title: RNA_EPI.8_WSB_AC635

data_processing:
RNA-seq reads were filtered for quality and 3' trimmed for adapter sequences.
Reads were aligned to mouse genome assembly mm10 using STAR (v2.4) with default parameters.
Read count tables were created using HTSeq v0.6.1.
Normalization and differential expression analysis was performed with DESeq2.
Assembly: mm10
Supplementary files format and content: Tab-delimited text file with un-normalized read counts over all genes.

growth_protocol: EpiSCs were cultured on irradiated MEFs and N2B27 containing 12.5 ng/mL hsFGF, 20 ng/mL Activin A, and 175 nM NVP-TNKS6561.  For DE, EpiSCs are plated at 110,000 cells/cm2 on laminin in chemically defined media (CDM) containing 0.7 ug/mL insulin, 1% KOSR, 2 uM thiazovivin, and the above hsFGF, Activin, and NVP. After 5 hours, the cells are washed and media replaced with CDM containing 3 uM CHIR and 40 ng/mL Activin A. After 16 hours, the cells are washed and media replaced with CDM supplemented with 100 ng/mL Activin A and 100 nM LDN-193189 for 24 additional hours1. Definitive endoderm cells were dissociated in Accutase, washed in FACS buffer, counted, and resuspended with rat anti-mouse CD184/CXCR4 Alexa Fluor 647-conjugated antibody at a concentration of 1 µl/106 cells and incubated for 1 h at 4°C. Cells were then washed with FACS buffer, centrifuged (200 g for 3 min at 4°C) and resuspended in a solution composed by 80% FACS buffer and 20% anti-rat IgG MicroBeads (Miltenyi Biotec, 130-048-501). Cells were incubated in the mixture for 15 min at 4°C, then washed and resuspended in 500 µl buffer. The MS column was placed in an OctoMACS separator (Miltenyi Biotec, 130-048-501) on the MACS multistand. The cell suspension was added to the column and rinsed three times with buffer. Finally, the column was removed from the separator and the magnetically labeled cells were collected.

extract_protocol:
Phase separation in cells lysed in TRIzol was induced with 50% isopropanol and 0.5% 2-mercaptoethanol and RNA was extracted from the aqueous phase using the MagMAX mirVana Total RNA Isolation Kit (Thermo Fisher, A27828) on a KingFisher Flex Magnetic Particle Processor according to the manufacturer's protocol with 1-2 million cells input.
EpiSC: 100-500 ng of total RNA with RIN values of 9.5-10 underwent polyA selection and TruSeq library preparation according to instructions provided by Illumina (TruSeq Stranded mRNA LT Kit), with eight cycles of PCR. Samples were barcoded and run on a NovaSeq 6000 in a PE100 run, using the NovaSeq 6000 S4 Reagent Kit (200 Cycles) (Illumina).  DE: 2 ng total RNA with RNA integrity numbers ranging from 7.7 to 10 underwent amplification using the SMART-Seq v4 Ultra Low Input RNA Kit, with 12 cycles of amplification. Subsequently, 10 ng of amplified cDNA was used to prepare libraries with the KAPA Hyper Prep Kit using eight cycles of PCR. Samples were barcoded and run on a NovaSeq 6000 in a PE100 run, using the NovaSeq 6000 S4 Reagent Kit (200 Cycles) (Illumina).

value: epiblast stem cells


title: FaDu cells, knockdown, CTL

status: Public on Jun 21 2025

type: SRA

city: Dalian

first: Wang

last: Yang

address: 9 Lvshun South Rd West Section, Lüshunkou District, Dalian, Liaoning, China

department: Institute of Cancer Stem Cell

country: China

institute: Dalian Medical University

library_source: transcriptomic

data_processing:
Raw data(raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data(clean reads) were obtained by removing reads containing adapter, reads on containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content of the clean data were calculated. All the down stream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Bowtie v2.0.6 and paired-end clean reads were aligned to the reference genome using TopHat v2.0.9.
The mapped reads of each sample were assembled by both Scripture (beta2) (Guttman et al. 2010) and Cufflinks (v2.1.1) (Trapnell et al. 2010) in a reference-based approach. Both methods use spliced reads to determine exons connectivity, but with two different approaches. Scripture uses a statistical segmentation model to distinguish expressed loci from experimental noise and uses spliced reads to assemble expressed segments. It reports all statistically expressed isoforms in a given locus. Cufflinks uses a probabilistic model to simultaneously assemble and quantify the expression level of a minimal set of isoforms that provides a maximum likelihood explanation of the expression data in a given locus (Cabili et al. 2011). Scripture was run with default parameters, Cufflinks was run with ‘min-frags-per-transfrag=0’ and ‘--library-type’, other parameters were set as default.
Cuffdiff (v2.1.1) was used to calculate FPKMs of both lncRNAs and coding genes in each sample (Trapnell, C. et al. 2010). Gene FPKMs were computed by summing the FPKMs of transcripts in each gene group. FPKM means fragments per kilo-base of exon per million fragments mapped, calculated based on the length of the fragments and reads count mapped to this fragment.
Cuffdiff provides statistical routines for determining differential expression in digital transcript or gene expression data using a model based on the negative binomial distribution (Trapnell, C. et al. 2010). Transcripts with an P-adjust <0.05 were assigned as differentially expressed.
Assembly: human NCBI genome build 38
Supplementary files format and content: Excel file includes raw counts for each Sample
Supplementary files format and content: Excel file includes FPKM values for each Sample

molecule: total RNA

organism: Homo sapiens

source_name: hypopharynx

growth_protocol: FaDu cells were maintained at standard culture conditions (37°C, 5% CO2) in MEM medium with 10% FBS, Sodium Pyruvate , Nonessential Amino Acids Solution .

extract_protocol:
RNA was harvested using TRIzol and cleaned using RNAeasy Kit (Qiagen). 4 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: tissue

value: hypopharynx

tag: cell line

value: FaDu

tag: cell type

value: Hypopharyngeal carcinoma

tag: genotype

value: knockdown CTL


title: RNA_EPI.9_C57B6_44

city: New York

first: Richard

last: Koche

state: New York

country: USA

institute: Memorial Sloan Kettering Cancer Center

organism: Mus musculus


title: ATAC_DE_NOD_AC595_S1_40k

library_source: genomic

data_processing:
Raw sequencing reads were trimmed and filtered for quality (Q>15) and adapter content using version 0.4.5 of TrimGalore and running version 1.15 of cutadapt and version 0.11.5 of FastQC.
Version 2.3.4.1 of bowtie2 was employed to align reads to mouse assembly mm10 and alignments were deduplicated using MarkDuplicates in Picard Tools v2.16.0.
Enriched regions were discovered using MACS2 with a p-value setting of 0.001 and then filtered for blacklisted regions.
The BEDTools suite was used to create normalized bigwig files scaled by the size factors from DESeq2 normalization of the raw counts matrix.
Assembly: mm10
Supplementary files format and content: normalized bigwigs

molecule: genomic DNA

growth_protocol: EpiSCs were cultured on irradiated MEFs and N2B27 containing 12.5 ng/mL hsFGF, 20 ng/mL Activin A, and 175 nM NVP-TNKS6561.  For DE, EpiSCs are plated at 110,000 cells/cm2 on laminin in chemically defined media (CDM) containing 0.7 ug/mL insulin, 1% KOSR, 2 uM thiazovivin, and the above hsFGF, Activin, and NVP. After 5 hours, the cells are washed and media replaced with CDM containing 3 uM CHIR and 40 ng/mL Activin A. After 16 hours, the cells are washed and media replaced with CDM supplemented with 100 ng/mL Activin A and 100 nM LDN-193189 for 24 additional hours. Definitive endoderm cells were dissociated in Accutase, washed in FACS buffer, counted, and resuspended with rat anti-mouse CD184/CXCR4 Alexa Fluor 647-conjugated antibody at a concentration of 1 µl/106 cells and incubated for 1 h at 4°C. Cells were then washed with FACS buffer, centrifuged (200 g for 3 min at 4°C) and resuspended in a solution composed by 80% FACS buffer and 20% anti-rat IgG MicroBeads (Miltenyi Biotec, 130-048-501). Cells were incubated in the mixture for 15 min at 4°C, then washed and resuspended in 500 µl buffer. The MS column was placed in an OctoMACS separator (Miltenyi Biotec, 130-048-501) on the MACS multistand. The cell suspension was added to the column and rinsed three times with buffer. Finally, the column was removed from the separator and the magnetically labeled cells were collected.

extract_protocol:
Briefly, cells were dissociated into single cells, filtered through a Flowmi cell strainer and the nuclei were isolated by incubation with lysis buffer [10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% NP-40, 0.01% digitonin and 1% bovine serum albumin (BSA)] for 5 min at 4°C; 20,000 and 40,000 nuclei were isolated. Two replicates of 20k and 40k nuclei were collected per biological sample.
Libraries were prepared for sequencing using standard Illumina protocols

value: definitive endoderm

value: NOD/ShiLtJ


title: RNA_EPI.7_NZO_AC671

value: NZO/HiLtJ


title: ATAC_EPI_C57B6_4_S2


title: FaDu cells, overexpress, HNRNPC

value: overexpress HNRNPC


title: FaDu cells, overexpress, ZCRB1

value: overexpress ZCRB1


title: RNA_EPI.5_NOD_AC595


title: U251, VASN, R&D systems

city: Guangzhou

first: Zixi

last: Qin

address: Department of Pathology, Medical College, Jinan University, Guangzhou, 510632, P. R. China

institute: Jinan university

data_processing:
We use FastQC performs basic statistics on the quality of the raw reads. Then, those read sequences produced by the Illumina pipeline in FASTQ format were preprocessed through Trimmomatic software. The remaining reads that passed all the filtering steps was counted as clean reads and all subsequent analyses were based on this. At last, we use FastQC performs basic statistics on the quality of the clean reads.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using BWA v0.7.12 (Burrows Wheeler Aligner) and clean reads were aligned to the reference genome using BWA mem v0.7.12.
All peak calling was performed with MACS2 using ‘macs2 -q 0.05 --call-summits --nomodel--shift -100 --extsize 200 --keep-dup all’. For simulations of peaks called per input read, aligned and de-duplicated BAM files were used without any additional filtering.
The position of peak summit around transcript start sites of genes can predict the interaction sites of protein and gene. PeakAnnotator was used to identify the nearest TSS of every peak and the distance distribution between peaks and TSS was shown. Besides, the distribution of peak summits on different function regions, such as 5’UTR, CDS, 3’UTR, was performed.
Peak related genes can be confirmed by PeakAnnotator.
Assembly: bigWig files were generated using the genomicRanges R package. Score represents the normalized coverage of DNA fragments at a given genomic coordinate. narrowPeak files were generated using MACS v2 with default settings.
Supplementary files format and content: hg38
Supplementary files format and content: bigWig, narrowPeak (except for Input sample)
Library strategy: CUT&Tag

source_name: Brain

growth_protocol: U251 glioma stem cell was cultured under clonal conditions in low-adherence plates and maintained in DMEM/F12 medium supplemented with 2% B27, 20 ng/mL basic fibroblast growth factor (bFGF), and 10 ng/mL epidermal growth factor (EGF) at 37℃ under 5% CO2.

extract_protocol:
Cells were harvested, counted and centrifuged for 5 min at 500× g at room temperature. CUT&Tag assay was performed according to the manufacturer’s instructions (NovoNGS® CUT&Tag 3.0 High-Sensitivity Kit, Novoprotein, N259-YH01). Briefly, the cells are bound to Concanavalin A-coated magnetic beads, and the cell membrane is permeabilized by Digitonin. The enzyme pA-Tn5 Transposase precisely binds the DNA sequence near the target protein under the antibody guidance and results in factor-targeted tagmentation. DNA sequence is tagmented, with adapters added at the same time at both ends, which can be enriched by PCR to form the sequencing-ready libraries. After the PCR reaction, libraries were purified with the AMPure beads and library quality was assessed on the Agilent Bioanalyzer 2100 system.
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. The library preparations were sequenced on Illumina Novaseq platform and 150bp paired-end reads were generated.

value: Brain

value: Glioma cell

tag: chip antibody

value: VASN (R&D systems, #MAB2140)


title: FaDu cells, overexpress, CTL

value: overexpress CTL


title: RNA_EPI.4_NOD_AC576


title: FaDu cells, overexpress, SF3B3

value: overexpress SF3B3


title: KO K562, H3K27ac, EpiCypher, rep 3

city: cambridge

first: Jessica

last: Corry

address: 80 Tennis court road

country: United Kingdom

institute: University of Cambridge

data_processing:
Adapter sequences trimmed and poor quality reads removed using Trimmomatic (v0.39)
Additional barcode sequences that could have been missed removed with kseq (v20190822)
Trimmed reads alligned to GRCH38, Gencode v31 using bowtie 2 (v2.4.2) with parameters "--very sensitive local" to assign multiple mapped reads to their best allignment and "--dovetail" to permit allignment of mate pairs that extend past one another
Reads alligned to spike in E.coli genome (K12-MG1655) using bowtie2 with additional parameters "--no overlap" and "-- no dovetail".
Aligned reads filtered using SAMtools (v0.8.1) and spike in normalisation performed as outlined in Skene et al, 2017 (PMID:28079019)
For peak calling, macs2 "callpeak" (v2.2.7.1) was used.
Assembly: GRC38, Gencode v31
Supplementary files format and content: peaks called for each replicate in form of narrowpeak file
Library strategy: CUT&RUN

growth_protocol: 10% FBS, RPMI-40, 10 mM glutamine and cultured at 37oC at 5% CO2.

