centrifuged at 300 g for 3 minutes at 4℃, with the procedure repeated twice. After obtaining the candidate cells, 50 μl of 0.04% BSA was used to prepare a cell suspension. Taipan blue staining was used to detect cell activity, which needed to be greater than 85%. The cell concentration, measured by an Automated Cell Counter, was between 700 and 1200 cells per microliter.
According to the manufacturer's guidelines for the Human Single-Cell Kit, single-cell suspensions were loaded onto 10x Chromium to capture individual cells. The steps for cDNA amplification and library construction that followed adhered to the designed protocol.

value: Ligamentum flavum

value: primary cell

value: fibroblast


title: LSS

description:
Library name: Sample1
10x Genomics


title: Colonic mucosa, Control1

description:
Library name: Sample1
Cont.1


title: PPP2

city: Ulm

first: Pallab

last: Maity

address: Meyerhofstrasse/James Franck Ring

department: Dermatology

country: Germany

institute: University of Ulm

description: Library name: 16_HS_SB_S16

data_processing:
Sequencing reads were next subjected to modified Tuxedo protocol (Pertea et al., 2016). Hisat2 was used to align the sequencing reads to human reference sequence (grch38), followed by conversion of aligned sequence slignment sap (SAM) files to indexed bam files by samtools (version 1.7). Transcript assembly, merging and quantification of transcripts of all samples were then performed by Stringtie (version 2.1.5). Differential expression analyses were performed by DESeq2 (version 1.34.0) in RStudio environment.
Assembly: grch38
Supplementary files format and content: tab delimited text file containg raw count data of each sample

molecule: polyA RNA

extract_protocol:
Total RNA was isolated from human skin biopsies using RNeasy Micro Kit (Qiagen).
200 ng of total RNA was used for cDNA library preparation using NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina (NEB).

value: skin


title: PPP_Healthy2

description: Library name: 21_HS_SB_S5


title: PPP_Healthy1

description: Library name: 19_HS_SB_S3


title: GPP_Healthy1

description: Library name: 18-HS-PS_S6


title: GPP_Healthy3

description: Library name: 21-HS-PS_S9


title: GPP2

description: Library name: 12_HS_SB_S12


title: GPP_Healthy2

description: Library name: 19-HS-PS_S7


title: PPP3

description: Library name: 17-HS-PS_S5


title: PPP1

description: Library name: 16-HS-PS_S4


title: CAGEseq_501mel shLuc 6D rep4

description: Library name: CAGEseq_501mel shLuc 6D rep4

value: melanoma


title: CNR_501mel_set1_dCas9_sgGFP_H3K27ac

description: Library name: sample 4

value: dCas9-KRAB, sgGFP


title: GPP3

description: Library name: 13_HS_SB_S13


title: KGN cells, treated with 77-39 for 72h(3)

city: shenzhen

first: jingxin

last: ma

address: Xueyuan Avenue, Nanshan District, Shenzhen

institute: shenzhen university

description: Library name: sample6

data_processing:
Illumina Casava2.19 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm10 whole genome using hisat2 v2.0.5 with parameters hisat2 [options]* -x <hisat2-idx> {-1 <m1> -2 <m2> | -U <r> | --sra-acc <SRA accession number>} [-S <hit>]
Fragments Per Kilobase of exon per Megabase of library size (FPKM) were calculated using a protocol from Trapnell,Cole et al., Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol, 2010. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Assembly: mm8
Supplementary files format and content: tab-delimited text file includes FPKM values for each Sample

source_name: ovarian

growth_protocol: Cells were grown in DMEM/F-12 medium without red phenol containing 10% heat inactivated FBS.Cells were grown at 37 °C in a humidified atmosphere with 5%  CO2, and medium was replaced twice per week.

extract_protocol:
Retinas were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: the cell were treated with 10μm/ml 77-39 for 72h

value: ovarian

value: KGN

value: granulosa cell tumor

value: WT


title: COV434 cells,control1

description: Library name: sample7


title: COV434 cells, treated with 77-39 for 72h(1)

description: Library name: sample10


title: COV434 cells, treated with 77-39 for 72h(3)

description: Library name: sample12


title: GPP1

description: Library name: 12-HS-PS_S1


title: COV434 cells, treated with 77-39 for 72h(2)

description: Library name: sample11


title: KGN cells,control1

description: Library name: sample1


title: PPP_Healthy3

description: Library name: 22_HS_SB_S6


title: COV434 cells,control3

description: Library name: sample9


title: COV434 cells,control2

description: Library name: sample8


title: Circle-seq of Human: adult male urine; PCa20

city: Hangzhou, Zhejiang

first: Zhimin

last: Zhou

address: No. 866, Yuhangtang Road, Xihu District, Hangzhou City, Zhejiang Province

institute: Zhejiang University

description: Library name: PCa20

data_processing:
Raw data quality control was conducted using FastQC (v0.11.9). Subsequent trimming was performed with Trim Galore (v0.6.7) to remove adapters and low-quality bases, setting an error tolerance rate of 0.1. Reads with lengths greater than 20 bp after trimming were retained (parameters: -q20, --stringency, --length 20, -e 0.1) and aligned to UCSC hg38 reference genome using the BWA software (v0.7.17-r1188) with the BWA-MEM algorithm and default parameters. The resulting BAM files were subjected to Circle-Map software (v1.1.4) to call eccDNAs.
Assembly: hg38
Supplementary files format and content: tab-delimited bed file includes eccDNA chromatin coordinate and related properties (e.g., split reads) for each Sample

source_name: Urine

extract_protocol:
Urine samples were immediately subjected to cfDNA extraction following the manual of the Quick-DNA™ Urine DNA Kit (ZYMO RESEARCH, D3061). To remove linear DNA, the purified cfDNA was treated with 5 units of Plasmid-Safe DNase (Epicentre, E3110K) at 37°C for 16 hours followed by incubation at 70°C for 30 min to inactivate the DNase. EccDNA was purified by 2 × DNA beads cleanup (Vazyme, N411) and eluted in 13 μl of TE buffer. Next, 10 μl of eccDNA was amplified at 30°C for 8 hours in a reaction mixture containing 5 μl of Reaction Buffer (10 ×), 1 μl of Phi29 MAX DNA Polymerase (Vazyme, N106), 2.5 μl of Exo-Resistant Random Primers (500 mM) (MCLAB, ERRP-110), 0.8 μl of dNTP Solution Mix (10 mM) (NEB, N0447S), and 26.5 μl of nuclease-free water. The amplified DNA was debranched by S1 nuclease (Thermo, EN0321), then purified by 2 × DNA beads cleanup, and finally eluted with 25 μl of TE buffer. 500 ng of debranched DNA was sheared by Covaris S220 with a targeted peak size of 400 bp.
PCR-free DNA library was constructed from the sheared DNA with VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, ND607). The quality of each library was examined by Bioanalyzer with Agilent DNF-915 Reagent Kit. All libraries were sequenced on the Illumina NovaSeq 6000 platform with paired-end 150 bp reads, generating an expected data of 10 Gb for each.

