title: A549 expressing FTLΔCC, rep2

city: Shanghai

first: Xiaohua

last: Hu

address: 2005 Songhu Rd.

institute: Fudan University

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence.
The expression of the transcript was calculated by FPKM (Fragments Per Kilobase of exon model per Million mapped reads).
Assembly: hg38
Supplementary files format and content: tab-delimited text files including FPKM values for each sample

source_name: lung

growth_protocol: A549 cells were cultured in 1640 and supplemented with 1× penicillin / streptomycin (Gibco), 10% FBS (HyClone), and 6 mmol/L L-glutamine (Gibco) and maintained at 37°C in 5% CO2.

extract_protocol:
Total RNA from the cells was extracted using TRIzol (Life Technologies), followed by purification using a RNeasy Mini Kit (Qiagen).
Illumina TruSeq RNA Sample Prep Kit was used with 1 ug of total RNA for the construction of sequencing libraries.

treatment_protocol: Cells were transfected in a 24-well plate with 500 ng of plasmid DNA using PolyJet (SignaGen Laboratories) or Lipofectamine 3000 reagent (Invitrogen) per the manufacturer’s instructions.

value: lung

value: cancer cell

value: FTLdeltaCC

value: deprived of serum for 2h


title: Linf_IL15_CCL20_11

status: Public on Aug 11 2026

type: RNA

city: Sao Paulo

first: Patricia

last: Severino

state: Sao Paulo

address: Rua Comendador Elias Jafet, 755

department: Albert Einstein Research and Education Institute

country: Brazil

institute: Hospital Israelita Albert Einstein

description: Gene expression data

hyb_protocol: 5.5 μg of the fragmented and labeled cDNA was hybridized to Human Clariom S array for 16 h at 45°C and 60 rpm in an Affymetrix Hybridization Oven. Arrays were washed and stained in an Affymetrix Fluidics Station 450 according to Affymettix protocol.

scan_protocol: The arrays were scanned using the Affymetrix Gene Chip Scanner 3000 7G and signal intensity files were generated by Affymetrix GeneChip Command Console Software (AGCC, Thermo Fisher Scientific).

data_processing: Array data quality analysis, normalization (RMA+DAGB) and statistical tests for differential expression (eBayes) were carried out with the Transcriptome Analysis Console software (TAC 4.0.1)

label: Biotin

molecule: total RNA

organism: Homo sapiens

label_protocol: labelled cRNAwas prepared according to the GeneChip Whole Transcript Expression Arrays protocol (Affymetrix, Thermo Fisher Scientific)

extract_protocol:
PBMC Isolation Method: Ficoll-Paque density gradient
T Lymphocyte Isolation: Magnetic separation (Anti-CD3)
RNeasy® Mini kit

tag: Sex

value: Fem

tag: age (years)

tag: health status

value: Healthy

tag: tissue

value: Whole Blood - PBMC


title: mPDAC, control, rep1

type: SRA

city: Houston

first: Ronald

middle: J

last: Parchem

state: TX

address: 1 Baylor Plaza

department: Molecular and Cellular Biology

country: USA

institute: Baylor College of Medicine

description: mRNA-seq

library_source: transcriptomic

data_processing:
Reads were trimmed with cutadapt, and aligned to mm10 with STAR
Assembly: mm10, hg38
Supplementary files format and content: Common-separated values of raw read counts of mPDAC
Supplementary files format and content: Common-separated values of raw read counts of MiaPaCa2 cells