extract_protocol:
Chromatin profiling for WT and ACK KO K562 cells was performed using CUTANA™ ChIC/CUT&RUN Kit (EpiCypher) according to the manufacturer’s protocol . Wash Buffer, Cell Permeabilization Buffer and Antibody Buffer were supplemented with 1% Triton X-100 and 0.05% SDS. Cells were washed twice with wash buffer, and resuspended in 410ul wash buffer. 100 µL of this solution was aliquoted into four reaction tubes i.e. 5 x 107 cells per CUT&RUN reaction. 100 µL of pre-activated ConA bead slurry was added to each reaction tube and the mixture incubated for 10 min at room temperature to allow cell-bead binding. Cell-bead solution was then buffer exchanged into Antibody Buffer and target-specific antibodies added to individual reaction tubes in the following amounts: 0.5 µg of anti-H3K4me3, 1 µg of anti-H3K27ac , 0.6 µg of anti-Stat5 and 0.5 µg of IgG as a negative control.  Antibody binding was performed at 4oC overnight with gentle mixing on a nutator. Binding of pAG-MNase, targeted chromatin digestion and DNA release were completed according to the manufacturer’s protocol. To reverse cross-links, CUT&RUN released DNA was supplemented with 0.8 µL of 10% SDS (Merck) and 1 µL of 20 µg/ul Proteinase K (Ambion), and incubated overnight at 55oC using a thermocycler. DNA purification was performed using DNA Cleanup Columns.
CUTnRUN libraries were prepared using the KAPA hyperprep kit (Roche) and multiplexed using 1.5uM custom TruSeq UDI-UMI adapters. Libraries were amplified using 14 cycles on the thermocycler.  Libraries were quantified on a fragment analyzer 5300 system (Agilent) and pooled equimolarly to a final 1.9 nM concentration and sequences using a 51bp pared end setting on a Illumina NovaSeq6000.

treatment_protocol: 1% formaldehyde treatment for 1 minute at RT before quenching in 125mM glycine.

value: Chronic myeloid leukamia

value: H3K27ac(EpiCypher, 13-0045)

value: ACK KO


title: RNA_EPI.3_C57B6_5.1

value: epiblast stem cells


title: KO K562, H3K27ac, EpiCypher, rep 2


title: RNA_EPI.19_C57B6_1


title: RNA_EPI.1_129_AC630


title: RNA_EPI.18_C57B6_4


title: RNA_EPI.2_129_AC681


title: KO K562, H3K4me3, EpiCypher, rep 2

value: H3K4me3(EpiCypher, 13-0041)


title: RNA_EPI.17_DBA_55


title: RNA_EPI.15_WSB_AC627

value: WSB/EiJ


title: KO K562, STAT5, SantaCruz, rep 3

value: STAT5 (SantaCruz, sc-271542)


title: KO K562, H3K4me3, EpiCypher, rep 1


title: KO K562, H3K27ac, EpiCypher, rep 1


title: RNA_EPI.14_PWK_AC695

value: PWK/PhJ


title: KO K562, STAT5, SantaCruz, rep 2


title: RNA_EPI.13_NZO_AC653


title: KO K562, IgG, EpiCypher, rep 3

value: IgG (EpiCypher, 13-0042)


title: RNA_EPI.16_PWK_ALT


title: RNA_EPI.6_PWK_AC698


title: KO K562, STAT5, SantaCruz, rep 1


title: RNA_EPI.12_C57B6_2


title: KO K562, IgG, EpiCypher, rep 1


title: KO K562, IgG, EpiCypher, rep 2


title: WT K562, H3K27ac, EpiCypher, rep 3

value: wildtype


title: RNA_EPI.11_CAST_WM078

value: CAST/EiJ


title: RNA_DE.X_NZO_AC653

value: definitive endoderm

value: NZO/HiLtJ


title: WT K562, H3K27ac, EpiCypher, rep 1


title: WT K562, H3K27ac, EpiCypher, rep 2


title: KO K562, H3K4me3, EpiCypher, rep 3


title: RNA_DE.O_WSB_AC627


title: RNA_DE.R_DBA_55


title: WT K562, H3K4me3, EpiCypher, rep 2


title: RNA_DE.Q_C57B6_1


title: RNA_DE.P_129_AC630


title: RNA_DE.N_PWK_ALT


title: RNA_DE.L_C57B6_5.1


title: WT K562, H3K4me3, EpiCypher, rep 1


title: RNA_DE.M_C57B6_4


title: WT K562, STAT5, SantaCruz, rep 3


title: WT K562, STAT5, SantaCruz, rep 1


title: WT K562, STAT5, SantaCruz, rep 2


title: RNA_DE.K_WSB_AC635


title: WT K562, IgG, EpiCypher, rep3


title: RNA_DE.I_PWK_AC698


title: RNA_DE.J_NOD_AC576

value: NOD/ShiLtJ


title: WT K562, IgG, EpiCypher, rep1


title: RNA_DE.H_PWK_AC695


title: WT K562, IgG, EpiCypher, rep2


title: RNA_EPI.10_CAST_WM080


title: WT K562, H3K4me3, EpiCypher, rep 3


title: K562, KO, biological replicate 2

data_processing:
Libraries were accessed using FastQC (v0.11.7), Qualimap (v2.2.2c) (PMID: 26428292) and SAMtools stats (v1.9).
Alignment was performed using STAR (version 2.7.2b) (PMID: 23104886) with alignment against the human genome (GRCh38, Ensembl v100)
Trimming of adapters was performed using NGmerge (v0.3) (PMID: 30572828)
A human transcriptome index consisting of cDNA and ncRNA entries from Ensembl v100) was generated and gene abundances were obtained using Salmon (v1.1.0) (PMID: 28263959). The bioinformatics workflow was organized using Nextflow workflow management system (v20.10) (PMID: 28398311) and Bioconda software management tool (PMID: 29967506).
For differential gene expression analysis, DESeq2 (v1.26.0) was used with “ashr” (PMID: 27756721) for fold change shrinkage, all in R (v3.6.1) (PMID: 25516281)
Assembly: GRC38
Supplementary files format and content: tab deliminated normalised counts (CPM) for each gene

growth_protocol: 10% FBS, RPMI-40, 10 mM glutamine and cultured at 37C at 5% CO2.

extract_protocol:
RNA was extracted using Agencourt RNAdvance Tissue kit including DnaseI (Ambion) treatment for 15 min at 37oC on an automated Biomek i7 Hybrid system  (Beckman Coulter).  RNA quantification and quality control was performed on Fragment Analyzer 5300 system and showed an RNA integrity number (RIN) of  10 for all the samples. 500ng of input RNA was used per sample
mRNA-seq library preparation was performed using KAPA Stranded mRNA-Seq Kit, with KAPA mRNA Capture Beads (Roche) following manufacturer’s protocol with 500 ng of input RNA, 5 min fragmentation time at 95oC, 10 PCR cycles, and multiplexed using TruSeq UD-UMI adapters. Resulting libraries were pooled equimolarly to a final concentration of 1.9 nM.

treatment_protocol: 1 million cells were collected as matches samples at the time of CUTnRUN sample preparation

value: Chronic myeloid leukaemia


title: K562, KO, biological replicate 1


title: RNA_DE.E_CAST_WM078


title: K562, WT, biological replicate 3

value: WT


title: K562, WT, biological replicate 2


title: RNA_DE.D_NOD_AC595


title: K562, WT, biological replicate 1


title: RNA_DE.B_NZO_AC671


title: RNA_DE.A_129_AC681


title: chick basilar papilla,day3_SM_BQ788_2

city: Kyoto

first: Mami

last: Matsunaga

state: Kyoto

address: Kawahara cho 54, Shogoin, Sakyo-ku

department: Department of Otolaryngology, Head and Neck Surgery

country: Japan

institute: Kyoto university

description: Library name: Sample 5

data_processing:
Illumina NovaSeq X Plus
Read quality was checked for each sample using fastp.
Read trimming and mapping/quantification were performed using CLC Genomics Workbench 12.0(QIAGEN Aarhus A/S).
Read trimming was performed by quality limit: 0.01.
Assembly: bgalgal1_mat_broiler_grcg7b
Supplementary files format and content: excel file include FPKM values for each sample

organism: Gallus gallus

source_name: auditory epithelium

growth_protocol: Cochlear ducts dissected from post-hatched day-1 chicks was maintained in DMEM with 1%FBS and incuvated with 5%CO2 at 37℃

extract_protocol:
Sensory epithelium (basilar papilla) were removed from the cochlea duct via thermolysin treatment (500 μg/ml in medium 199, Sigma–Aldrich) and RNA was extracted from the collected sensory epithelia using RNeasy Micro Kit (QIAGEN) and processed according to the manufacturer’s protocol.
400ng of total RNA was subjected to library synthesis using the NEBNext® Ultra II  RNA Library Prep Kit for Illumina®.

treatment_protocol: Cochlear ducts dissected from post-hatched day-1 chicks are exposed to streptomycin (SM), an ototoxic antibiotic, for 48 h resulting in total deletion of HCs. After that, during the regeneration of hair cells from supporting cells, BQ788, an inhibitor of EDNRB added to the explant culture medium, and examined the effect of the EDNRB signal for hair cell regeneration.

value: auditory epithelium


title: chick basilar papilla,day3_SM_BQ788_3

description: Library name: Sample 6


title: KO_3

city: Baltimore

first: Angelika

last: Doetzlhofer

state: MD

address: 855 North Wolfe St

department: Neuroscience

institute: Johns Hopkins University School of Medicine

data_processing:
psuedoalignment and quantification using kallisto
QC and differential testing using Sleuth
Assembly: Ensembl v96 reference transcriptome
Supplementary files format and content: h5 abundance files

source_name: cochlear organoid culture

growth_protocol: organoids were incubated for 7 days in expansion medium (DMEM/F12 , N2 supplement , B-27 supplement, EGF, FGF2, CHIR99021, 616452, VPA, and penicillin).

extract_protocol:
Cells were pelleted and RNA was extracted using the RNeasy Micro Kit (Qiagen cat. 74004).
These samples were processed using Illumina's TruSeq stranded Total RNA kit, per manufacturer's recommendations, using the UDI indexes.  The samples were sequenced on the Illumina NovaSeq 6000, paired end, 2x50 bp reads

value: cochlear organoid culture

value: Jag1 Knockout


title: pVAX1-HA2-fimA + CpG ODN rep1

city: zunyi

first: Huijie

last: zhang

address: No. 1, Daxue Road, Honghuagang District, Zunyi City

institute: ZunyiMedicalUniversity

data_processing:
Casava1.8 software
Sequenced reads were trimmed for adaptor sequence,and masked for low-complexity or low-quality sequence.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5.
Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.16.1). DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate.Genes with an adjusted P-value <0.05 found by DESeq2 were assigned as differentially expressed.
Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package, in which gene length bias wascorrected. GO terms with corrected Pvalue less than 0.05 were considered significantly enriched by differential expressed genes.We used clusterProfiler R package to test the statistical enrichment of differential expression genes in KEGG pathwa
PPI analysis of differentially expressed genes was based on the STRING database, which known and predicted Protein-Protein Interactions
Supplementary files format and content: rn6
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

organism: Rattus norvegicus

source_name: spleen

extract_protocol:
RNA was harvested using the Animal Tissue Total RNA Extraction Kit (DP451, TIANGEN, Beijing, China). 1ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using NanoPhotometer®spectrophotometer (IMPLEN,   CA, USA) following manufacturer's protocols.