value: Urine


title: KGN cells,control3

description: Library name: sample3


title: KGN cells,control2

description: Library name: sample2


title: Circle-seq of Human: adult male urine; PCa16

description: Library name: PCa16


title: Circle-seq of Human: adult male urine; PCa13

description: Library name: PCa13


title: Circle-seq of Human: adult male urine; PCa12

description: Library name: PCa12


title: Circle-seq of Human: adult male urine; PCa21

description: Library name: PCa21


title: Circle-seq of Human: adult male urine; PCa19

description: Library name: PCa19


title: Circle-seq of Human: adult male urine; PCa15

description: Library name: PCa15


title: Circle-seq of Human: adult male urine; PCa18

description: Library name: PCa18


title: Circle-seq of Human: adult male urine; PCa7

description: Library name: PCa7


title: Circle-seq of Human: adult male urine; PCa5

description: Library name: PCa5


title: Circle-seq of Human: adult male urine; PCa17

description: Library name: PCa17


title: Circle-seq of Human: adult male urine; PCa4

description: Library name: PCa4


title: Circle-seq of Human: adult male urine; PCa11

description: Library name: PCa11


title: Circle-seq of Human: adult male urine; PCa9

description: Library name: PCa9


title: KGN cells, treated with 77-39 for 72h(2)

description: Library name: sample5


title: KGN cells, treated with 77-39 for 72h(1)

description: Library name: sample4


title: Circle-seq of Human: adult male urine; PCa8

description: Library name: PCa8


title: Circle-seq of Human: adult female urine; NC16

description: Library name: NC16


title: Circle-seq of Human: adult female urine; NC14

description: Library name: NC14


title: Circle-seq of Human: adult male urine; NC13

description: Library name: NC13


title: Circle-seq of Human: adult female urine; NC15

description: Library name: NC15


title: Circle-seq of Human: adult female urine; NC12

description: Library name: NC12


title: Circle-seq of Human: adult male urine; NC11

description: Library name: NC11


title: Circle-seq of Human: adult male urine; PCa3

description: Library name: PCa3


title: Circle-seq of Human: adult male urine; PCa6

description: Library name: PCa6


title: Circle-seq of Human: adult male urine; PCa10

description: Library name: PCa10


title: Circle-seq of Human: adult male urine; PCa2

description: Library name: PCa2


title: Circle-seq of Human: adult male urine; PCa1

description: Library name: PCa1


title: Circle-seq of Human: adult male urine; NC6

description: Library name: NC6


title: Circle-seq of Human: adult female urine; NC10

description: Library name: NC10


title: Circle-seq of Human: adult male urine; PCa14

description: Library name: PCa14


title: Circle-seq of Human: adult male urine; NC5

description: Library name: NC5


title: Circle-seq of Human: adult female urine; NC2

description: Library name: NC2


title: Circle-seq of Human: adult female urine; NC1

description: Library name: NC1


title: Circle-seq of Human: adult female urine; NC8

description: Library name: NC8


title: Circle-seq of Human: adult male urine; NC7

description: Library name: NC7


title: R-loop CUT&Tag  of ISO induced PC-3; Human; adult male; Replicate2

description: Library name: ISO-induced_PC-3_replicate2

data_processing:
Raw data quality control was conducted using FastQC (v0.11.9). Subsequent trimming was performed with Trim Galore (v0.6.7) to remove adapters and low-quality bases, setting an error tolerance rate of 0.1. Reads with lengths greater than 20 bp after trimming were retained (parameters: -q20, --stringency, --length 20, -e 0.1) and aligned to UCSC hg38 reference genome using the Bowtie2 (v2.5.1). The resulting BAM files were subjected to  MACS2 (v2.2.9.1) to call R-loops.
Assembly: hg38
Supplementary files format and content: tab-delimited bed file includes peak chromatin coordinate and related properties (e.g., signalValue) for each Sample

extract_protocol:
PC-3 cells were treated with 30 μM ISO or an equal volume of DMSO (mock) for 24 hours and then collected for CUT&Tag using Hyperactive Universal CUT&Tag Assay Kit for Illumina kit (Vazyme, TD903) as previously described with minor modifications [25]. In brief, 5 × 104 cells were incubated with ConA beads for 10 minutes at room temperature. Cells were lysed with digitonin and then incubated overnight at 4°C with Anti-DNA-RNA Hybrid Antibody (clone S9.6) (Millipore, MABE1095). Following incubation with anti-mouse secondary antibody, pA/G-Tnp was added to digest DNA.
The fragmented DNA was purified with ChIP DNA Clean &concentrator kit (ZYMO RESEARCH, D5205) and amplified by PCR with 12 cycles. Final libraries were sequenced on Illumina NovaSeq 6000 platform.


title: MCF7H_39_Ad_CMV_GFP_72h_Replicate2

city: Bethesda

first: David

middle: J.

last: Clark

state: MD

address: 6 Center Drive

department: NICHD

institute: NIH

description: Library name: MCF7H_39_Ad_GFP_72h_R2

data_processing:
We used salmon/1.10.0 to calculate the expression level for each transcript with parameters: -l A --validateMappings.
The transcript reference is the pc_transcripts v43 transcripts of hg38 assembly from GENCODE.
The tab-delimited text files from salmon contains the expression level in TPM.
Assembly: hg38
Supplementary files format and content: tab-delimited text files include TPM values for each Sample

source_name: Breast; Mammary gland

extract_protocol:
RNA was extracted using Direct-zol RNA MiniPrep (Qiagen). 1.5-2 ug of total RNA was used for the construction of sequencing libraries.
Libraries for RNA-seq were prepared using TruSeq Stranded mRNA LT Sample Prep Kit following manufacturer's protocols.

value: Breast; Mammary gland

value: Epithelial cell

tag: time

value: Day 3


title: MCF7H_43_Ad_CMV_Dam_3HA_GFP_72h_Replicate3

description: Library name: MCF7H_43_Ad_Dam_GFP_72h_R3


title: MCF7H_42_Ad_CMV_Dam_3HA_GFP_72h_Replicate2

description: Library name: MCF7H_42_Ad_Dam_GFP_72h_R2


title: MCF7H_41_Ad_CMV_Dam_3HA_GFP_72h_Replicate1

description: Library name: MCF7H_41_Ad_Dam_GFP_72h_R1


title: MCF7H_0min_Replicate3

description: Library name: MCF7H_0min_R3

value: Day 0


title: MCF7H_40_Ad_CMV_GFP_72h_Replicate3

description: Library name: MCF7H_40_Ad_GFP_72h_R3


title: MCF7H_0min_Replicate2

description: Library name: MCF7H_0min_R2


title: MCF7H_0min_Replicate1

description: Library name: MCF7H_0min_R1


title: Circle-seq of Human: adult male urine; NC9

description: Library name: NC9


title: MCF7H_38_Ad_CMV_GFP_72h_Replicate1

description: Library name: MCF7H_38_Ad_GFP_72h_R1


title: Circle-seq of Human: adult female urine; NC3

description: Library name: NC3


title: H4K12la-rep2

city: Tianjin

first: zilong

last: fan

address: 22 Weidai Road, Heping District

institute: Tianjin Medical University

description: Library name: Sample5

data_processing:
FastQC tool was used to examine metrics, such as sequencing error rate, sequencing depth, and fragment length distribution
Aligned the preprocessed reads to a reference genome using alignment tool (Bowtie2)
Peak calling was performed using the model-based analysis of ChIP–seq (MACS2)
BigWig files were generated through Deeptools
Assembly: hg38
Supplementary files format and content: bigWig narrowPeak
Library strategy: CUT&Tag