organism: Mus musculus

source_name: pancreas

growth_protocol: 10% FBS+1xglutamax in DMEM/F12

extract_protocol:
Trizol total RNA extraction
illumina Truseq stranded mRNA

value: pancreas

tag: cell line

value: mPDAC

tag: cell type

value: Pancreatic cancer cell

tag: genotype

value: KrasWT, Ago2WT


title: CLR316

description: Library name: RPS2_WT_IP2

data_processing:
UMI removal and cut adapt:  umi_tools extract   --random-seed 1   --bc-pattern NNNNNNNNNN   --stdin 23145FL-08-01-37_S37_L001_R1_001.fastq.gz   --log /dev/null | cutadapt   -j 8   --match-read-wildcards   --times 1   -e 0.1   --quality-cutoff 6   -m 18   -a a_adapters.fasta   -O 1   -   --report minimal   2> CLR314.cut.adapt.log | cutadapt   -j 8  --match-read-wildcards   --times 1   -e 0.1   --quality-cutoff 6   -m 18   -a a_adapters.fasta   -O 5   -   -o CLR314.trim.gz   --report minimal   >>CLR314.cut.adapt.log
Repeat mapping (takes adapter-trimmed fastq files, and maps to a repeat database to filter repeat elements prior to genome mapping:     STAR     --runMode alignReads     --runThreadN 8     --alignEndsType EndToEnd     --genomeDir mus_musculus_repbase_v2     --genomeLoad NoSharedMemory     --outBAMcompression 10     --outFileNamePrefix CLR314.repeat.map     --outFilterMultimapNmax 100     --outFilterMultimapScoreRange 1     --outFilterScoreMin 10     --outFilterType BySJout     --outReadsUnmapped Fastx    --outSAMattrRGline ID:foo     --outSAMattributes All     --outSAMmode Full     --outSAMtype BAM Unsorted     --outSAMunmapped None     --outStd Log     --readFilesCommand zcat     --readFilesIn CLR314.trim.gz
Unique genomic mapping (takes repeat-removed reads and maps to the reference genome: STAR   --runMode alignReads   --runThreadN 8   --alignEndsType EndToEnd   --genomeDir /storage/vannostrand/genomes/mm10/star_2_4_0i_gencode15_sjdb --genomeLoad NoSharedMemory   --outBAMcompression 10   --outFileNamePrefix CLR314.genome.map   --outFilterMultimapNmax 1   --outFilterMultimapScoreRange 1   --outFilterScoreMin 10   --outFilterType BySJout   --outReadsUnmapped Fastx   --outSAMattrRGline ID:foo   --outSAMattributes All   --outSAMmode Full   --outSAMtype BAM Unsorted   --outSAMunmapped None   --outStd Log   --readFilesIn CLR314.repeat.mapUnmapped.out.mate1
Sort mapped reads: samtools sort -@ 8 -m 2G -o CLR314.genome.map.bam *Aligned.out.bam
Remove PCR duplicate reads: samtools index CLR314.genome.map.bam umi_tools dedup   --random-seed 1   --stdin CLR314.genome.map.bam   --method unique   --stdout CLR314.bam
Make bigwigs: samtools index CLR314.bam b2bw CLR314.bam  --cpus 1
Get per-gene read counts for each dataset: featureCounts -a gencode.vM20.chr_patch_hapl_scaff.annotation.gtf -s 1 -T 8 -o WT_KRAS.feature.count CLR314.bam CLR316.bam CLR315.bam CLR317.bam
Get the Protein coding only regions of the featurecounts : perl pull_proteincodinggenes.pl WT_KRAS.feature.count.tsv mm10
Quantify translation efficiency with deseq2: deseq WT_KRAS.feature.count.tsv.proteincodingonly.txt  WT_KRAS.experiment.design.tsv WT_KRAS.feature.count.tsv.proteincodingonly.deseq.txt
Assembly: mm10/GRCm38
Supplementary files format and content: *.bw files are standard genome coverage bigwig files containing read density for each genomic position *.feature.count.tsv.proteincodingonly.deseq.txt files contain per-gene read counts
Library strategy: Ribo-Seq

source_name: WT Mouse pancreatic cancer cells

growth_protocol: 10% FBS +1xglutamax in DMEM/F12

extract_protocol:
Ribosome-associated RNAs were quantified with Ribo-eCLIP (Li. YH, Starner AS, et al. In preparation), a modified variation of the previously described eCLIP protocol (Blue, SM. et al, Nat Protocols 2022) to profile RPS2-associated RNA. Briefly, 20 million mPDAC iKRASG12D cells with constant and no dox were UV crosslinked (254 nM, 400 mJ/cm2), snap frozen, and lysed as previously described.
Limited RNA fragmentation was performed, 2% of the sample was removed for total RNA isolation, and the remainder was subjected to immunoprecipitation of RPS2-bound RNA with anti-RPS2 antibody (A303-794A, Fortis Life Sciences). Dephosphorylation, RNA adapter ligation, SDS-PAGE electrophoresis and nitrocellulose membrane transfer, RNA isolation, reverse transcription, and PCR amplification were performed as previously described (Blue, SM. et al, Nat Protocols 2022). After pooling, ribosomal RNA-containing fragments were removed by CRISPR/Cas9 depletion (CRISPRClean Ribodepletion, Jumpcode Genomics). Samples were sequenced on the Illumina Novaseq X Plus platform.