value: spleen

tag: time

value: 7th week

tag: strain

value: Sprague-Dawley

tag: treatment

value: pVAX1-HA2-fimA + CpG ODN


title: K562, KO, biological replicate 3

value: ACK KO


title: KO_2


title: pVAX1-HA2-fimA + CpG ODN rep2


title: pVAX1-HA2-fimA + CpG ODN rep3


title: pVAX1-HA2-fimA control group rep 2

value: pVAX1-HA2-fimA control group


title: pVAX1-HA2-fimA control group rep 3


title: KO_1


title: pVAX1-HA2-fimA control group rep 1


title: chick basilar papilla,day3_SM_3,ctrl

description: Library name: Sample 3


title: chick basilar papilla,day3_SM_2,ctrl

description: Library name: Sample 2


title: RNA_DE.G_C57B6_44


title: RNA_DE.F_C57B6_2


title: Control_2


title: blank group rep 3

value: blank group


title: RNA_DE.C_PWK_AC699

value: PWK/PhJ


title: blank group rep 2


title: blank group rep 1


title: Control_1


title: chick basilar papilla,day3_SM_1,ctrl

description: Library name: Sample 1


title: chick basilar papilla,day3_SM_BQ788_1

description: Library name: Sample 4


title: In_Vitro_Activation_KO+AOX_Activated_5

status: Public on Jun 20 2025

city: Chicago

first: Rogan

middle: A

last: Grant

state: IL

address: 303 E Superior St, Simpson Querrey Biomedical Research Center 5-522

department: Pulmonary and Critical Care Medicine

institute: Northwestern University

description: Library name: 969-KA-A

data_processing:
FASTQ files were generated using bcl2fastq 2.20 (Illumina) for NextSeq500 output or bcl-convert 4.0.3 (Illumina) for NextSeq2000 output using default parameters.
To facilitate reproducible analysis, samples were processed using the publicly available nf-core/RNA-seq pipeline v.3.12.0 implemented in Nextflow 23.10.0 using Singularity 3.8.1 with the minimal command nextflow run nf-core/rnaseq \ -r ‘3.12.0’ \ -profile nu_genomics \–additional_fasta ‘transgenes.fa’ \–star_index false \–three_prime_clip_r2 3 \–genome ‘GRCm38’ for SMART-seq samples. For NEBnext samples, 3′ clipping was not performed. In brief, lane-level reads were trimmed using trimGalore! 0.6.7 and aligned to the hybrid genome described above using STAR 2.6.1d. Gene-level assignment was then performed using salmon 1.10.1.
Assembly: GRCm38
Supplementary files format and content: Raw counts from nf-core/rnaseq output and relevant metadata for DEA

molecule: polyA RNA

source_name: CD8+ T cells

extract_protocol:
RNA was isolated using the AllPrep DNA/RNA Mini Kit (Qiagen) following the manufacturer’s instructions
For 40dpi samples, RNA quality and quantity were assessed using TapeStation 4200 RNA tapes (Agilent) and RNA-seq libraries were prepared from 0.2-6.0ng total RNA using the SMART-seq v4 Ultra Low Input RNA Kit (Takara 634773). For all other samples, libraries were prepared from 15ng total RNA using the NEBNext Ultra DNA Library Prep kit for Illumina (NEB E7370L). Library quality control was then performed using TapeStation 4200 High-Sensitivity DNA tapes (Agilent). Dual-indexed libraries were pooled and sequenced on a NextSeq500 instrument (Illumina) for 40dpi samples or a NextSeq2000 instrument for all other samples (Illumina) 75 or 100 cycles, respectively, single-end, to an average sequencing depth of 14.1 million reads per sample.

treatment_protocol: All mice were housed and used in accordance with the Institutional Animal Care and Use Committee (IACUC) at Northwestern University. RISPfl/fl (Uqcrfsfl/fl) mice4, QPCfl/fl (Uqcrqfl/fl) mice53, and AOXLSL 19 mice were previously described. GzmBCreERT2 mice generated by Douglas Fearon39, were a gift from Kamal Khanna at University of Connecticut. CD4-Cre (Stock #022071), P14 transgenic (Stock #004694), Thy1.1 (Stock #000406) and Sox2Cre (Stock# 008454) mice were purchased from The Jackson Laboratory, backcrossed in house when necessary and bred to relevant gene combinations. All mouselines were screened for C57BL/6J genetic background purity by Illumina SNP chip (DartMouse). For all experiments, male and female mice between 1.5 and 6 months old were age-matched and assigned to experimental groups by pre-experimental genotyping. Sex matching was used when required for adoptive transfer experiments. For inducible Cre recombination, tamoxifen was administered by chow (Envigo).  P14 immune chimeras were generated by transferring 5×104 congenically marked P14 CD8 T cells from donors bearing the appropriate genotypes into naive recipient mice by tail vein injection. At least 12hr later, recipients were infected with 2×105 PFU LCMV Armstrong via intraperitoneal (i.p) injection. For endogenous studies naive recipient mice were infected with 2×105 PFU LCMV Armstrong i.p. LCMV Armstrong was a gift from David Masopust at University of Minnesota. For acute infections and in vivo tumor killing assays, adoptive transfer recipients of CD4-Cre RISP AOX P14 CD8 T cells were wildtype mice carrying an unexpressed AOX gene (e.g. Cre-/-, AOXL/W), to enable central tolerance to the AOX protein. One group of such recipients was also used for GzmbCreERT2 QPC AOX P14 CD8 T cell recipients as noted in the legend.  For LCMV clone 13 (Cl13) studies, naïve CD8+ T cells were isolated from the spleens of donor CD90.1+ P14 mice of the indicated genotypes using the EasySep™ Mouse Naïve CD8+ T Cell Isolation Kit (STEMCELL; Cat: 19858). A total of 2,500 naïve P14 CD8+ T cells were intravenously transferred into naïve C57BL/6 recipient mice, followed by LCMV Cl13 infection. To establish chronic infection, each mouse received an intravenous injection of 2 × 10^6 PFU LCMV Cl13. The virus was propagated on BHK21 cells, and viral titers were quantified using a plaque formation assay on Vero cells.  For in vivo cytotoxicity assays, MC38-gp33 tumor cells were a gift from Ananda Goldrath at UCSD and upon arrival were independently confirmed mycoplasma negative and gp33 expressing. For tumor administration, 5x10^5 MC38-gp33 tumor cells were injected subcutaneously into the recipient mouse’s shaved flank. Isolated P14 CD8 T cells from donors bearing the indicated genotypes were activated in vitro for 3 days using 1:1 anti-CD3/anti-CD28 T cell expansion beads (Miltenyi 130-093-627)) and 50U/ml IL2 (NIH Repository) prior to i.v. adoptive transfer of 10^6 P14, into mice with established palpable tumors 5 days after tumor cell administration. Tumor bearing mice were monitored and tumors were measured with calipers in accordance with the relevant approved IACUC protocols.  Restimulation of P14 CD8 T cells was performed in 96-well flat-bottom plates (MIDSCI; Cat: TP92096). A total of 2 × 10⁶ splenocytes were seeded in complete RPMI and incubated at 37°C for 4 hours in the presence of 0.133 µg/mL wild-type GP33 peptide (KAVYNFATC; GenScript; Cat: RP20257), 5.0 µg/mL Brefeldin A Solution (BioLegend; Cat: 420601) and 0.2 µg/mL anti-CD107a antibody.  Isolated CD8 T cells  (see above methods) were labeled with Cell Trace Violet for proliferation assays according to manufacturer instructions (Thermo Fisher C34557) and loaded into a round bottom 96well plate with anti-CD3e/anti-CD28 beads (Miltenyi 130-093-627) at a ratio of 3 beads per cell with 50U/ml IL-2 for 96hr in adapted mouse plasma like media for T cells (MPLMT, see table 1)54 or complete RPMI (see table 2) then stained and assessed by flow cytometry.  For in vitro memory formation assays CD8 T cells were similarly isolated and activated in vitro with anti-CD3e/anti-CD28 beads at 1:1 ratio and 50U/ml IL2 for 3 days. On the third day the cells were removed from the beads and cultured with IL-15 and IL-7 for 3 more days. All in vitro memory formation assays were performed in complete RPMI. On day 6 cells were stained directly surface, intracellular (BD cytofix/cytoperm, # BDB554714) or intranuclear (BioLegend True Nuclear Kit, 424401) antibodies (see Table. 4) before acquisition on the flow cytometer.

value: CD8+ T cells

tag: group

value: KO + AOX

tag: state

value: Activated

tag: dpi

tag: donor genotype

tag: protocol

tag: host genotype

value: Cd4Cre(+), RISP(Fx/Fx), AOX(LSL/WT)

tag: qpc

tag: risp

value: KO

tag: aox status

value: AOX


title: In_Vitro_Activation_KO+AOX_Naïve_5

description: Library name: 969-KA-N

value: Naïve

value: WT


title: In_Vitro_Activation_KO_Activated_4

description: Library name: 943-K-A

value: Cd4Cre(+), RISP(Fx/Fx), AOX(WT/WT)


title: In_Vitro_Activation_KO+AOX_Activated_4

description: Library name: 968-KA-A


title: In_Vitro_Activation_WT_Activated_5

description: Library name: 947-W-A

value: Cd4Cre(+), RISP(WT/WT), AOX(WT/WT)


title: In_Vitro_Activation_WT_Activated_4

description: Library name: 942-W-A


title: In_Vitro_Activation_WT_Naïve_5

description: Library name: 942-W-N


title: In_Vitro_Activation_KO_Naïve_5

description: Library name: 943-K-N


title: In_Vitro_Activation_KO_Naïve_3

description: Library name: 765-K-N


title: In_Vitro_Activation_WT_Naïve_6

description: Library name: 947-W-N


title: In_Vitro_Activation_WT_Activated_3

description: Library name: 764-W-A


title: In_Vitro_Activation_KO_Activated_2

description: Library name: 765-K-A


title: In_Vitro_Activation_KO_Activated_3

description: Library name: 788-K-A


title: In_Vitro_Activation_KO_Naïve_2

description: Library name: 756-K-N


title: In_Vitro_Activation_WT_Naïve_4

description: Library name: 764-W-N


title: In_Vitro_Activation_WT+AOX_Naïve_4

description: Library name: 761-WA-N

value: WT + AOX

value: Cd4Cre(+), RISP(WT/WT), AOX(LSL/WT)


title: In_Vitro_Activation_WT+AOX_Activated_3

description: Library name: 761-WA-A


title: In_Vitro_Activation_KO+AOX_Naïve_3

description: Library name: 727-KA-N


title: DRG, CT6, control mice, pool of 3 mice, biological replicate 1

city: Toulouse

first: Rüçhan

last: Ekren

address: CHU Purpan – BP 3028 –  Toulouse Cedex, 3

department: INFINITY

country: France

institute: Inserm UMR 1291

description: TruSeq Stranded mRNA

data_processing:
Libraries quantification and sequencing were performed at the GeT-PlaGe core facility (INRAE, Toulouse, France). Libraries were quantified by qPCR using the KAPA Library Quantification Kit (Roche) to obtain an accurate quantification. Libraries were equimolarly pooled and RNA sequencing was then performed on one SP lane of the Illumina NovaSeq 6000 instrument (Illumina), using the NovaSeq 6000 SP v1 Reagent Kit (300 cycles), and a paired-end 2 x 150 pb strategy.
FastQC was performed on sequence reads to evaluate sequencing quality. Reads were then aligned on GRCm39 (mm10) as a reference genome using STAR aligner (v2.4) and counted with Htseq (v0.9.1), and a counting matrix was generated.
Assembly: GRCm39 (mm10)
Supplementary files format and content: mtx files, HTseq count output of each sample

source_name: DRG

growth_protocol: Mice were bred and housed in the local animal facilities of CREFRE (Toulouse, France) in pathogen-free sanitary conditions, with free access to water and food, and were submitted to alternating cycles of 12 hours of light and darkness at an ambient temperature of 22°C. Both age-matched male and female mice were used in experiments. All animal care and experimentation were conducted in France in compliance with the guidelines of the European Union (86/609/EEC) and the French Committee of Ethics (87/848) policies and with the specific approval from the local ministry-approved committee on ethics in animal experimentation (Ethics Committee UMS006 CEEA-122, project n°21938-2019090417341270v5).

extract_protocol:
DRG from adult mice were stored immediately in RNALater then dissociated in trizol with the Miltenyi gentleMACS Dissociator. Tissue RNA was extracted using the RNeasy Qiagen Mini Kit (Qiagen) according to the manufacturer’s guidelines.
RNA concentration and purity were determined using a ND-2000 Spectrophotometer (ThermoFisher Scientific). Integrity of RNA was checked with a Fragment Analyzer (Agilent Technologies), using the RNA Standard Sensitivity Kit. 260/280 purity ratios were all > 1.8. Integrity indices revealed acceptable and homogenous values for the RNA Integrity Number (8.2-8.6) and 28S/18S ratios (0.8-1.2). RNA-seq paired-end libraries were prepared at the GeT-Santé facility according to Illumina’s protocol with some adjustments, using the TruSeq Stranded mRNA library prep Kit (Illumina). Briefly, mRNAs were first selected from either 3000 ng (DRG sequencing) or 500 ng (sorted fetal MC sequencing) of total RNA using poly-T beads. Then, RNAs were fragmented for 2' and retrotranscribed to generate double stranded cDNA. Compatible adaptors were ligated, allowing the barcoding of the samples with unique single indices. 11 cycles of PCR were applied to amplify libraries, and an additional double-sized purification step allowed to obtain 280-1000 pb fragments. Libraries quality was assessed using the HS NGS kit on the Fragment Analyzer (Agilent Technologies).