source_name: liver

extract_protocol:
HepG2 cells were collected and counted for incubation with pre-treated ConA beads. Subsequently, cells are resuspended in an Antibody Buffer and incubated overnight at 4 °C with the corresponding primary antibody . The secondary antibodies were diluted in appropriate proportions and incubate them with the cells at room temperature. Subsequently, pA/G-Tnp transposons were rotated and incubated with the samples for 1 h to activate the translocase fragment DNA and extract DNA.
DNA library was constructed with TruePreP Index Kit V2 for Illumina (TD202, Vazyme Biotech). The library was purified by VAHTS DNA Clean Beads (N411, Vazyme Biotech)

value: liver

value: HepG2 cells

value: Hepatocellular Carcinoma


title: ENO1-rep1

description: Library name: Sample1


title: HDAC1-rep2

description: Library name: Sample6


title: ENO1-rep2

description: Library name: Sample4


title: HDAC1-rep1

description: Library name: Sample3


title: H4K12la-rep1

description: Library name: Sample2


title: R-loop CUT&Tag  of ISO induced PC-3; Human; adult male; Replicate1

description: Library name: ISO-induced_PC-3_replicate1


title: Circle-seq of Human: adult male urine; NC4

description: Library name: NC4


title: Nalm-6 SF3B1 K700E MT cells Rep 2

city: Greenwood

first: Austin

last: Herbert

state: South Carolina

address: 114 Gregor Mendell Circle

department: Genetics and Biochemistry

institute: Clemson University

description:
Library name: SF3B1 K700E 2
Column name K72 in gene_CPM.txt

data_processing:
Quality control, read filtering, and adapter trimming was performed with bbduk from bbmap
Reads were mapped to Hg38 using Hisat2
Counts per million were generated with FeatureCounts from SubRead package to produce the gene_CPM.txt
rMATs was performed for differential splicing analysis to produce the rMATS_for_GEO_submission.txt
Assembly: Hg38, gencode v39 annotation
Supplementary files format and content: rMATS_for_GEO_submittion.txt contains alternative splicing analysis performed with rMATs between SF3B1 K700E replicates and SF3B1 wildtype replicates
Supplementary files format and content: gene_CPM.txt is a counts per million table containing genes as rows, samples as columns and values as counts per million.

source_name: Peripheral blood

growth_protocol: Nalm-6 cells were cultured in RPMI-1640 and maintained at 37 C and 5% CO2. Cells were split every three days.

extract_protocol:
Trizol, phenol chlorophorm
QiaSelect -rRNA depletion, Super Script IV RT with random nonomers, Nextera standard DNA library prep.

value: Peripheral blood

value: SF3B1 K700E


title: Huh7,NC-3

city: Shanghai

first: Yujun

last: Hao

address: No. 25, Lane 2200, Xietu Road

institute: Medicine for Cancer, Shanghai Cancer Institute, School of Medicine, Shanghai Jiao Tong University

description: Library name: NC-3

data_processing:
After library preparation and pooling of different samples, the samples were subjected for NGS platform by Berry Genomics Co., Ltd
Paired-end clean reads were aligned to the reference genome using HISAT2 software. Featurecount was used to count the reads numbers of each gene.
the expression level of each transcript was calculated according to FPKM
Assembly: GRCh38
Supplementary files format and content: tab-delimited text files include FPKM values for each sample.
Supplementary files format and content: txt files include FPKM value for each sample

extract_protocol:
Total RNA was extracted from the sample using TRIzol® Reagent according the manufacturer’s instructions (Invitrogen)
RNA libraries were prepared for sequencing using standard Illumina protocols.

value: Human Hepatocellular carcinoma cell

value: NC


title: Huh7,NC-2

description: Library name: NC-2


title: Nalm-6 SF3B1 K700E MT cells Rep 3

description:
Library name: SF3B1 K700E 3
Column name K73 in gene_CPM.txt


title: Huh7,ABD circular RNA treated-3

description: Library name: circABD-3

value: ABD circular RNA treated


title: Huh7,NC-1

description: Library name: NC-1


title: Huh7,ABD circular RNA treated-2

description: Library name: circABD-2


title: Huh7,ABD circular RNA treated-1

description: Library name: circABD-1


title: Nalm-6 SF3B1 WT cells Rep 2

description:
Library name: WT 2
Column name WT2 in gene_CPM.txt


title: Nalm-6 SF3B1 WT cells Rep 3

description:
Library name: WT 3
Column name WT2 in gene_CPM.txt


title: Nalm-6 SF3B1 K700E MT cells Rep 1

description:
Library name: SF3B1 K700E 1
Column name K71 in gene_CPM.txt


title: Nalm-6 SF3B1 WT cells Rep 1

description:
Library name: WT 1
Column name WT1 in gene_CPM.txt


title: PD_R_neg GEX

city: La Jolla

first: Emil

last: Johansson

state: California

address: 9420 Athena Cir

department: Center for Autoimmunity and Inflammation

institute: La Jolla Institute for Immunology

description:
Library name: PD_R_neg_GEX
GEX

data_processing:
The CellRanger (v7.1) software suite was used to convert Illumina sequencer's base call files to FASTQ files, to align sequencing reads to the GRCh38 human reference genome and generate count matrix, and to demultiplex samples based on the TotalSeq B hashtag sequences.
Assembly: refdata-gex-GRCh38-2020-A
Supplementary files format and content: Cellranger output files: count barcodes.tsv.gz, count features.tsv.gz, count matrix.mtx.gz
Supplementary files format and content: Cellranger output files: CITE_barcodes.tsv.gz, CITE_features.tsv.gz, CITE_matrix.mtx.gz

source_name: Peripheral blood mononuclear cells

extract_protocol:
A total of 20 unique samples were hashed using BioLegend Total-Seq B antibodies. Cells were sorted and approximately 12,000 cells/sample were targeted and loaded on the 10X Chromium Controller (10X Genomics).
We used the Chromium Next GEM Single Cell 3' HT Reagent Kits v3.1 (Dual Index) with Feature Barcode technology for Cell Surface Protein and Cell Multiplexing and performed cDNA synthesis, amplification, and library preparation for gene expression and hashtag antibodies according to manufacturer's protocol. cDNA and final libraries were quality checked for fragment size by capillary electrophoresis using the Tapestation 4200 (Agilent), and quantified using the Qubit Flex assay (Thermo Fisher), before pooling and sequencing on a NovaSeq 6000 S4 v1.5 Flowcell; Illumina) to a depth of > 20,000 reads/cell for gene expression and > 5,000 reads/cell for hashtag antibody libraries.