treatment_protocol: Ribo eCLIP

value: WT Mouse pancreatic cancer cells

tag: treatment

value: IP


title: A549 expressing FTLΔCC, rep1

country: China


title: KrasG12D,chase12h,rep3

description: SLAM-seq

data_processing:
Slamdunk
Assembly: mm10
Supplementary files format and content: Tab-separated values for total read count and T>C read count for each sample
Library strategy: SLAM-seq

molecule: polyA RNA

extract_protocol:
Trizol total RNA extraction
QuantSeq 3’ mRNA-Seq V2 Library Prep Kit FWD with UDI set B1 by Lexogen

value: pancreatic cancer cell

value: DMSO, s4U, doxycycline


title: CLR315

description: Library name: RPS2_WT_IN1

data_processing:
UMI removal and cut adapt:  umi_tools extract   --random-seed 1   --bc-pattern NNNNNNNNNN   --stdin 23145FL-08-01-37_S37_L001_R1_001.fastq.gz   --log /dev/null | cutadapt   -j 8   --match-read-wildcards   --times 1   -e 0.1   --quality-cutoff 6   -m 18   -a a_adapters.fasta   -O 1   -   --report minimal   2> CLR314.cut.adapt.log | cutadapt   -j 8  --match-read-wildcards   --times 1   -e 0.1   --quality-cutoff 6   -m 18   -a a_adapters.fasta   -O 5   -   -o CLR314.trim.gz   --report minimal   >>CLR314.cut.adapt.log
Repeat mapping (takes adapter-trimmed fastq files, and maps to a repeat database to filter repeat elements prior to genome mapping:     STAR     --runMode alignReads     --runThreadN 8     --alignEndsType EndToEnd     --genomeDir mus_musculus_repbase_v2     --genomeLoad NoSharedMemory     --outBAMcompression 10     --outFileNamePrefix CLR314.repeat.map     --outFilterMultimapNmax 100     --outFilterMultimapScoreRange 1     --outFilterScoreMin 10     --outFilterType BySJout     --outReadsUnmapped Fastx    --outSAMattrRGline ID:foo     --outSAMattributes All     --outSAMmode Full     --outSAMtype BAM Unsorted     --outSAMunmapped None     --outStd Log     --readFilesCommand zcat     --readFilesIn CLR314.trim.gz
Unique genomic mapping (takes repeat-removed reads and maps to the reference genome: STAR   --runMode alignReads   --runThreadN 8   --alignEndsType EndToEnd   --genomeDir /storage/vannostrand/genomes/mm10/star_2_4_0i_gencode15_sjdb --genomeLoad NoSharedMemory   --outBAMcompression 10   --outFileNamePrefix CLR314.genome.map   --outFilterMultimapNmax 1   --outFilterMultimapScoreRange 1   --outFilterScoreMin 10   --outFilterType BySJout   --outReadsUnmapped Fastx   --outSAMattrRGline ID:foo   --outSAMattributes All   --outSAMmode Full   --outSAMtype BAM Unsorted   --outSAMunmapped None   --outStd Log   --readFilesIn CLR314.repeat.mapUnmapped.out.mate1
Sort mapped reads: samtools sort -@ 8 -m 2G -o CLR314.genome.map.bam *Aligned.out.bam
Remove PCR duplicate reads: samtools index CLR314.genome.map.bam umi_tools dedup   --random-seed 1   --stdin CLR314.genome.map.bam   --method unique   --stdout CLR314.bam
Make bigwigs: samtools index CLR314.bam b2bw CLR314.bam  --cpus 1
Get per-gene read counts for each dataset: featureCounts -a gencode.vM20.chr_patch_hapl_scaff.annotation.gtf -s 1 -T 8 -o WT_KRAS.feature.count CLR314.bam CLR316.bam CLR315.bam CLR317.bam
Get the Protein coding only regions of the featurecounts : perl pull_proteincodinggenes.pl WT_KRAS.feature.count.tsv mm10
Quantify translation efficiency with deseq2: deseq WT_KRAS.feature.count.tsv.proteincodingonly.txt  WT_KRAS.experiment.design.tsv WT_KRAS.feature.count.tsv.proteincodingonly.deseq.txt
Assembly: mm10/GRCm38
Supplementary files format and content: *.bw files are standard genome coverage bigwig files containing read density for each genomic position *.feature.count.tsv.proteincodingonly.deseq.txt files contain per-gene read counts

value: input


title: Linf_IL15_11

description: Gene expression data


title: Linf_Control_11

description: Gene expression data

source_name: Control


title: A549 expressing FTL, rep2

value: FTL


title: CLR314

description: Library name: RPS2_WT_IP1


title: KrasG12D,chase12h,rep2

description: SLAM-seq


title: not4abc_AZD_3

city: Birmimngham

first: Daniel

middle: J.