treatment_protocol: Eight- to 10-week-old male and female mice were subjected to timed pregnancies. Pregnant dams were then subject to chronic restraint stress in transparent drilled 50-ml tubes, put under a bright light (200W), 3 times a day for 30 minutes each, from embryonic day (E)-13 until E-18. Control (CT) pregnant females were left undisturbed. After birth, all pups were kept in the same cage with their biological mother. Pups were then weaned at the age of 21 to 28 days in distinct gender groups in new cages and introduced to a new diet. Dorsal root ganglia (DRG) were collected from 8-week old (W8) control (CT) and PS offspring (3 mice per group), and bulk RNA sequencing was conducted.

tag: tissue

value: DRG

tag: age

value: 8-weeks old

value: Control offspring


title: In_Vitro_Activation_KO_Activated_1

description: Library name: 756-K-A

tag: cell type


title: In_Vitro_Activation_WT+AOX_Naïve_2

description: Library name: 714-WA-N


title: In_Vitro_Activation_KO_Naïve_1

description: Library name: 745-K-N


title: In_Vitro_Activation_KO+AOX_Naïve_4

description: Library name: 968-KA-N


title: In_Vitro_Activation_KO_Naïve_4

description: Library name: 788-K-N


title: In_Vitro_Activation_WT_Naïve_2

description: Library name: 704-W-N


title: In_Vitro_Activation_WT_Naïve_3

description: Library name: 735-W-N


title: In_Vitro_Activation_WT_Activated_2

description: Library name: 704-W-A


title: In_Vitro_Activation_KO+AOX_Activated_3

description: Library name: 727-KA-A


title: In_Vitro_Activation_WT+AOX_Naïve_1

description: Library name: 711-WA-N


title: Adoptive_Transfer_WT+AOX_Naïve_5

description: Library name: 4WAN

value: GzmbCreERT2(+), QPC(Fx/WT), P14(+), Thy(1.1/1.1), AOX(LSL/WT)


title: In_Vitro_Activation_WT+AOX_Activated_1

description: Library name: 714-WA-A


title: Adoptive_Transfer_KO+AOX_Naïve_3

description: Library name: 3RN

value: GzmbCreERT2(+), QPC(Fx/Fx), P14(+), Thy(1.1/1.1), AOX(LSL/WT)


title: In_Vitro_Activation_WT_Naïve_1

description: Library name: 702-W-N

value: CD8+ T cells


title: In_Vitro_Activation_KO+AOX_Naïve_2

description: Library name: 700-KA-N

value: KO + AOX


title: In_Vitro_Activation_KO+AOX_Activated_2

description: Library name: 700-KA-A

value: Activated


title: Adoptive_Transfer_KO_Naïve_5

description: Library name: 4KN


title: In_Vitro_Activation_WT+AOX_Activated_2

description: Library name: 716-WA-A


title: In_Vitro_Activation_KO+AOX_Activated_1

description: Library name: 698-KA-A

value: Cd4Cre(+), RISP(Fx/Fx), AOX(LSL/WT)


title: Adoptive_Transfer_WT+AOX_Naïve_4

description: Library name: 3WAN


title: Adoptive_Transfer_WT_Naïve_3

description: Library name: 1WN

value: GzmbCreERT2(+), QPC(Fx,WT), P14(+), Thy(1.1/1.1), AOX(WT/WT)


title: Adoptive_Transfer_WT+AOX_Naïve_3

description: Library name: 1WAN

value: AOX


title: Adoptive_Transfer_WT+AOX_Activated_4

description: Library name: 3WAP


title: In_Vitro_Activation_WT+AOX_Naïve_3

description: Library name: 716-WA-N

value: Naïve


title: Adoptive_Transfer_WT+AOX_Activated_3

description: Library name: 1WAP

value: WT


title: Adoptive_Transfer_KO+AOX_Naïve_2

description: Library name: 1RN

value: KO


title: Adoptive_Transfer_KO+AOX_Activated_4

description: Library name: 4RP


title: Adoptive_Transfer_WT_Naïve_5

description: Library name: 5WN


title: Adoptive_Transfer_WT_Naïve_4

description: Library name: 4WN


title: Adoptive_Transfer_WT+AOX_Activated_5

description: Library name: 5WAP


title: In_Vitro_Activation_WT_Activated_1

description: Library name: 702-W-A

value: Cd4Cre(+), RISP(WT/WT), AOX(WT/WT)


title: Adoptive_Transfer_WT_Activated_5

description: Library name: 5WP


title: Adoptive_Transfer_KO+AOX_Activated_3

description: Library name: 2RP


title: Adoptive_Transfer_WT_Activated_4

description: Library name: 3WP


title: Adoptive_Transfer_KO_Activated_3

description: Library name: 1KP


title: In_Vitro_Activation_KO+AOX_Naïve_1

description: Library name: 698-KA-N


title: ATAC_DE_WSB_AC635_20k40k

tag: cell line

value: definitive endoderm

tag: genotype

value: WSB/EiJ


title: Adoptive_Transfer_WT+AOX_Naïve_1

description: Library name: 2WAN

value: WT + AOX


title: Adoptive_Transfer_KO_Naïve_4

description: Library name: 2KN


title: Adoptive_Transfer_WT_Activated_3

description: Library name: 2WP


title: Adoptive_Transfer_KO+AOX_Naïve_1

description: Library name: 4RN


title: Adoptive_Transfer_KO_Naïve_3

description: Library name: 5KN


title: Adoptive_Transfer_WT+AOX_Naïve_2

description: Library name: 5WAN


title: Adoptive_Transfer_KO_Naïve_2

description: Library name: 3KN


title: Adoptive_Transfer_WT_Naïve_2

description: Library name: 3WN


title: Adoptive_Transfer_WT+AOX_Activated_1

description: Library name: 2WAP


title: Adoptive_Transfer_KO_Naïve_1

description: Library name: 1KN


title: Adoptive_Transfer_WT_Naïve_1

description: Library name: 2WN


title: Adoptive_Transfer_KO_Activated_4

description: Library name: 4KP


title: Adoptive_Transfer_KO+AOX_Activated_2

description: Library name: 3RP


title: Adoptive_Transfer_WT_Activated_2

description: Library name: 4WP


title: Adoptive_Transfer_WT+AOX_Activated_2

description: Library name: 4WAP


title: Adoptive_Transfer_KO+AOX_Activated_1

description: Library name: 1RP


title: DHX36/FANCJ double knockout mESC labelled with double pulse of EdU plate 3

city: Utrecht

first: Jeroen

last: van den Berg

state: Utrecht

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

data_processing:
scEdU-seq Libraries were sequenced 2x100 bp on Illumina NextSeq2000
See van den Berg et al.,(scEdU-seq, 2022)
For scEdU-seq, ChiC-seq,  see van den Berg et al.,(scEdU-seq, 2022),
Assembly: GRCh38 (Gencode)
Supplementary files format and content: deduplicated single cell count tables (tsv.gz)
Library strategy: scEdU-seq

source_name: embryonic stem cell

growth_protocol: mouse Embryonic Stem Cells were cultured

extract_protocol: van den Berg et al.,(scEdU-seq, 2022)

treatment_protocol: there was no treatment outside of double pulse scEdU-seq labeling

value: embryonic stem cell


title: Adoptive_Transfer_KO_Activated_1

description: Library name: 2KP


title: Adoptive_Transfer_WT_Activated_1

description: Library name: 1WP


title: Adoptive_Transfer_KO_Activated_2

description: Library name: 3KP


title: FANCJ knockout mESC labelled with double pulse of EdU plate 2

value: FANCJKO


title: DHX36 knockout mESC labelled with double pulse of EdU plate 4


title: DHX36/FANCJ double knockout mESC labelled with double pulse of EdU plate 4


title: DHX36/FANCJ double knockout mESC labelled with double pulse of EdU plate 2


title: FANCJ knockout mESC labelled with double pulse of EdU plate 4


title: DHX36 knockout mESC labelled with double pulse of EdU plate 1


title: DHX36/FANCJ double knockout mESC labelled with double pulse of EdU plate 1


title: DHX36 knockout mESC labelled with double pulse of EdU plate 2


title: FANCJ knockout mESC labelled with double pulse of EdU plate 1


title: DHX36 knockout mESC labelled with double pulse of EdU plate 3


title: WT mESC labelled with double pulse of EdU plate 4


title: PIEZO2 positive fibroblast

city: Yachiyo-City

first: Kota

last: Kokubo

state: Chiba

address: Yachiyodai-minami 1-10-12

department: Department of Immunology

institute: Chiba University

data_processing:
For the data analyses, read sequences (50 bp) were aligned to the mm10 mouse reference genome (University of California, Santa Cruz, CA, USA; December 2011) using the Bowtie 2 (version 2.3.4) and TopHat (version 1.3.2) software programs. Fragments per kilobase of exon per million mapped reads for each gene were calculated using the Cufflinks (version 2.2.1) software programs.
Assembly: GRCh38
Supplementary files format and content: csv file that includes FPKM values for each sample

source_name: Skin

extract_protocol:
Keroid tissues were dissociated in a gentleMACS C-tube (Miltenyi Biotec) with digestion buffer composed of DMEM (Gibco) and Multi Tissue Dissociation Kit 1 using a gentle MACS Octo dissociator (Miltenyi Biotec). After running the program “37°C_multi_H”, cell suspensions were passed through a 70-μm nylon mesh sheet.
Total cellular RNA was extracted with TRIzol reagent (Invitrogen). For cDNA library construction, we used a SMARTer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian (TaKaRa, Shiga, Japan) following the manufacturer’s protocol.

value: Skin

value: fibroblast


title: H1975 S310F_3

city: Tianjin

first: Feng

middle: Yuan

last: Ying

address: Tianjin

institute: Tianjin Medical University Cancer Institute and Hospital

description: Library name: Sample 6

data_processing:
The sequencing platform used was NovaSeq X Plus (Illumina, USA) with a PE150 sequencing strategy.
Assembly: Homo Sapiens(GRCh38/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

source_name: Lung

growth_protocol: H1975 EV/S310F were cultured in RPMI 1640 medium with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a humidified incubator with 5% CO2.

extract_protocol:
RNA was harvested using TRNzol Universal Reagent.
RNA libraries for RNA-seq were prepared using Fast RNA-seg Lib Prep Kit V2 (Cat.No.RK20306) following manufacturer's protocols.

treatment_protocol: empty vector and HER2 S310F mutant

value: Lung

value: HER2 S310F mutant


title: PIEZO2 negative fibroblast


title: H1975 S310F_1

description: Library name: Sample 4


title: H1975 EV_3

description: Library name: Sample 3

value: empty vector


title: H1975 S310F_2

description: Library name: Sample 5


title: H1975 EV_1

description: Library name: Sample 1


title: Horse sperm 3 (Small RNA-seq)

city: Madrid

first: Alfonso

last: Gutiérrez-Adán

address: Av. Puerta de Hierro 28

department: Animal Reproduction

country: Spain

institute: INIA

data_processing:
Raw reads were trimmed using Trimmomatic
Mapping, miRNA detection and quantification were performed by miRDeep2 using miRBase database
Assembly: Equus Caballus EquCab3.0
Supplementary files format and content: CSV file containing novel and annotated miRNA read counts

organism: Equus caballus

source_name: Sperm

extract_protocol:
Small RNA was isolated using TRIzol reagent, treated with Dnase for 15 min and purified by phenol:chloroform extraction for sperm and treated with Dnase for 1 hour for testes.
Small RNA libraries were prepared by adapter ligation, reverse transcription, PCR enrichment, purification and size selection.