value: Peripheral blood mononuclear cells


title: PD_R_neg CITE

description:
Library name: PD_R_neg_CITE
CITE

library_source: other

data_processing:
The CellRanger (v7.1) software suite was used to convert Illumina sequencer's base call files to FASTQ files, to align sequencing reads to the GRCh38 human reference genome and generate count matrix, and to demultiplex samples based on the TotalSeq B hashtag sequences.
Assembly: refdata-gex-GRCh38-2020-A
Supplementary files format and content: Cellranger output files: count barcodes.tsv.gz, count features.tsv.gz, count matrix.mtx.gz
Supplementary files format and content: Cellranger output files: CITE_barcodes.tsv.gz, CITE_features.tsv.gz, CITE_matrix.mtx.gz
Library strategy: CITE-seq

molecule: protein


title: PD_R_pos CITE

description:
Library name: PD_R_pos_CITE
CITE


title: HC_NR_pos CITE

description:
Library name: HC_NR_pos_CITE
CITE


title: HC_NR_pos GEX

description:
Library name: HC_NR_pos_GEX
GEX


title: PD_R_pos GEX

description:
Library name: PD_R_pos_GEX
GEX


title: human pulmonary artery smooth muscle cells, hypoxia, H3K27Ac (ChIP)

city: daqing

first: Xiangrui

last: Zhu

state: heilongjiang

address: 39 Xinyang Road

institute: Harbin Medical University

description: Library name: human pulmonary artery smooth muscle cells_hypoxia, IP

data_processing:
The libraries were then sequenced on the Illumina NovaSeq 6000 platform by Epibiotek Co., Ltd. in Guangzhou.
Assembly: hg38
Supplementary files format and content: RNA-Seq: raw counts for each Sample
Supplementary files format and content: ChIP-Seq: bw file

growth_protocol: Human pulmonary artery smooth muscle cells were maintained in smooth muscle cell culture medium (1101, ScienCell, USA) with 10% FBS and 1% penicillin-streptomycin. They were placed in a CO2 incubator (Thermo Fisher, USA) at a temperature of 37°C, with 5% CO2 and 100% relative humidity for cultivation.

extract_protocol:
Total RNA was isolated using TRIzol reagent (Thermo Fisher, USA) according to the manufacturer's protocol.
VAHTS Stranded mRNA-seq Library Prep Kit for Illumina V2 (NR612-02, Vazyme Biotech, China) was used for library preparation according to the instructions. ChIP DNA was processed for library generation using the QIAseq Ultralow Input Library Kit (180497, QIAGEN, Germany) following the manufacturer’s protocol.

treatment_protocol: Human pulmonary artery smooth muscle cells were subjected to hypoxic treatment using a tri-gas incubator (Thermo Fisher, USA) with an atmosphere containing 3% O2, 5% CO2, and 92% N2.

value: hypoxia

value: 24 hours

tag: chip antibody

value: H3K27Ac (Cell Signaling, #8173)


title: HC_NR_neg GEX

description:
Library name: HC_NR_neg_GEX
GEX


title: HC_NR_neg CITE

description:
Library name: HC_NR_neg_CITE
CITE


title: human pulmonary artery smooth muscle cells, hypoxia (Input)

description: Library name: human pulmonary artery smooth muscle cells_hypoxia, Input


title: human pulmonary artery smooth muscle cells, normoxia, H3K27Ac (ChIP)

description: Library name: human pulmonary artery smooth muscle cells_normoxia, IP

value: normoxia


title: human pulmonary artery smooth muscle cells, normoxia (Input)

description: Library name: human pulmonary artery smooth muscle cells_normoxia, Input


title: human pulmonary artery smooth muscle cells, normoxia 3

description: Library name: SAMPLES 3


title: human pulmonary artery smooth muscle cells, hypoxia 3

description: Library name: SAMPLES 6


title: human pulmonary artery smooth muscle cells, normoxia 2

description: Library name: SAMPLES 2


title: human pulmonary artery smooth muscle cells, normoxia 1

description: Library name: SAMPLES 1


title: IMR-32_ATAC

city: Suzhou

first: Gen

last: Li

state: Jiangsu

address: No. 92 Zhongnan Street

institute: Children's Hospital of Soochow University

description: Library name: IMR-32_ATAC

data_processing:
Basecalls were performed using bcl2fastq v2.17 for Novaseq output. ATAC-seq reads were trimmed using Fastp v0.23.2 with default settings, and were aligned to the UCSC hg38 genome using Bowtie2 version v2.4.4 with parameter --very-sensitive -X 2000.
Peaks were identified using MACS3 (v3.0.0a6) with the significance cut-off FDR<0.01
bigWig files were generated using bedGraphToBigWig tool.
Assembly: hg38
Supplementary files format and content: bigwig files

source_name: cell line

extract_protocol:
ATAC-seq was performed with Hyperactive ATAC-Seq Library Prep Kit for Illumina (Vazyme, China) according to the manufacturer’s protocol.
Library construction was performed using TruePrep® DNA Library Prep Kit V2 for Illumina (Vazyme) and TruePrep® Index Kit V2 for Illumina (Vazyme).

value: cell line

value: neuroblastoma


title: IMR-32 cells, shControl-1

description:
Library name: Sample1
Column name in shNeuroD1 vs shControl_all.txt: shControl_1

data_processing:
In order to remove technical sequences, including adapters, polymerase chain reaction (PCR) primers, or fragments thereof, and quality of bases lower than 20, pass filter data of fastq format were processed by Cutadapt (V1.9.1) to be high quality clean data.
Reference genome sequences and gene model annotation files of relative species were downloaded from genome website, such as UCSC, NCBI, ENSEMBL. Secondly, Hisat2 (v2.0.1) was used to index reference genome sequence. Finally, clean data were aligned to reference genome via software Hisat2 (v2.0.1).
In the beginning transcripts in fasta format are converted from known gff annotation file and indexed properly. Then, with the file as a reference gene file, HTSeq (v0.6.1) estimated gene and isoform expression levels from the pair-end clean data.
Differential expression analysis used the DESeq2 Bioconductor package, a model based on the negative binomial distribution. the estimates of dispersion and logarithmic fold changes incorporate data-driven prior distributions, Padj of genes were setted <0.05 to detect differential expressed ones.
Assembly: hg38
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample
Supplementary files format and content: csv files include FPKM values for each Sample

extract_protocol:
Total RNA of each sample was extracted using TRIzol Reagent. Total RNA of each sample was quantified and qualified by NanoDrop (Thermo Fisher Scientific Inc.). 1 μg total RNA was used for following library preparation.
The poly(A) mRNA isolation was performed using Poly(A) mRNA Magnetic Isolation Module or rRNA removal Kit. The mRNA fragmentation and priming was performed using First Strand Synthesis Reaction Buffer and Random Primers. First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase and the second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix. The purified double-stranded cDNA by beads was then treated with End Prep Enzyme Mix to repair both ends and add a dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performed using beads, and fragments of ~400 bp (with the approximate insert size of 300 bp) were recovered. Each sample was then amplified by PCR using P5 and P7 primers, with both primers carrying sequences which can anneal with flow cell to perform bridge PCR andP5/ P7 primer carrying index allowing for multiplexing. The PCR products were cleaned up using beads, validated using an Qsep100 (Bioptic, Taiwan, China), and quantified by Qubit3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA).