value: Sperm

tag: developmental stage


title: FANCJ knockout mESC labelled with double pulse of EdU plate 3


title: Horse sperm 4 (Small RNA-seq)


title: Horse sperm 2 (Small RNA-seq)


title: Direct_Infection_KO_Naïve_5

description: Library name: NC_21_174

value: CD8+ T cells

value: Cd4Cre(+), RISP(Fx/Fx), AOX(WT/WT)


title: Horse sperm 1 (Small RNA-seq)


title: Adult testis 4 (Small RNA-seq)

source_name: Testis

value: Testis

value: Adult


title: Adult testis 33 (Small RNA-seq)


title: Direct_Infection_KO_Naïve_3

description: Library name: NC_21_172

value: KO

value: Naïve

value: WT


title: Adult testis 20 (Small RNA-seq)


title: WT mESC labelled with double pulse of EdU plate 1


title: Puber testis 19 (Small RNA-seq)

value: Puber


title: WT mESC labelled with double pulse of EdU plate 2


title: WT mESC labelled with double pulse of EdU plate 3


title: Direct_Infection_KO_Activated_5

description: Library name: NC_21_169

value: Activated


title: Direct_Infection_KO_Naïve_1

description: Library name: NC_21_170


title: Adult testis 13 (Small RNA-seq)


title: Direct_Infection_KO_Naïve_2

description: Library name: NC_21_171


title: Direct_Infection_KO_Activated_4

description: Library name: NC_21_168


title: Puber testis 11 (Small RNA-seq)


title: H1975 EV_2

description: Library name: Sample 2


title: Direct_Infection_KO_Activated_3

description: Library name: NC_21_167


title: set3.urt1_rep3

city: København N

first: Peter

last: Brodersen

address: Ole Maaløes Vej 5

country: Denmark

institute: University of Copenhagen

data_processing:
Fastq files were sorted based on their gene-specific primer used in the 2ndPCR reaction, which is forMYB33: 5’-AAGAATTCTCGTCGCCTGAA-3’,AFB2_5’CF: 5’-GGTCTCCTTACAGACCAAGT-3’ andAFB2_3’CF: 5’-GATTCTTCTGTGAAAGCCATTCTG-3’.
read 2 fastq files beginning with the 3’-adapter delimiter sequence followed by 15 random bases (Unique Molecular Identifier, UMI) were kept and were deduplicated using the UMI.
the 3’-end of the deduplicated reads were trimmed usingCutadapt v.2.4(Martin, 2011) for adapter leftovers (TTCAGGCGACGAGAATTCTT).
The final output is a .txt file wherein each line represents a tail of a unique read. To obtain such a file, two rounds ofCutadaptwith special command lines were used to first trim away the gene body until the CS (in the case ofAFB23’-CF the gene body was cleaved until the end of the cDNA sequence given in TAIR). Unspecific reads included due to off-targets of the nested gene-specific primers were removed in the same trimming step by the command-- discard-untrimmed.In the second round ofCutadapttrimming,AFB2_5’CF andMYB33_5’CF reads that have not been cleaved were removed by searching for reads with a sequence running over the CS followed by the command-- discard-trimmed.
the .txt files were imported in R and deduplicated once again with a custom R script that only allows UMI that differ from each other in minimum 13 of the 15 random bases. The pipeline together with a R script of the downstream analyses of the .txt files can be found on Github:https://github.com/MariaLouisaVigh/HESO1URT1
Assembly: not mapped to genome but reads were filtered depending on the nested PCR
Supplementary files format and content: txt files of the end extremeties. Each line represents a unique molecule tail

organism: Arabidopsis thaliana

source_name: inflorescences

growth_protocol: All seeds were sterilized and germinated on plates containing 1x Murashige & Skoog medium (#M0222.0050, Saveen og Werner ApS, Denmark), supplemented with 1% (w/v) sucrose and 0.8% (w/v) agar. Seedlings were transferred to soil (Plugg/Såjord [seedcompost]; SW Horto, Bramming, Denmark) 10 days after germination (DAG). After transfer to soil, plants were grown in a Percival growth chamber for an additional 4 weeks under the following conditions: 16 h light (Master TL-D 36W/840 and 18W/840 bulbs (Philips); 130 mmol m−2 s−1, 21°C, 60% relative humidity)/ 8 h darkness (16°C, 60% relative humidity).

extract_protocol:
Inflorescences and rosettes for northern blots, western blots and sequencing libraries were collected and snap-frozen in liquid nitrogen. Total RNA from inflorescences was extracted with TRI Reagent (#T9424, Sigma-Aldrich Denmark A/S) according to the manufacturer’s instructions. The RNA was once again resuspended and purified with phenol:chloroform:isoamyl alcohol. The RNA was dissolved in nuclease-free water and RNA integrity was assessed on a Bioanalyser RNA Nano Chip (Agilent Technologies).
10 μg of total inflorescence RNA was used as input in each library. Twenty pmol of a 3’-ssDNA adapter (custom order, IDT), previously described in (Zuber et al., 2018) was ligated to 10 μg of total RNA. To separate excess adapter from successfully ligated RNA, samples were purified using NucleoSpin® RNA Clean-up columns (Macherey Nagel). Next, 2 x 20 μl reactions of 2 μg of denatured RNA incubated with 50 pmole of 3’-RT oligo, 10 mM dNTP mix, 200 U of SuperScript IV reverse transcriptase (InvitrogenTM), 1X SuperScript RT buffer, 0.1 M DTT and 40 U Ribolock (ThermoScientific) were incubated at 50°C for 10 minutes. The reverse transcriptase was then inactivated at 80°C for 10 min. The cDNA was purified with phenol:chloroform:isoamyl alcohol and the aqueous phase was EtOH-precipitated. The cDNA was resuspended in 8μl of water. Two rounds of nested PCR reactions were then performed. The first PCR contained 2 μl of cDNA, 10 pmol of a gene-specific primer (MYB33_5’CF/AFB2_5’CF/AFB2_3’CF), 10 pmol of RACEseq_rev1 primer, 12.5 nmol dNTP mix, 1U of Phusion DNA Polymerase and 1 x Phusion HF buffer (ThermoScientific). The PCR specifications were 94 °C 30 sec, (94°C 20 sec, 50°C 20 sec, 72°C 30 sec) x 20 cycles, 72 °C 30 sec. Thereafter, 4 x 20 μl reactions for each gene-specific PCR reaction were set up using 1 μl of PCR reaction 1, 10 pmole of a new gene-specific primer, 10 pmole of TRUseq RPI primer, 12.5 nmole of dNTP mix, 1 U Phusion DNA Polymerase and 1 x Phusion HF buffer (ThermoScientific). The PCR specifications were 94°C 1 min, (94°C 30 sec, 56°C 20 sec, 72°C 30 sec) x 25 cycles, 72 °C 30 sec. The four identical PCR reactions were pooled, and the libraries were PCR purified using Beckman CoulterTM Agencourt AMPure XP beads following the manual from the provider. The libraries were assessed with Agilent Bioanalyser on a DNA 1000 chip and the concentration was determined with Qubit HS DNA chemistry (InvitrogenTM). Libraries were pooled in equimolar ratios and sequenced on a NextSeq (v3 chemistry) MidOutput flow cell with 41 x 111 bp settings. 50% of phiX was added to compensate for the poor read diversity of poly(A)-tails. Sequences of all adapters and primers used for the library construction are listed in Dataset EV3 in https://doi.org/10.1101/2024.05.27.596008.

value: inflorescences


title: Puber testis 10 (Small RNA-seq)


title: Pre-puber testis 14 (Small RNA-seq)

value: Pre-puber


title: set3.urt1_rep2


title: Direct_Infection_KO_Activated_2

description: Library name: NC_21_166


title: set3.heso1urt1_rep4


title: Direct_Infection_KO_Activated_1

description: Library name: NC_21_165


title: set3.heso1urt1_rep2


title: set3.heso1urt1_rep1


title: Pre-puber testis 3a (Small RNA-seq)


title: set3.heso1_rep3


title: Infant testis 30 (Small RNA-seq)

value: Infant


title: Pre-puber testis 3b (Small RNA-seq)


title: Direct_Infection_KO_Naïve_4

description: Library name: NC_21_173


title: set3.heso1_rep1


title: Direct_Infection_KO+AOX_Naïve_3

description: Library name: NC_21_162

value: KO + AOX

value: Cd4Cre(+), RISP(Fx/Fx), AOX(LSL/WT)

value: AOX


title: Direct_Infection_KO+AOX_Naïve_4

description: Library name: NC_21_163


title: Infant testis 31b (Small RNA-seq)


title: set3.WT_rep4

value: Col-0 WT


title: set3.WT_rep3


title: Direct_Infection_KO+AOX_Naïve_1

description: Library name: NC_21_160


title: Infant testis 31a (Small RNA-seq)


title: set3.WT_rep2


title: set3.heso1_rep2


title: set2.urt1_rep3


title: Direct_Infection_KO+AOX_Activated_4

description: Library name: NC_21_158


title: set2.rrp4_rep3


title: Horse sperm 3 (RNA-seq)

data_processing:
Raw reads were trimmed using Trimmomatic, and just for sperm samples rRNA was filtered using Bowtie2
Using HISAT2 the fragments are mapped to the reference genome
Assembly and quantification was performed by StringTie
Assembly: Equus Caballus EquCab3.0
Supplementary files format and content: Ctab files contain coverage data per transcript for each sample, as obtained by StringTie

extract_protocol:
Total RNA was isolated using TRIzol reagent, treated with Dnase for 15 min and purified by phenol:chloroform extraction for sperm and treated with Dnase for 1 hour for testes.
Library preparation includes rRNA removal and dUTP method for strand-sepcific library

value: Sperm


title: set3.heso1_rep4


title: set2.rrp4_rep2


title: Direct_Infection_KO+AOX_Activated_1

description: Library name: NC_21_155

value: CD8+ T cells


title: Horse sperm 2 (RNA-seq)


title: Direct_Infection_WT_Naïve_5

description: Library name: NC_21_154

value: Cd4Cre(+), RISP(WT/WT), AOX(WT/WT)


title: set2.MIR393_rep3


title: set2.MIR393_rep2


title: set2.MIR393_rep1


title: Direct_Infection_WT_Naïve_4

description: Library name: NC_21_153


title: Adult testis 4 (RNA-seq)

value: Testis


title: 5UTR-MaP of HEK293 cells under ATP-depleted conditions, replicate 2

city: Groningen

first: Danny

last: Incarnato

state: Netherlands

address: Nijenborgh 7

department: Molecular Genetics

institute: University of Groningen

description:
Library name: 5UTR-MaP_ATPdepleted_rep2
Instrument: MGI DNBSEQ-T7

data_processing:
Following sequencing, paired-end reads were clipped of sequencing adapters using Cutadapt (parameters: -A AGATCGGAAG -a AGATCGGAAG -m 75:75 -O 1), and merged using PEAR (parameters: -n 75 -q 20 -u 0 -e -y 10G -z). Merged reads were then combined with R1 and the reverse-complemented R2 for read pairs that could not be merged. Reads were then mapped to the MANE v1.2 reference (plus the 18S and 28S rRNA sequences) using the rf-map tool of the RNA Framework and Bowtie2, after clipping terminal bases with Phred quality < 20, and discarding reads containing internal Ns (parameters: -b2 -cq5 20 -ctn -cmn 0 -mp "--very-sensitive-local" -bnr). Alignments in SAM format were sorted and converted to BAM format using Samtools. BAM alignments were then processed using RNA Framework’s rf-count to generate both RC files (containing per-base mutations and coverage) and MM files (containing a map of mutated positions per read). Aligned reads spanning less than 100 nt of a transcript, as well as reads having more than 10% mutated bases, or less than 2 mutations, were discarded. Insertions and ambiguously aligned deletions, as well as deletions longer than 1 nt, were ignored. Mutations were considered only if both the mutated base and the two surrounding bases had Phred quality > 20, and consecutive mutations falling within 3 nt from each other were ignored (parameters: -m -mm -wl 2000 -ds 100 -es -na -ni -md 1 -dc 3 -me 0.1). DRACO analysis was performed with a window size of 90 nt, slid in 5 nt increments, requiring a minimum base coverage of 2,000X and a minimum of 2,000 reads post-filtering to perform the eigen deconvolution, and by repeating the graph-cut procedure 30 times (parameters: --absWinLen 90 --absWinOffset 5 --minBaseCoverage 2000 --minFilteredReads 2000 --minPermutations 10 --maxPermutations 50 --firstEigengapShift 0.95 --lookaheadEigengaps 1 --softClusteringIters 30 --softClusteringInits 500 --softClusteringWeightModule 0.005).
Supplementary files format and content: Wiggle files of bulk normalized DMS-MaPseq mutation frequencies

molecule: cytoplasmic RNA

growth_protocol: Human HEK293 cells were cultured in high glucose DMEM medium (cat. L0104, Biowest), supplemented with 10% fetal bovine serum (FBS) (cat. H1138, Merck), 25 U/mL penicillin and 25 μg/mL streptomycin, at 37°C and 5% CO2. For ATP depletion, cells were washed twice in PBS, and then kept for 20 min in glucose-free DMEM (cat. 11966025, ThermoFisher), supplemented with 10% FBS, 1 mM sodium pyruvate, 25 U/ml penicillin, 25 μg/ml streptomycin, 10 mM 2-deoxy-D-glucose (cat. 25972-M, Merck), 10 mM sodium azide (cat. 71289, Merck), at 37°C and 5% CO2.

extract_protocol:
Cell pellets were immediately lysed by direct addition of 1 mL ice-cold TRIzol™ Reagent (cat. 15596018, ThermoFisher Scientific). RNA was extracted as per manufacturer instructions. Residual gDNA was removed by digestion with TURBO DNase I (cat. AM2239, ThermoFisher Scientific) at 37°C for 30 min.
Prior to library preparation, ~1.5 μg of polyA+ RNA were enriched per sample using Oligo d(T)25 Magnetic Beads (cat. S1419S, New England Biolabs). RNA was directly eluted from the beads by fragmentation in 4 mM MgCl2 for 5.5 min at 94°C, and then cleaned up on Monarch® Spin RNA Cleanup columns. Endogenous 5′-phosphate groups, as well as 2′-3′-cyclic phosphates generated by chemical fragmentation, were removed by treatment with 1 U Shrimp Alkaline Phosphatase (rSAP) (cat. M0371L, New England Biolabs), in a final volume of 20 μl, at 37°C for 30 min, followed by cleanup on Monarch® Spin RNA Cleanup columns. Decapping of 5′-capped RNA fragments was performed by treating the RNA with 5 U Cap-Clip™ Acid Pyrophosphatase (cat. C-CC15011H, CellScript), in a final volume of 20 μl, at 37°C for 1 h, followed by clean up on Monarch® Spin RNA Cleanup columns. Decapped RNA fragments were then ligated to an RNA adapter (CUACACGACGCUCUUCCGAUCU) harboring a 5′-biotin-TEG modification. Decapped RNA fragments and the RNA adapter (1 μl of a 10 μM dilution) were first denatured by incubation at 70°C for 5 min, after which the samples were snap-cooled on ice for 1 min. Samples were then supplemented with 30 U T4 RNA Ligase 1 (ssRNA Ligase), High Concentration (cat. M0437M, New England Biolabs), and ligation was performed in a final volume of 20 μl, at 25°C for 2 h, in the presence of 12.5% PEG-8000 and 1 mM ATP. 10 min before the end of the incubation, 20 μl Dynabeads™ MyOne™ Streptavidin T1 beads (cat. 65601, ThermoFisher Scientific) were aliquoted in a 2 mL tube, washed twice in 100 μl 2X Binding & Wash Buffer [10 mM Tris-HCl pH 7.5; 1 mM EDTA; 2 M NaCl], and then resuspended in 40 μl of the same buffer. RNA samples were then supplemented with 20 μl nuclease-free water to dilute the PEG, and then transferred to the washed beads. Samples were extensively vortexed, then incubated for 15 min at 22°C in a thermomixer, with constant shaking (1,000 RPM). Samples were then placed on the magnet, supernatant discarded, and beads were washed twice with 500 μl 1X Binding & Wash Buffer, by extensive vortexing. Two additional washes were then performed with 500 μl nuclease-free water, by incubating at 80°C for 2 min. We found this step to be critical when preparing libraries from DMS-treated samples, but not 2A3-treated samples, as DMS modifications introduce positive charges on the RNA that, due to the negative charge of the phosphate backbone, cause the RNA to aggregate. Heat denaturation at this stage allows washing away non-5′-cap-derived fragments. Ligated RNA fragments were then eluted by incubating the beads in 50 μl Formamide Elution Buffer [95% Formamide; 10 mM EDTA] at 95°C for 3 min, then cleaned up on Monarch® Spin RNA Cleanup columns. Eluted RNA fragments were then ligated to a 5′-pre-adenylated and C3 spacer 3′-blocked DNA 3′ adapter (rApp-AGATCGGAAGAGCACACGTCT-SpC3). RNA fragments and the adapter (1 μl of a 10 μM dilution) were first denatured by incubation at 70°C for 5 min, after which the samples were snap-cooled on ice for 1 min. Samples were then supplemented with 200 U T4 RNA Ligase 2, truncated KQ (cat. M0373L, New England Biolabs), and ligation was performed in a final volume of 20 μl, at 25°C for 2 h, in the presence of 12.5% PEG-8000. Samples were cleaned up on Monarch® Spin RNA Cleanup columns. For 2A3-treated samples RNA was eluted in 8 μl nuclease-free water, supplemented with 2 μl 10 μM RT primer and 1 μl 10 mM dNTPs, while for DMS-treated samples RNA was eluted in 9 μl nuclease-free water, supplemented with 2 μl 10 μM RT primer and 2 μl 10 mM dNTPs. RNA was incubated at 70°C for 5 min, and then snap-cooled on ice for 1 min. RT reactions were performed in a final volume of 20 μl. For 2A3-treated samples reactions were supplemented with 4 μl 5X RT Buffer [250 mM Tris-HCl pH 8.0; 375 mM KCl], 2 μl DTT 0.1 M, 20 U SUPERase•In™ RNase Inhibitor, 200 U SuperScript™ II Reverse Transcriptase (cat. 18064022, ThermoFisher Scientific), 6 mM final MnCl2 and incubated 1.5 h at 42°C, 10 min at 50°C, 10 min at 55°C, 10 min at 60°C and 15 min at 75°C. For DMS-treated samples reactions were supplemented with 4 μl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl; 15 mM MgCl2], 1 μl DTT 0.1 M, 20 U SUPERase•In™ RNase Inhibitor, 200 U TGIRT™-III Enzyme, and incubated 10 min at 42°C, 1 h at 57°C and 1 h at 60°C. The TGIRT™-III-RNA-cDNA complex was destroyed by addition of 1 μl 10 M NaOH, followed by incubation at 95°C for 3 min. Reactions were cleaned up on Monarch® Spin RNA Cleanup columns, using 1 volume RNA Binding Buffer and 1 volume 100% ethanol, to only recover fragments ≥ 200 nt. Barcoding was performed by PCR, using the NEBNext® Ultra™ II Q5® Master Mix (cat. M0544X, New England Biolabs), as per manufacturer instructions.

treatment_protocol: Dimethyl sulfate (DMS; cat. D186309, Merck) from a fresh 1:4 dilution in ethanol (~2.64 M) was directly added to the cells at a final concentration of 150 mM. Probing was conducted for 2 min at 37°C. Reactions were then quenched by addition of 1 volume 1 M DTT, after which cells were collected by centrifugation at 5,000g for 1 min.


title: Direct_Infection_WT_Naïve_3

description: Library name: NC_21_152


title: 5UTR-MaP of HEK293 cells under ATP-depleted conditions, replicate 1

description:
Library name: 5UTR-MaP_ATPdepleted_rep1
Instrument: MGI DNBSEQ-T7


title: Adult testis 33 (RNA-seq)

value: Adult


title: Direct_Infection_WT_Naïve_2

description: Library name: NC_21_151


title: Horse sperm 4 (RNA-seq)


title: set2.WT_rep1


title: set2.rrp4_rep1


title: Adult testis 20 (RNA-seq)


title: set2.WT_rep2


title: Direct_Infection_KO+AOX_Activated_3

description: Library name: NC_21_157

value: KO


title: set2.urt1_rep2


title: Horse sperm 1 (RNA-seq)


title: Direct_Infection_WT_Naïve_1

description: Library name: NC_21_150

value: Naïve


title: 5UTR-MaP of HEK293 cells under standard culture conditions, replicate 2

description:
Library name: 5UTR-MaP_Standard_rep2
Instrument: MGI DNBSEQ-T7

growth_protocol: Human HEK293 cells were cultured in high glucose DMEM medium (cat. L0104, Biowest), supplemented with 10% fetal bovine serum (FBS) (cat. H1138, Merck), 25 U/mL penicillin and 25 μg/mL streptomycin, at 37°C and 5% CO2.


title: set2.urt1_rep1


title: Adult testis 13 (RNA-seq)


title: Direct_Infection_WT_Activated_5

description: Library name: NC_21_149

value: WT


title: 5UTR-MaP of HEK293 cells under standard culture conditions, replicate 1

description:
Library name: 5UTR-MaP_Standard_rep1
Instrument: MGI DNBSEQ-T7


title: set1.urt1_rep3


title: Puber testis 19 (RNA-seq)

value: Puber


title: set2.WT_rep3


title: Direct_Infection_WT_Activated_4

description: Library name: NC_21_148

value: Activated


title: set1.urt1_rep2

value: inflorescences


title: NPY 3.(NPYtet/tet).507.male

city: Birmingham

first: Alex

middle: Dwight

last: Dawson

state: Alabama

address: 902, 14th st. South

department: Biology

institute: University Of Alabama At Birmingham

description:
Library name: Sample 8 NPY 508
NPY_507

data_processing:
Following seqencing, reads with adapter contamination, reads with ambigious nuceotides above 10% of the whole read, and reads with low quality nucleotides reaching more than 50% of the whole read were removed. Basecalling was performed using CASAVA base recognition
Alignment was performed using HISAT2 (2.0.5) with default perameters, quantification was done using FeatureCounts v1.5.0-p3 using default perameters. Differential analysis was performed using DEseq2 v.1.20.0 with filtering set at |log2(foldchange)| >1 & padj <=0.05
Assembly: mm39
Supplementary files format and content: gene_count.xls
Supplementary files format and content: gene_fkpm.xls

source_name: Whole skin

growth_protocol: Mice were reared at standard conditions and euthanized at 35 weeks for harvest of whole skin

extract_protocol:
Whole skin was pulverized using a Biopulverizer (Biospec #59012MS), followed by a short homogenization using a bead homogenizer (MP biomedicals). Homogenized skin was phase separated using chloroform and precipitated using ethanol. Precipiated RNA was purified using the qiagen RNeasy mini kit. RNA was packaged using dry ice and submitted to Novogene for RNA-sequencing
RNA with RIN values >7 were submitted to Novogene for library construction and sequencing .The following protocol was provided by Novogene "Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers."  Our library construction was performed using non-directional strands generated via second strand synthesis with dTTP. "For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification"

treatment_protocol: No biological treatment was applied, mice were aged to 35 weeks old

value: Whole skin

tag: Sex

value: male

value: (NPY tet/tet)

value: aged to 35 weeks old


title: NPY 2.(NPYtet/tet).506.male

description:
Library name: Sample 7 NPY 507
NPY_506


title: Puber testis 11 (RNA-seq)


title: set1.urt1_rep1


title: Direct_Infection_WT_Activated_3

description: Library name: NC_21_147


title: Puber testis 10 (RNA-seq)


title: NPY 1.(NPYtet/tet).505.male

description:
Library name: Sample 6 NPY 506
NPY_505


title: Direct_Infection_WT_Activated_2

description: Library name: NC_21_146


title: set1.heso1urt1_rep2


title: WT 5.(NPY+/+).504.male

description:
Library name: Sample 5 WT 505
WT_504


title: Direct_Infection_WT_Activated_1

description: Library name: NC_21_145


title: Pre-puber testis 14 (RNA-seq)

value: Pre-puber


title: Pre-puber testis 3b (RNA-seq)


title: WT 3.(NPY+/+).502.male

description:
Library name: Sample 3 WT 503
WT_502


title: set1.heso1urt1_rep1


title: WT 2.(NPY+/+).501.male

description:
Library name: Sample 2 WT 501
WT_501


title: Exp3_WT_MPP3_S11

city: Paris

first: Evelyne

last: LAURET

address: 22 rue Méchain

institute: INSERM

data_processing:
STAR Version 2.7.6a
STAR --genomeDir [/path/to/reference/index] --sjdbGTFfile [/path/to/reference/gtf] --runThreadN [number of threads] 20 --readFilesIn [all fastq.gz archives] --outFileNamePrefix [prefix for this sample] --twopassMode Basic --outReadsUnmapped None --sjdbOverhang [read size minus 1] 74 --outSAMtype BAM SortedByCoordinate --readFilesCommand zcat --outSAMunmapped Within --outFilterType BySJout --quantMode TranscriptomeSAM
rsem-calculate-expression -p 20 --paired-end --bam --no-bam-output /path/to/AlignedToTranscriptome/file /path/to/rsem-prepare-reference/files [sample-rsem-suffix]
R version 3.6.3 (2020-02-29) Platform: x86_64-pc-linux-gnu (64-bit) Running under: Ubuntu 18.04.5 LTS
factoextra_1.0.7, ggrepel_0.8.2, pheatmap_1.0.12, RColorBrewer_1.1-2, ggplot2_3.3.2, reshape_0.8.8, DESeq2_1.26.0, SummarizedExperiment_1.16.1, DelayedArray_0.12.3, BiocParallel_1.20.1, matrixStats_0.57.0, Biobase_2.46.0, GenomicRanges_1.38.0, GenomeInfoDb_1.22.1, IRanges_2.20.2, S4Vectors_0.24.4, BiocGenerics_0.32.0, readr_1.4.0,     tximport_1.14.2, biomaRt_2.47.3
Assembly: GRCm38 from Ensembl release 101
Supplementary files format and content: normalized matrix in a tab file

source_name: bone marrow

extract_protocol:
BM was harvested, red blood cells were lysed, lin- cells were enriched usig Dynabeads. The differents cell compartments (HSC/MPP1 or MPP3) were purified on an AriaIII cell sorter according to their cell surface markers. HSC/MPP1 : lin-Sca+kit+CD150+CD48- and MPP3 : lin-Sca+kit+CD150-CD48-CD135-
To construct the libraries, 10 ng of high-qualitytotal RNA sample (RIN >6.8) was processed using NEBNext low input RNA library prep kit (NEB) according to manufacturer instructions

value: bone marrow

value: HSPC


title: WT 4.(NPY+/+).503.male

description:
Library name: Sample 4 WT 504
WT_503


title: Infant testis 30 (RNA-seq)

value: Infant


title: Exp3_KO_MPP3_S12


title: set1.heso1_rep1


title: set1.WT_rep3

value: Col-0 WT


title: set1.heso1_rep2


title: Pre-puber testis 3a (RNA-seq)

value: Testis


title: Infant testis 31b (RNA-seq)


title: set1.WT_rep2


title: WT 1.(NPY+/+).500.male

description:
Library name: Sample 1 WT 500
WT_500


title: Exp3_KO_HSC_MPP1_S10


title: Central mCherry- hindlimb, H11^hZRS404G>A-mCherry, rep2

city: Irvine

first: Evgeny

last: Kvon

state: CA

address: 836 Health Sciences Road

institute: University of California, Irvine

description: Library name: Sample4

data_processing:
Paired-end reads were aligned to the mm10 reference genome using STAR, version 2.7.9a with default parameters
Expression values were counted using HTSeq and differential expression analysis performed using DESeq2, version 3.16
Assembly: mm10
Supplementary files format and content: csv file countains raw count values for each Sample

source_name: FVB

extract_protocol:
Hindlimb cells were dissected, dissociated, sorted, and then snap-frozen. RNA extraction was performed by RNeasy extraction kit.
cDNA library construction was performed by Novogene and pair-end sequenced to an average depth of 25M reads per sample.