treatment_protocol: IMR-32 cells were stably transduced with shNCshNeuroD1. RNA interference was induced by treating cells with Doxycycline (1 μg/mL, 48 h).

value: Neuroblastoma cell line

value: WT


title: IMR-32_H3K27Ac, CUT&Tag

description: Library name: IMR-32_H3K27Ac

data_processing:
Basecalls were performed using bcl2fastq v2.17 for Novaseq output. CUT&Tag reads were trimmed using Fastp v0.23.2 with default settings, and were aligned to the UCSC hg38 genome using Bowtie2 version v2.4.4 with parameter --end-to-end --very-sensitive --no-mixed --no-discordant --phred33 -I 10 -X 700.
Peaks were identified using MACS3 (v3.0.0a6) with the significance cut-off FDR<0.01
bigWig files were generated using bedGraphToBigWig tool.
Assembly: hg38
Supplementary files format and content: bigwig files
Library strategy: CUT&Tag

extract_protocol:
CUT&Tag was performed with Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme) according to the manufacturer’s protocol.
Library construction was performed using TruePrep® DNA Library Prep Kit V2 for Illumina (Vazyme) and TruePrep® Index Kit V2 for Illumina (Vazyme).

value: H3K27Ac(Abcam, #ab4729)


title: IMR-32 cells, shControl-2

description:
Library name: Sample2
Column name in shNeuroD1 vs shControl_all.txt: shControl_2


title: IMR-32 cells, shNeuroD1-1

description:
Library name: Sample3
Column name in shNeuroD1 vs shControl_all.txt: shNeuroD1_1

value: NeuroD1 knockdown


title: TEP Stage_IFNA+G_Exp_2_Rep_3

city: Palo Alto

first: Abdulvasey

last: Mohammed

state: CA

address: 240 Pasteur Dr

department: Pediatrics

institute: Stanford

data_processing:
Sequencing data were processed using the nf-core/rnaseq v3.14.0 pipeline using Latch Bio's automated workflow (v 3.14.0-9a3a87-wip-adbeff)
Pipeline details: Sequencing data were trimmed using Trim Galore v 0.6.7. Transcript quantification using Salmon v1.10.1, with human genome GRCh38.p14 used to construct the reference transcriptome.
Transcript counts were summed for gene count quantification.
Assembly: GRCh38.p14
Supplementary files format and content: Count matrix of number of reads mapping to genes across all samples.

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-26

extract_protocol:
After the TEP stage, cells were dissociated into a single-cell suspension using Accutase (AT104) for 2–3 cycles of 5-minute incubations. Subsequently, cells were stained with EpCAM-APC, and the EpCAM-high population was sorted using a BD FACS Aria II SORP. Cell viability was assessed using 7-AAD staining. Total RNA was extracted from the sorted cells using the Qiagen RNeasy Kit.
Illumina Stranded mRNA Prep
50-200ng Total RNA input were used to construct libraries followed by pooling libraries into NovaSeq6000 lanes at 2 x 100 bp read length.

treatment_protocol: Recombinant human IFNG (50 ng/mL, Peprotech 300-02) and IFNA2 (20 ng/mL, Peprotech 300-02AA) were added for the duration of the TEP stage, depending on the experimental treatment.

value: iPSC

value: IFNA2+ IFNG+


title: TEP Stage_IFNA+G_Exp_2_Rep_2

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-25


title: IMR-32_IgG, CUT&Tag

description: Library name: IMR-32_IgG

value: IgG(Abcam, #ab172730)


title: TEP Stage_IFNA+G_Exp_2_Rep_1

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-24


title: TEP Stage_IFNG_Exp_2_Rep_3

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-23

value: IFNG+


title: TEP Stage_IFNA_Exp_2_Rep_3

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-20


title: TEP Stage_IFNG_Exp_2_Rep_2

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-22


title: TEP Stage_IFNA_Exp_2_Rep_2

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-19


title: TEP Stage_IFNG_Exp_2_Rep_1

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-21


title: TEP Stage_IFNA_Exp_2_Rep_1

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-18


title: TEP Stage_No_IFN_Exp_2_Rep_2

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-16

value: Control


title: TEP Stage_No_IFN_Exp_2_Rep_3

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-17


title: human pulmonary artery smooth muscle cells, hypoxia 1

description: Library name: SAMPLES 4


title: human pulmonary artery smooth muscle cells, hypoxia 2

description: Library name: SAMPLES 5


title: TEP Stage_IFNA+G_Exp_1_Rep_3

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-14


title: TEP Stage_IFNA+G_Exp_1_Rep_2

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-13


title: TEP Stage_IFNA+G_Exp_1_Rep_1

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-12


title: TEP Stage_No_IFN_Exp_2_Rep_1

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-15


title: TEP Stage_IFNG_Exp_1_Rep_2

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-10


title: TEP Stage_IFNG_Exp_1_Rep_3

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-11


title: TEP Stage_IFNA_Exp_1_Rep_3

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-8


title: TEP Stage_IFNA_Exp_1_Rep_2

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-7


title: TEP Stage_IFNA_Exp_1_Rep_1

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-6


title: IMR-32 cells, shNeuroD1-2

description:
Library name: Sample4
Column name inshNeuroD1 vs shControl_all.txt: shNeuroD1_2


title: TEP Stage_No_IFN_Exp_1_Rep_3

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-5


title: TEP Stage_No_IFN_Exp_1_Rep_2

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-4


title: 5 Days Postnatal Thymus Medullary Thymic Epithelial Cells (mTECI)

source_name: Postnatal Thymus

extract_protocol:
Human fetal thymus samples, collected at postnatal day 5, 3 months, and 4 months, were mechanically dissected into fragments smaller than 2 mm³ using surgical scissors. Each sample was then enzymatically digested in a digestion medium (PBS) containing DNase (0.095 mg/mL) and Liberase TM (0.095 mg/mL, Sigma-Aldrich) on a rotating device to ensure continuous mixing. After 2–3 rounds of digestion (25 minutes per round), the cell suspension was centrifuged at 110 G for 2 minutes. The supernatant was collected for staining of thymic stromal immune cells prior to sorting.  The remaining cell pellet, containing epithelial cells, underwent additional rounds of digestion if necessary until there were no visible fragments. This cell suspension was then subjected to density centrifugation using Histopaque-1077 (Sigma-Aldrich) to enrich for epithelial cells in the low-density fraction (LDF). Cells from the LDF were then processed to deplete hCD45+ cells using Human CD45 MicroBeads/LD depletion columns (Miltenyi Biotec). Immune cells were stained using the following antibodies: CD19/20-BV650, CD5/CD7-FITC, CD123-PE, CD1c/CD11c-PE-Cy7, CD45-APC, and HLA-DR-APC-Cy7. Immune cells were sorted on a BD FACS Aria II SORP as follows: Thymocytes (CD45+, CD5+, CD7+), B Cells (CD45+, CD19+, CD20+), mDC (CD45+, CD1c+, CD11c+, HLA-DR+), and pDC (CD45+, CD123+, HLA-DR+). Epithelial cells were stained using the following antibodies: CD205-PE, HLA-DR-PE-Cy7, EPCAM-APC, and CD45-APC-Cy7. Epithelial cells were sorted as follows: cTEC (EPCAM+, CD205+, HLA-DR-, CD45-), mTECI (EPCAM+, CD205-, HLA-DR-, CD45-), and mTECII (EPCAM+, CD205-, HLA-DR+, CD45-). Cell viability was assessed using 7-AAD staining. Total RNA was extracted from sorted cells using the Qiagen RNeasy Kit.
Illumina Stranded mRNA Prep
50-200ng Total RNA input were used to construct libraries followed by pooling libraries into NovaSeq6000 lanes at 2 x 100 bp read length.