value: FVB

value: Central

value: wild-type

tag: reporter


title: Exp3_WT_HSC_MPP1_S9

value: WT


title: Central mCherry- hindlimb, H11^hZRS404G>A-mCherry, rep1

description: Library name: Sample3


title: Exp2_KO_MPP3_S8

value: KO


title: P301S_hom_rep3

city: Bethesda

first: Kory

middle: R

last: Johnson

address: 10/3B01, 9000 Rockville Pike

department: DIR IT & Bioinformatics

institute: NINDS/NIH

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

source_name: spinal cord

extract_protocol:
RNA was harvested using Qiagen RNeasy Mini Kit. 500ng of total RNA was used for the construction of sequencing libraries.
Directional RNA Library prep kit was used in conjunction with the polyA mRNA workflow following the manufacturer’s standard protocol.

value: spinal cord

value: P301S homozygous


title: TRPML3, Starved 3

city: Delhi

first: Tapan

middle: Kumar

last: Nayak

address: IIT Delhi, Hauz Khas

department: KSBS

country: India

institute: Indian Institute of Technology Delhi

description:
Library name: TS3
Column name same as library name in Normcounts_all_withIDs.csv.gz and Rawcounts_all_withIDs.csv.gz files

data_processing:
Quality of raw fastq files was assessed with FastQC. Adapters were trimmed with fastp
Reads were mapped to reference genome (NCBI GRCh38) with HISAT2. Transcript assembly was performed with Stringtie. prepDE.py script was used to extract raw counts
raw counts were filtered and normalized with DESeq2
Assembly: NCBI GRCh38
Supplementary files format and content: Normalized counts of genes in all samples are provided in 'Normcounts_all_withIDs.csv.gz', a compressed CSV file. Genes with total counts<12 across all samples were omitted
Supplementary files format and content: Raw counts of genes in all samples are provided in 'Rawcounts_all_withIDs.csv.gz', a compressed CSV file

growth_protocol: HEK293T cells were maintained in DMEM/F12-Ham supplemented with 10% (v/v) Fetal Bovine Serum, 1.2g/L NaHCO3 and 1% (v/v) Penicillin-Streptomycin

extract_protocol:
Whole RNA from ~1x10E6 cells per sample was extracted using RNEasy Plus and Qiashredder kits (Qiagen). Total 12 samples were prepared (biological triplicate samples per condition)
500ng of RNA was used per sample for library preparation. polyA RNA was enriched. Libraries were prepared using KAPA hyper prep Kit

treatment_protocol: HEK cells were transfected with 1ug of control Vector/TRPML3 using Effectene (Qiagen). Prior to RNA extraction, transfected cells were either left in serum-fed (10%) conditions or serum-starved (0%) for 4hrs to induce autophagy. This resulted in 4 experimental conditions: Vector control fed (V), Vector control starved (VS), TRPML3 fed (T), and TRPML3 starved (TS)

value: Embryonic Kidney

tag: overexpression

tag: treatment

value: Serum-Starved


title: Exp2_WT_MPP3_S7


title: Anterior mCherry+ hindlimb, H11^hZRS404G>A-mCherry, rep2

description: Library name: Sample2

value: Anterior


title: P301S_hom_rep2


title: TRPML3, Starved 2

description:
Library name: TS2
Column name same as library name in Normcounts_all_withIDs.csv.gz and Rawcounts_all_withIDs.csv.gz files


title: Anterior mCherry+ hindlimb, H11^hZRS404G>A-mCherry, rep1

description: Library name: Sample1


title: TRPML3, Starved 1

description:
Library name: TS1
Column name same as library name in Normcounts_all_withIDs.csv.gz and Rawcounts_all_withIDs.csv.gz files


title: P301S_hom_rep1


title: Exp2_KO_HSC_MPP1_S6


title: Posterior hindlimb, ZIC3, rep2

description: Library name: Sample4

data_processing:
Fastq files were aligned to the mm10 genome using Bowtie2, version 2.5.1 with the following options: -local --very-sensitive- local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700
Samtools, version 1.15.1 was used to generate, sort, and index bam files, from which bigwig files were generated using deeptools, version 3.5.1
Assembly: mm10
Supplementary files format and content: bigwig, peaks.txt
Library strategy: CUT&Run

extract_protocol:
Hindlimb domains were dissected, dissociated into single cells, and bound to ConA beads to enable binding of ZIC3 antibody to the DNA followed by Mnase-based digestion of DNA.
Extracted DNA was constructed into sequencable libraries using Epicypher's CUT&RUN library kit. DNA was end-repaired, ligated to adapters, and uracils were selectively excised. Resulting DNA was indexed with i5/i7 primers before being sequenced for 100M reads on an Illumina NovaSeq 6000.

tag: antibody


title: Exp2_WT_HSC_MPP1_S5


title: P301S_het_rep3

value: P301S heterozygous


title: Exp1_WT_MPP3_S3


title: Anterior hindlimb, ZIC3, rep2

description: Library name: Sample2


title: set1.WT_rep1

value: inflorescences


title: P301S_het_rep2


title: TRPML3, Fed 3

description:
Library name: T3
Column name same as library name in Normcounts_all_withIDs.csv.gz and Rawcounts_all_withIDs.csv.gz files

value: Serum-Fed


title: Exp1_KO_MPP3_S4

value: bone marrow

value: HSPC


title: Posterior hindlimb, ZIC3, rep1

description: Library name: Sample3


title: Infant testis 31a (RNA-seq)

value: Infant


title: TRPML3, Fed 1

description:
Library name: T1
Column name same as library name in Normcounts_all_withIDs.csv.gz and Rawcounts_all_withIDs.csv.gz files


title: Anterior hindlimb, ZIC3, rep1

description: Library name: Sample1


title: TRPML3, Fed 2

description:
Library name: T2
Column name same as library name in Normcounts_all_withIDs.csv.gz and Rawcounts_all_withIDs.csv.gz files


title: Exp1_KO_HSC_MPP1_S2


title: Posterior hindlimb, wild-type-hZRS404G>A, H3K27ac, rep2

description: Library name: Sample18

data_processing:
Paired-end reads were aligned to the mm10 reference genome using Bowtie2 with the following parameters: -end-to-end --very-sensitive --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 70.
Samtools, version 1.15.1 was used to generate, sort and index bam files, from which bigwig files were generated using deeptools, version 3.5.1.
Assembly: Wild-type samples = mm10 reference genome Wild-type + reporter samples = A custom mm10 genome (mm10 reference and an extra chromosome for the hZRS transgene reporter). Knock-in + reporter samples = A custom mm10 genome (mm10 with variant mouse ZRS allele replacing the wild type ZRS at chromosome 5 and an extra chrosome for the hZRS transgene reporter)
Supplementary files format and content: bigwig
Library strategy: CUT&Tag

extract_protocol:
Hindlimb cells were dissected, dissociated, sorted, and then immobilized onto ConA-magnetic beads. Following the CUT&Tag-direct protocol (version 4), bead-bound cells were incubated with primary and secondary antibodies then Tn5 for transposition. DNA libraries were then barcoded and indexed before being sequenced.
cDNA library construction was performed by Novogene and pair-end sequenced to an average depth of 25M reads per sample.

tag: batch


title: Exp1_WT_HSC_MPP1_S1


title: Vector control, Starved 3

description:
Library name: VS3
Column name same as library name in Normcounts_all_withIDs.csv.gz and Rawcounts_all_withIDs.csv.gz files

value: Control Vector


title: WT_rep2


title: WT_rep3


title: Vector control, Starved 2

description:
Library name: VS2
Column name same as library name in Normcounts_all_withIDs.csv.gz and Rawcounts_all_withIDs.csv.gz files


title: KP1-59185

city: Barcelona

first: Júlia

last: Perera-Bel

address: Doctor Aiguader, 88

department: MARData-BU

institute: Hospital del Mar Research Institute

data_processing:
Reads were mapped with STAR software (v.2.7.1a) against the moouse reference genome mm10.
The table of counts was obtained with featureCounts function in the package subread (v.1.6.4).
Counts were normalized with log2 counts per million (CPM) with the edgeR package with a prior.count=3. Genes with more than 10 counts in at least 2 samples were kept.
Assembly: mm10
Supplementary files format and content: counts

growth_protocol: Mouse SCLC cells (KP1 and 5B) (1 million) were collected, resuspended in sterile PBS with Matrigel (354234, Corning, 1:1) and injected into the right flank of 6 to 12-week old B6129SF1/J mice.

extract_protocol:
Mouse tumor fragments were immediately frozen in liquid nitrogen after isolation. RNA was isolated using the RNeasy Mini Kit (Qiagen), and RNA integrity (RIN) was determined by Bioanalyzer (Agilent). All samples exhibited acceptable quality (RIN > 7) for RNAseq with polyA capture.
NGS libraries, prepared according to the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolab) (version 4.0, 4/21) manual by MARGenomics Core Facility at Hospital del Mar Research Institute, yielded a final library size of ~40M counts per sample. Sequencing was conducted on the Illumina Nextseq 500 High-output platform with 2x75bp read length at the UPF Genomics Core Facility.

treatment_protocol: Group allocation was randomized using RandoMice software (https://doi.org/10.1371/journal.pone.0237096) three days after tumor cell inoculation. Mice were distributed into three treatment arms: cisplatin (Cis), cisplatin plus anti-PD-L1 (Cis+D-D), or cisplatin plus anti-PD-L1 and savolitinib (Cis+S+D-S+D).  Schedule administration was the following a) cisplatin: 4 mg/kg i.p. on days 0 and 7 of treatment; b) anti-mouse PD-L1 [clone 80] or NIP228 mIgG1 D265A isotype: 10 mg/kg i.p. twice a week; c) savolitinib: 30 mg/kg orally daily.

value: Cis+S+D-S+D

tag: mouse model

tag: strain


title: Posterior hindlimb, wild-type-hZRS404G>A, H3K27ac, rep1

description: Library name: Sample17

value: FVB


title: WT_rep1


title: Vector control, Starved 1

description:
Library name: VS1
Column name same as library name in Normcounts_all_withIDs.csv.gz and Rawcounts_all_withIDs.csv.gz files


title: KP1-57453

value: Cis


title: P301S_het_rep1


title: Central hindlimb, wild-type-hZRS404G>A, H3K27ac, rep2

description: Library name: Sample16

value: wild-type


title: KP1-57432

value: Cis+D-D


title: Vector control, Fed 3

description:
Library name: V3
Column name same as library name in Normcounts_all_withIDs.csv.gz and Rawcounts_all_withIDs.csv.gz files


title: Vector control, Fed 1

description:
Library name: V1
Column name same as library name in Normcounts_all_withIDs.csv.gz and Rawcounts_all_withIDs.csv.gz files


title: Central hindlimb, wild-type-hZRS404G>A, H3K27ac, rep1

description: Library name: Sample15


title: KP1-57245


title: 4741_Control_2_RIBOseq

city: Rochester

first: Yi

last: Pan

state: NY

address: 601 Elmwood Ave

department: Department of Biochemistry & Biophysics

institute: University of Rochester Medical Center

description:
Library name: Ribo-2R
Column name in AllGeneExpressionsTable.csv: Ribo-2R

data_processing:
Sequence elements added during library construction were trimmed with Cutadapt, and reads shorter than 15 nt after trimming were discarded.
Trimmed reads were then mapped to the S. cerevisiae genome reference R64-3-1 and the mCherry reporter cassette sequence by STAR.
RIBO-seq metagene statistical analysis was performed for quality control, 3-periodicity in coverage, and P-site offset determination.
Assembly: R64-3-1
Supplementary files format and content: A csv file including the raw read counts and calculated rpkm
Library strategy: Ribo-Seq

organism: Saccharomyces cerevisiae

source_name: haploid

growth_protocol: Yeast cells were grown in synthetic dextrose -leucine -uracil liquid medium

extract_protocol:
For RIBO-seq, lysates were prepared according to the procedure and were digested with RNase I for 45 min at room temperature as previously described in McGlincy and Ingolia, 2017. Digestion was stopped with SuperaseIN and monosomes purified by size exclusion chromatography on MicroSpin S-400 HR columns (GE Healthcare) as described in Aeschimann et al., 2015. Size selection of footprints with length 25-33 nt was performed by electrophoresis on 15% TBE-urea gels. For RNA-seq, total RNA was purified from an aliquot of cell lysate and rRNA depleted using NEB NEBNext rRNA depletion kit v2 following manufacturer instructions. mRNA fragmentation was conducted for 20 min at 94°C to generate RNA fragments of sizes similar to those of the ribosome footprints.
Illumina ready RIBO-seq libraries were prepared using a SMARTer smRNA-seq kit (TakaraBio).

value: haploid

value: control gRNAs transformed


title: 4741_Treated_2_RNAseq

description:
Library name: RNA-4R
Column name in AllGeneExpressionsTable.csv: RNA-4R

data_processing:
Sequence elements added during library construction were trimmed with Cutadapt, and reads shorter than 15 nt after trimming were discarded.
Trimmed reads were then mapped to the S. cerevisiae genome reference R64-3-1 and the mCherry reporter cassette sequence by STAR.
RIBO-seq metagene statistical analysis was performed for quality control, 3-periodicity in coverage, and P-site offset determination.
Assembly: R64-3-1
Supplementary files format and content: A csv file including the raw read counts and calculated rpkm

value: targeted gRNAs transformed


title: KP1-57414


title: 4741_Treated_2_RIBOseq

description:
Library name: Ribo-4R
Column name in AllGeneExpressionsTable.csv: Ribo-4R


title: Anterior hindlimb, wild-type-hZRS404G>A, H3K27ac, rep1

description: Library name: Sample13


title: 4741_Treated_1_RIBOseq

description:
Library name: Ribo-3R
Column name in AllGeneExpressionsTable.csv: Ribo-3R


title: Vector control, Fed 2

description:
Library name: V2
Column name same as library name in Normcounts_all_withIDs.csv.gz and Rawcounts_all_withIDs.csv.gz files

value: Embryonic Kidney


title: Posterior hindlimb, wild-type-hZRS404G>A, H3K4me1, rep1

description: Library name: Sample11


title: KP1-57243


title: Anterior hindlimb, wild-type-hZRS404G>A, H3K27ac, rep2

description: Library name: Sample14


title: HHL5 cells treated with OA/PA for 24 h, replicate 2, input

city: Shanghai

first: Jin

last: Hou

address: 800 Xiangyin Road

institute: Second Military Medical University

description:
Library name: O2_Input
Methylated_RNA_sites.mRNA.xlsx, sheet2

data_processing:
Raw reads (Raw Data) are generated after sequencing on a sequencer, image analysis, base identification and QC. Q30 was first used for quality control. Then, cutadapt software (v1.9.3) was used to remove splice information and remove low quality reads to obtain high quality clean reads. The clean reads were matched to the reference genome using Hisat2 (v2.0.4). Methylated genes in each sample were then identified using MACS (1.4.2). Differential methylation gene identification was performed using diffReps (1.55.6). The peaks located mRNA were screened using our own programs and annotated accordingly. GO and Pathway analyses were performed for differentially methylatedgene.
Assembly: hg38
Supplementary files format and content: methylation sites, methylated RNA and enrichment information
Library strategy: MeRIP-Seq

source_name: liver

extract_protocol:
RNA was randomly fragmented to ~200 nt using NEBNext® Magnesium RNA Fragmentation Module. Protein A/G beads were conjugated with the m5C antibody by rotating at room temperature for 1 h. The RNA fragments were then incubated with the bead-linked antibodies and rotated at 4 °C for 4 h. Following incubation, the RNA/antibody complexes were washed for several times, and the captured RNA was eluted from the complexes and purified for sequencing or qRT-PCR. RNA libraries for the IP samples were constructed with GenSeq® Low Input Whole RNA Library Prep Kit (GenSeq, Inc.) following the manufacturer’s instructions. The quality of the libraries was qualified using Agilent 2100 bioanalyzer (Agilent), and the libraries were subsequently sequenced.
The m5C-MeRIP-seq service and subsequent bioinformatic analysis were conducted by CloudSeq Biotech Inc. (Shanghai, China).

value: liver

value: HHL5 cell line

value: embryonic liver cells

value: WT

value: OA/PA 24h


title: 4741_Control_1_RIBOseq

description:
Library name: Ribo-1R
Column name in AllGeneExpressionsTable.csv: Ribo-1R


title: HHL5 cells treated with OA/PA for 24 h, replicate 2, m5C-MeRIP-seq

description:
Library name: O2_IP
Methylated_RNA_sites.mRNA.xlsx, sheet2


title: HHL5 cells treated with OA/PA for 24 h, replicate 1, input

description:
Library name: O1_Input
Methylated_RNA_sites.mRNA.xlsx, sheet1


title: 4741_Treated_1_RNAseq

description:
Library name: RNA-3R
Column name in AllGeneExpressionsTable.csv: RNA-3R


title: HHL5 cells treated with OA/PA for 24 h, replicate 1, m5C-MeRIP-seq

description:
Library name: O1_IP
Methylated_RNA_sites.mRNA.xlsx, sheet1


title: Posterior hindlimb, wild-type-hZRS404G>A, H3K4me1, rep2

description: Library name: Sample12


title: 5B-6484


title: 5B-6483


title: Central hindlimb, wild-type-hZRS404G>A, H3K4me1, rep2

description: Library name: Sample10


title: 4741_Control_2_RNAseq

description:
Library name: RNA-2R
Column name in AllGeneExpressionsTable.csv: RNA-2R


title: 4741_Control_1_RNAseq

description:
Library name: RNA-1R
Column name in AllGeneExpressionsTable.csv: RNA-1R


title: Central hindlimb, wild-type-hZRS404G>A, H3K4me1, rep1

description: Library name: Sample9


title: iCell-RPE, AgeD, experiment 4

city: Aachen

first: Teodora

last: Piskova

address: Wendlingweg 2

country: Germany

institute: RWTH Aachen University

description:
Library name: AgeD-iRPE 4
Sample name in salmon.merged.gene_counts.csv: DE55NGSUKBR144392

data_processing:
The raw data was processed using the nf-core/rnaseq v3.12.0 (https://doi.org/10.5281/zenodo.1400710)
Sequence reads were quality checked using FastQC before and after adapter trimming.
Adapter sequences and low-quality bases were trimmed using Cutadapt (3' adapter: AAAAAAAA).
Trimmed reads were aligned to the GENCODE GRCh38 v44 genome using STAR (key parameters: max intron size 1,000,000; max mismatches 10%; max multimaps 20).
Alignment quality was assessed using Samtools, RSeQC, and QualiMap.
Gene-level quantification was performed using featureCounts (grouped by gene_type).
Assembly: GRCh38 (GENCODE v44)
Supplementary files format and content: .csv file contains raw gene counts for all samples

source_name: iCell-RPE (Fujifilm 01279)

growth_protocol: HiPSCs-derived RPE cells (Fujifilm Cellular Dynamics, R1102) were seeded on 4 kPa polyacrylamide hydrogels with a basement membrane-like coating and were grown for 10 days in a medium recommended by the manufacturer.

extract_protocol:
RNA isolation was performed using the RNeasy Micro RNA isolation kit (Qiagen, 74004) with DNase treatment. 100 ng RNA was used for the library preparation.
RNA libraries for RNAseq were prepared using the QuantSeq 3’-mRNA Library Prep/ Lexogen enrichment.

treatment_protocol: On day 10 after seeding, AAV5 were used at 7x106 infectious units per cell for 24 h to transduce the cells with FKBP-casp8 containing plasmid (VectorBuilder, VB230127-1043eaq) in addition to a non-transduced control. On day 15, FKBP-casp8 was dimerised by addition of 100 nM AP20187 in medium (Sigma Aldrich, SML2838) for 24 h. Paired non-transduced cells  were also subjected to the AP20187 treatment. The RNA isolation was performed on day 20, or four days after apoptosis treatment finalisation.

value: iCell-RPE (Fujifilm 01279)

value: retinal pigment epithelial cells

value: apoptosis via 100 nM AP20187


title: 5B-5833

value: Cis+S+D-S+D


title: YG_yfeCKO_24hr_rep2

city: La Jolla

first: Heera

last: Bajpe

address: 9500 Gilman Dr.

institute: UC San Diego

description: Library name: YG_yfeCKO_24hr_2

data_processing:
Raw sequencing reads were mapped to the reference genome using bowtie v1.1.2 with parameters -X 1000 -3 3 -n 2 -S
Read counts were generated using featureCounts
Assembly: NC_000913.2
Supplementary files format and content: Comma-delimited text files containing TPM values for each sample

organism: Escherichia coli str. K-12 substr. MG1655

source_name: K-12 substr. MG1655

growth_protocol: Glycerol stocks of E. coli strains were inoculated into M9 minimal media with the glucose M9 minimal medium for each candidate TF. The concentration of carbon sources was 0.2% (w/v). M9 minimal media was also supplemented with 1 ml trace element solution (100X). The culture was incubated at 37 ℃ overnight with agitation, and then was used to inoculate the fresh media (initial OD600 = 0.05). The fresh culture was incubated at 37 ℃ with agitation for additional 24 hrs.

extract_protocol:
Three milliliters of cells from mid-log phase culture were mixed with 6 ml RNAprotect Bacteria Reagent (Qiagen), mixed immediately by vortexing for 5 seconds, incubated for 5 minutes at room temperature, and then centrifuged at 5000g for 10 minutes. The supernatant was decanted and any residual supernatant was removed, then samples were frozen at -80℃. Next, RNA was harvested using RNeasy Plus Mini kit (Qiagen) in accordance with the manufacturer’s instruction. Illumina Ribo-Zero rRNA removal kit (bacteria) (MRZMB126) was used with 1 ug of total RNA for the removal of rRNA procedure. Then KAPA RNA HyperPrep kits(Cat.No.08098107702) was used with rRNA-depleted RNA samples for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: 24 hours incubation for the strains

value: K-12 substr. MG1655

value: yfeC deletion

value: 24 hours incubation


title: iCell-RPE, AgeD, experiment 3

description:
Library name: AgeD-iRPE 3
Sample name in salmon.merged.gene_counts.csv: DE39NGSUKBR144389


title: Anterior hindlimb, wild-type-hZRS404G>A, H3K4me1, rep2

description: Library name: Sample8

value: FVB

value: wild-type


title: 5B-6485


title: Anterior hindlimb, wild-type-hZRS404G>A, H3K4me1, rep1

description: Library name: Sample7


title: 5B-2975

value: Cis+D-D


title: iCell-RPE, AgeD, experiment 1

description:
Library name: AgeD-iRPE 1
Sample name in salmon.merged.gene_counts.csv: DE07NGSUKBR144383


title: 5B-4402


title: YG_MG1655_24hr_rep3

description: Library name: YG_MG1655_24hr_3


title: 5B-5275

value: Cis


title: YG_MG1655_24hr_rep2

description: Library name: YG_MG1655_24hr_2


title: Posterior hindlimb, wild-type-hZRS404G>A, ATAC, rep1

description: Library name: Sample5

data_processing:
Paired-end reads were first trimmed using Fastp, version 0.23.2 and then mapped using Bowtie2, version 2.5.1 with the following parameters: --local --very-sensitive --no-mixed --no-discordant -I 25 -X 700.
Bam files were sorted and indexed using samtools, version 1.15.1, and converted to bigwig files for genome visualization using deeptools, version 3.5.1
Assembly: Wild-type + reporter samples = A custom mm10 genome (mm10 reference and an extra chromosome for the hZRS transgene reporter). Knock-in + reporter samples = A custom mm10 genome (mm10 with variant mouse ZRS allele replacing the wild type ZRS at chromosome 5 and an extra chrosome for the hZRS transgene reporter)
Supplementary files format and content: bigwig

extract_protocol:
Hindlimb cells were dissected, dissociated, sorted, and then lysed into nuclei. Nuclei were then incubated with Tn5 for the transposition reaction.
Transposased DNA was extracted and then indexed before being sequenced for 50M reads.


title: Central hindlimb, wild-type-hZRS404G>A, ATAC, rep2

description: Library name: Sample4


title: Central hindlimb, wild-type-hZRS404G>A, ATAC, rep1

description: Library name: Sample3


title: 5B-2974


title: iCell-RPE, Ctrl, experiment 4

description:
Library name: iRPE 4
Sample name in salmon.merged.gene_counts.csv: DE12NGSUKBR144390


title: Posterior hindlimb, wild-type-hZRS404G>A, ATAC, rep2

description: Library name: Sample6


title: Anterior hindlimb, wild-type-hZRS404G>A, ATAC, rep2

description: Library name: Sample2