value: Postnatal Thymus


title: 5 Days Postnatal Thymus Plasmacytoid Dendritic Cells


title: 5 Days Postnatal Thymus Myeloid Dendritic Cells


title: 5 Days Postnatal Thymus Medullary Thymic Epithelial Cells (mTECII)


title: 5 Days Postnatal Thymus Thymocytes


title: 5 Days Postnatal Thymus Cortical Thymic Epithelial Cells


title: 5 Days Postnatal Thymus B Cells


title: 4 Month Postnatal Myeloid Dendritic Cells


title: 4 Month Postnatal B Cells


title: 3 Month Postnatal Medullary Thymic Epithelial Cells (mTECI)


title: 3 Month Postnatal Medullary Thymic Epithelial Cells (mTECII)


title: 3 Month Postnatal Cortical Thymic Epithelial Cells


title: ACSS2-KO-3,2

city: Xianyang

first: ZHUANGLI

middle: ZHUANGLI

last: ZHUANGLI

address: No. 3, Taicheng Road, Yangling District,Xianyang 712100，Shaanxi Province

institute: Northwest A&F University

data_processing:
Reads obtained from the sequencing machines includes raw reads containing adapters or low quality bases which will affect the following assembly and analysis. Thus, to get high quality clean reads, reads were further filtered by Cutadapt (https://cutadapt.readthedocs.io/en/stable/,version:cutadapt-1.9). We aligned reads of all samples to the research species reference genome using HISAT2 (https://daehwankimlab.github.io/hisat2/,version:hisat2-2.2.1) package, which initially remove a portion of the reads based on quality information accompanying each read and then maps the reads to the reference genome.
The mapped reads of each sample were assembled using StringTie (http://ccb.jhu.edu/software/stringtie/,version:stringtie-2.1.6) with default parameters. Then, all transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using gffcompare software(http://ccb.jhu.edu/software/stringtie/gffcompare.shtml,version:gffcompare-0.9.8). After the final transcriptome was generated, StringTie and ballgown (http://www.bioconductor.org/packages/release/bioc/html/ballgown.html) were used to estimate the expression levels of all transcripts and perform expression abundance for mRNAs by calculating FPKM (fragment per kilobase of transcript per million mapped reads) value.
Genes differential expression analysis was performed by DESeq2 software between two different groups (and by edgeR between two samples). The genes with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change ≥ 2 were considered differentially expressed genes. Differentially expressed genes were then subjected to enrichment analysis of GO functions and KEGG pathways.
Assembly: GRCh38
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

extract_protocol:
Total RNA was extracted using Trizol reagent (thermofisher, 15596018) following the manufacturer's procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA, 5067-1511) , high-quality RNA samples with RIN number > 7.0 were used to construct sequencing library.
mRNA was purified from total RNA (5ug) using Dynabeads Oligo (dT) (Thermo Fisher, CA, USA) with two rounds of purification. Following purification, the mRNA was fragmented into short fragments using divalent cations under elevated temperature (Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 94℃ 5-7min). Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA). An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA librarys were 300±50 bp. At last, we performed the 2×150bp paired-end sequencing (PE150) on an Illumina NovaseqTM 6000 following the vendor's recommended protocol.

value: cancer


title: TEP Stage_IFNG_Exp_1_Rep_1

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-9


title: ACSS2-KO-2,1


title: ACSS2-KO-2,2


title: nc,3

value: NC


title: ACSS2-KO-3,3


title: nc,1


title: ACSS2-KO-3,1


title: TEP Stage_No_IFN_Exp_1_Rep_1

growth_protocol:
Human iPSCs (SUN4 line, generated and shared with us by Prof. Hiromitsu Nakauchi’s laboratory at Stanford) cells were seeded at a density of 62.5k cells per well in a 24-well tissue culture plate (Corning 09-761-146). In brief, the protocol includes 4 stages and uses the following factors. Stage 1: For definitive endoderm induction, we used Activin A on days 1-4 and a WNT agonist on day 1. Stage 2: For anterior foregut endoderm induction, Activin A was used on day 5, and retinoic acid (RA) at 0.25 μM was used on days 5-7. BMP4 and a TGF-β inhibitor were used on days 6-7. For stage 3, induction of ventral pharyngeal endoderm (VPE), and stage 4, induction of TEPs, the following factors were used: RA at 0.1μM on days 8-13, a WNT agonist on days 8-13, BMP4 on days 8-13, a sonic hedgehog inhibitor on days 8-13, FGF8 on days 8-13, and a TGF-β inhibitor on days 8 and 9 (VPE only).
This differentiation protocol was adapted in 2D with minor modifications from the publication: Zeleniak, A., Wiegand, C., Liu, W. *et al.* De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids. *Nat Methods* **19**, 1306–1319 (2022). https://doi.org/10.1038/s41592-022-01583-3


title: nc,2


title: P1 Tracheal, never-smoker

city: London

first: Ahmed

last: Alhendi

address: 5 University St

country: United Kingdom

institute: UCL

description: 10x Genomics

data_processing:
The 10x raw data were processed using Cellranger v7.1.0
Assembly: human genome ex-GRCh38-2020-A
Supplementary files format and content: Tab-separated files and matrix files

source_name: Tracheal

extract_protocol:
Single cell suspensions were obtained from the tracheal brushes as per previously described protocols (dx.doi.org/10.17504/protocols.io.qubdwsn)
Single cell RNA libraries were prepared using 10x Chromium 5' V2 Gene Expression kit. The single cell suspension was loaded with the master mix, partitioning oil and gel beads into the chip to generate GEMs (gel bead-in emulsion), containing 10x barcoded cDNA from poly-A RNA. The pooled cDNA was then cleaned up using Silane Dynabeads, amplified by PCR and separated by size for 5' gene expression library construction.

value: Tracheal

value: Airway epithelial cells

tag: age

value: Never smoker


title: On-Acc166

city: Houston

first: Sanghoon

last: Lee

state: TX

address: 1881 East Road

institute: The University of Texas MD Anderson Cancer Center

data_processing:
RNA-seq FASTQ files were processed through FastQC (v0.11.5) and and RNA-SeQC (v1.1.8).
STAR 2-pass alignment (v2.7.0f) was performed with default parameters to generate RNA- seq BAM files.
HTSeq-count (v0.11.0) tool was applied to aligned RNA-seq BAM files to count for each gene how many aligned reads overlap with its exons.
Assembly: GRCh38
Supplementary files format and content: Tab-delimitated text file including raw counts for each sample

source_name: ovarian clear cell carcinoma

extract_protocol:
RNA extraction was perfromed using Qiagen RNeasy Mini Kit per the manufacturer's protocol.
Stranded mRNA libraries were prepared using the KAPA Stranded mRNA-Seq Kit (Roche).

value: ovarian clear cell carcinoma

tag: genetic backgrounds

value: PPP2R1AMUT AKTWT

tag: occc sample id

value: On


title: P3 Tracheal, never-smoker

description: 10x Genomics


title: On-Acc149


title: On-Acc150

value: PPP2R1AWT AKTWT


title: On-Acc160


title: On-Acc162


title: On-Acc139


title: On-Acc137


title: On-Acc121

value: PPP2R1AWT AKTALT


title: On-Acc38


title: 4 Month Postnatal Plasmacytoid Dendritic Cells


title: ACSS2-KO-2,3


title: On-Acc36


title: 4 Month Postnatal Thymus Thymocytes


title: Pre-Acc166

value: Pre


title: On-Acc31


title: Pre-Acc162


title: On-Acc19


title: Pre-Acc163


title: Pre-Acc149


title: Pre-Acc151


title: Pre-Acc160


title: Pre-Acc141


title: Pre-Acc143


title: Pre-Acc139


title: Pre-Acc137


title: Pre-Acc129


title: Pre-Acc135


title: Pre-Acc130


title: Pre-Acc125


title: On-Acc124


title: Pre-Acc122


title: Pre-Acc124


title: On-Acc119


title: Pre-Acc121


title: Pre-Acc69


title: Pre-Acc44


title: Pre-Acc115


title: Pre-Acc150


title: Pre-Acc119


title: Pre-Acc36


title: Pre-Acc38


title: Pre-Acc31


title: Pre-Acc128


title: Pre-Acc19


title: Pre-Acc29


title: HeLa cells for control, biol rep 3

city: Beijing

first: Xinyuan

last: Fan

address: No.292 Chengfu Road, Haidian District

institute: Peking University

data_processing:
Raw reads data in fastq format were processed by Fastp (version 0.23.1) software, which removed reads containing adapter, reads containing ploy-N and low-quality reads.
Assembly: Homo sapiens GRCh38/hg38
Supplementary files format and content: Microsoft office Excel .xlsx file including raw counts for each sample
Supplementary files format and content: Microsoft office Excel .xlsx file including FKPM values for each sample

source_name: cervix

growth_protocol: HeLa cells were maintained in DMEM (Gibco) supplemented with 10% FBS (v/v., Gibco), 100 IU/ml penicillin and 100 mg/ml streptomycin (Thermo Fisher) and cultured in an incubator at 37°C under 5% CO2.

extract_protocol:
RNA were extracted using VeZol reagent kit (Vazyme, R411-01).
mRNA was purified from total RNA using poly-T oligo-attached magnetic beads, and the sequencing libraries were prepared using NEB Next Ultra RNA Library Prep Kit for Illumina (NEB, USA, E7530L) according to the manufacture’s protocol.

treatment_protocol: HeLa cells cultured in 10-cm dish (Corning) was stimulated with 0.1 µM rapamycin in DMEM (with 10% FBS) for 24 h. Samples omitting rapamycin were used as control.

value: cervix

value: HeLa

value: epithelial cell

value: WT

value: no treatment


title: HeLa cells treated with 100 nM rapamycin, biol rep 3

value: 100 nM rapamycin


title: HeLa cells for control, biol rep 2


title: HeLa cells treated with 100 nM rapamycin, biol rep 1


title: HeLa cells for control, biol rep 1


title: HeLa cells treated with 100 nM rapamycin, biol rep 2


title: Pre-Acc126


title: Exp-T1K_4A,Cell-HaCaT,Treatment-sham

city: Bremen

first: Jyoti

last: Jyoti

state: Bremen

address: Campus Ring 6

department: School of Science

institute: Constructor University

description: 5gTemp_HaCat_NormalizedCounts_EDASeq.csv - T1K_4A

data_processing:
CLC Genomics Workbench 20.0.4 (CLC bio, Qiagen)
The resulting reads were reduced to succesful reads (Q30 bases run quality limit 0.15) and trimmed to 75 bp.
Trimmed sequence reads were mapped to human_GRCh38.p7_NCBI_20160606 (mismatch cost: 2; insertion/deletion cost: 3; length fraction: 0.8; similarity fraction: 0.8)
Assembly: GRCh38.p7
Supplementary files format and content: comma separated .csv files include normalized gene expression values. One file contains samples belonging to one batch of experiments.

source_name: HaCaT

growth_protocol: Cells were cultured in DMEM, high glucose, supplemented with 10% FBS and 1% penicillin streptomycin in an incubator with 37°C, 5% CO2 and saturated humidity.

extract_protocol:
To keep the homogeneity of the transmitting power flux density or SAR as high as possible, the cells outside a defined area were excluded during harvesting. After exposure, the medium of the cell dishes was discarded and a template made with a 3D printer (at 27 GHz an ellipse with a radius of 24 mm/23 mm; at 40.5 GHz a capped circle with a radius of 24 mm and a distance of the capped area to the center of 19 mm) was applied to the outside of the bottom of the dish and the areas to be omitted were marked. The cells were removed according to the marking with a cell scraper and the cell monolayer was then rinsed twice with PBS.
Cells were harvested by washing the remaining cell monolayer once with PBS and transferring 350 ul lysis buffer to each of the dishes. The cell lysates were collected using a cell scraper and transferring them into reaction tubes. The lysates were frozen at -80C until further processing. DNA and RNA extraction was done using the DNA/RNA Allprep Mini Kit (Qiagen) or the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol.
The transcriptome library was generated using TruSeq Stranded mRNA technology according to the manufacturer's protocol (Illumina).

treatment_protocol:
For each exposure experiment 120,000 HDF or 200,000 HaCaT cells were seeded onto a 60 mm cell culture dish. Cells were used in passages 6-7 (HDF) and 35 (HaCaT). One day (48h experiments) or three days (2h experiments) after seeding, the dishes were transferred to the exposure incubators and cells were exposed or sham-exposed for 2h or 48h, with a frequency of 27 GHz or 40.5 GHz, and a power flux density of 1 mW/cm2 or 10 mW/cm2. In all experiments, the temperature was measured continuously using a fiber optic sensor. The internal temperature of the incubator in which the exposure takes place was reduced in the 10 mW/cm2 experiments manually, so that an average temperature of 37C could be maintained in the petri dishes and a lid heater was used to prevent condensation in the exposed petri dish and maintain blinding conditions.
Temperature controls: Cells were exposed or sham exposed at 40.5 GHz and 10 mW/cm2 for 48h without compensating for the temperature increase. The starting temperature of the medium was 37.0C in these experiments and increased to an average of 38.3C in the exposed samples. No lid heaters were used, so that the heating of the medium in the exposed samples led to medium condensation.
UV controls: Three replicates each of HaCaT and HDF cells were exposed or sham-exposed at a wavelength of 254 nm and an illuminance of 1 mW/cm2. The medium was removed and collected, rinsed twice with PBS and the cells were thinly coated with 600 ul PBS per dish. Exposure was performed without the dish lid for 20 sec, for sham exposure the dishes were stored in the dark at room temperature. The PBS was then aspirated, the cells were layered with the previously collected medium, and incubated for 2h at 37C and 5%CO2.

value: HaCaT

value: sham

tag: date


title: Exp-T1K_2B,Cell-HaCaT,Treatment-sham

description: 5gTemp_HaCat_NormalizedCounts_EDASeq.csv - T1K_2B


title: Exp-T1K_1B,Cell-HaCaT,Treatment-exposed

description: 5gTemp_HaCat_NormalizedCounts_EDASeq.csv - T1K_1B

value: exposed


title: Exp-T1K_1A,Cell-HaCaT,Treatment-sham

description: 5gTemp_HaCat_NormalizedCounts_EDASeq.csv - T1K_1A


title: Exp-T1K_2A,Cell-HaCaT,Treatment-exposed

description: 5gTemp_HaCat_NormalizedCounts_EDASeq.csv - T1K_2A


title: Exp-UVC_K3S,Cell-HaCaT,Treatment-Control

description: UV_HaCat_NormalizedCounts_DESeq2.csv - UVC_K3S

value: Control


title: Exp-UVC_K1S,Cell-HaCaT,Treatment-Control

description: UV_HaCat_NormalizedCounts_DESeq2.csv - UVC_K1S


title: Exp-UVC_K3V,Cell-HaCaT,Treatment-UV

description: UV_HaCat_NormalizedCounts_DESeq2.csv - UVC_K3V

value: UV


title: Exp-UVC_K2V,Cell-HaCaT,Treatment-UV

description: UV_HaCat_NormalizedCounts_DESeq2.csv - UVC_K2V


title: Exp-T1F_2B,Cell-HDF,Treatment-exposed

description: 5gTemp_HDF_NormalizedCounts_EDASeq.csv - T1F_2B

source_name: HDF

value: HDF


title: Exp-T1F_3B,Cell-HDF,Treatment-sham

description: 5gTemp_HDF_NormalizedCounts_EDASeq.csv - T1F_3B


title: Exp-UVC_F3V,Cell-HDF,Treatment-UV

description: UV_HDF_NormalizedCounts_DESeq2.csv - UVC_F3V


title: Exp-UVC_F3S,Cell-HDF,Treatment-Control

description: UV_HDF_NormalizedCounts_DESeq2.csv - UVC_F3S


title: Exp-T1F_1A,Cell-HDF,Treatment-exposed

description: 5gTemp_HDF_NormalizedCounts_EDASeq.csv - T1F_1A


title: Exp-V7F_5BWdh,Cell-HDF,Power-10,Frequency-27,Time-2,Treatment-sham

description: P10_HDF_NormalizedCounts_DESeq2.csv - S116

tag: trial

tag: incubator

value: B

tag: frequency [ghz]

tag: power.density [mw/cm2]

tag: exposure.time [h]

tag: aliquot

tag: passage

value: b

value: c


title: Exp-V7F_5AWdh,Cell-HDF,Power-10,Frequency-27,Time-2,Treatment-exposed

description: P10_HDF_NormalizedCounts_DESeq2.csv - S115

tag: cell type

value: A


title: Exp-UVC_F2V,Cell-HDF,Treatment-UV

description: UV_HDF_NormalizedCounts_DESeq2.csv - UVC_F2V


title: Exp-UVC_K1V,Cell-HaCaT,Treatment-UV

description: UV_HaCat_NormalizedCounts_DESeq2.csv - UVC_K1V

tag: cell line


title: Exp-T1F_3A,Cell-HDF,Treatment-exposed

description: 5gTemp_HDF_NormalizedCounts_EDASeq.csv - T1F_3A


title: Exp-V6F_4AWdh,Cell-HDF,Power-10,Frequency-40.5,Time-2,Treatment-exposed

description: P10_HDF_NormalizedCounts_DESeq2.csv - S113

value: a


title: Exp-T1F_1B,Cell-HDF,Treatment-sham

description: 5gTemp_HDF_NormalizedCounts_EDASeq.csv - T1F_1B


title: Exp-T1F_2A,Cell-HDF,Treatment-sham

description: 5gTemp_HDF_NormalizedCounts_EDASeq.csv - T1F_2A


title: Exp-UVC_F1V,Cell-HDF,Treatment-UV

description: UV_HDF_NormalizedCounts_DESeq2.csv - UVC_F1V


title: Exp-V6F_4BWdh,Cell-HDF,Power-10,Frequency-40.5,Time-2,Treatment-sham

description: P10_HDF_NormalizedCounts_DESeq2.csv - S114


title: Exp-UVC_F1S,Cell-HDF,Treatment-Control

description: UV_HDF_NormalizedCounts_DESeq2.csv - UVC_F1S


title: Exp-V5F_6B,Cell-HDF,Power-10,Frequency-40.5,Time-48,Treatment-sham

description: P10_HDF_NormalizedCounts_DESeq2.csv - S99


title: Exp-V6F_3AWdh,Cell-HDF,Power-10,Frequency-40.5,Time-2,Treatment-exposed

description: P10_HDF_NormalizedCounts_DESeq2.csv - S111


title: Exp-V5F_5B,Cell-HDF,Power-10,Frequency-40.5,Time-48,Treatment-sham

description: P10_HDF_NormalizedCounts_DESeq2.csv - S97


title: Exp-V5F_6A,Cell-HDF,Power-10,Frequency-40.5,Time-48,Treatment-exposed

description: P10_HDF_NormalizedCounts_DESeq2.csv - S98


title: Exp-V8F_3B,Cell-HDF,Power-10,Frequency-27,Time-48,Treatment-exposed

description: P10_HDF_NormalizedCounts_DESeq2.csv - S95


title: Exp-T1K_4B,Cell-HaCaT,Treatment-exposed

description: 5gTemp_HaCat_NormalizedCounts_EDASeq.csv - T1K_4B


title: Exp-V5F_5A,Cell-HDF,Power-10,Frequency-40.5,Time-48,Treatment-exposed

description: P10_HDF_NormalizedCounts_DESeq2.csv - S96


title: Exp-V8F_3A,Cell-HDF,Power-10,Frequency-27,Time-48,Treatment-sham

description: P10_HDF_NormalizedCounts_DESeq2.csv - S94


title: Exp-V8F_2B,Cell-HDF,Power-10,Frequency-27,Time-48,Treatment-exposed

description: P10_HDF_NormalizedCounts_DESeq2.csv - S93


title: Exp-V6F_3BWdh,Cell-HDF,Power-10,Frequency-40.5,Time-2,Treatment-sham

description: P10_HDF_NormalizedCounts_DESeq2.csv - S112


title: Exp-V8F_2A,Cell-HDF,Power-10,Frequency-27,Time-48,Treatment-sham

description: P10_HDF_NormalizedCounts_DESeq2.csv - S92


title: Exp-V8F_1B,Cell-HDF,Power-10,Frequency-27,Time-48,Treatment-exposed

description: P10_HDF_NormalizedCounts_DESeq2.csv - S91