last: Gibbs

address: School of Biosciences,University of Birmingham

department: School of Biosciences

country: United Kingdom

institute: University of Birmingham

description:
Library name: Sample 12
E3_n4abc_azd

data_processing:
Data processing, mapping and quantification of gene expression level. The resulting paired-end sequencing data (raw reads) were first processed through in-house perl scripts. In short, low quality and poly-N reads were filtered and adapter sequences were trimmed from each read pair (trimmed reads).
Trimmed reads were then aligned to the indexed A.thaliana genome (TAIR10) using Hisat2 v2.0.5
Mapped read counts to each locus were quantified using FeatureCounts v1.5.0- p3. Gene expression levels were then estimated by calculating the FPKM (Fragments Per Kilobase of transcript per Million mapped reads) for each gene
Differential expression analysis between two conditions (with three biological replicates per condition) was conducted using the DESeq2 R package (v1.20.0)
Assembly: Trimmed reads were then aligned to the indexed A.thaliana genome (TAIR10) using Hisat2 v2.0.5

organism: Arabidopsis thaliana

source_name: 12-day-old seedlings

growth_protocol: RNA was extracted from biological triplicates of pooled 12-day-old seedlings grown vertically on ½ MS plates

extract_protocol:
total RNA was extracted using the RNeasy Plant Mini Kit (QIAGEN) following the manufacturer’s protocol. On column DNAse I treatment was performed using the Qiagen DNAse I Set (50) kit according to manufactures protocol. High-quality RNA was subsequently eluted in RNase-free water or Elution Buffer (EB
mRNA was purified from total RNA using poly-T oligo attached magnetic beads. After fragmentation, the first cDNA strand was synthesized using random hexamer primers, followed by the second cDNA strand synthesis using either dUTP for direction library or dTTP for non-directional library

treatment_protocol: Seedlings were placed in wells with liquid ½ MS media containing the appropriate concentration of the chemical. After the indicated incubation time, the seedlings were taken from the liquid treatments for further analyses

value: 12-day-old seedlings

value: T-DNA insertion line

value: TOR inhibitor (AZD8055)


title: KrasG12D,chase12h,rep1

description: SLAM-seq


title: CLR328

description: Library name: AGO_KRAS_IP2

data_processing:
Remove 10nt UMI from 5' end of read: umi_tools extract --random-seed 1 --bc-pattern NNNNNNNNNN --stdin /storage/vannostrand/path/to/clip1.fastq.gz
First adapter trimming: cutadapt -j 8 --match-read-wildcards --times 3 -e 0.1 --quality-cutoff 6 -m 18 -a file:/storage/vannostrand/software/eclip/data/se.2.round.adapters.fasta -O 1 - --report minimal
Second adapter trimming (removes additional adapter-concatamers): cutadapt   -j 8  --match-read-wildcards   --times 1   -e 0.1   --quality-cutoff 6   -m 18   -a $adapter_file   -O 5   -   -o ${x}.trim.gz
Repeat mapping (takes adapter-trimmed fastq files, and maps to a repeat database to filter repeat elements prior to genome mapping and peak calling, in order to reduce peak artifacts): STAR --runMode alignReads --runThreadN 8 --alignEndsType EndToEnd --genomeDir /storage/vannostrand/genomes/repbase/species_specific/homo_sapiens_repbase_v2 --genomeLoad LoadAndKeep --outBAMcompression 10 --outFileNamePrefix clip1.repeat.map/ --outFilterMultimapNmax 100 --outFilterMultimapScoreRange 1 --outFilterScoreMin 10 --outFilterType BySJout --outReadsUnmapped Fastx --outSAMattrRGline ID:foo --outSAMattributes All --outSAMmode Full --outSAMtype BAM Unsorted --outSAMunmapped None --outStd Log --readFilesCommand zcat --readFilesIn ${x}.trim.fastq.gz
Unique genomic mapping (takes repeat-removed reads and maps to the reference genome. Use unmapped reads for next step): STAR --runMode alignReads --runThreadN 8 --alignEndsType EndToEnd --genomeDir /storage/vannostrand/genomes/hg19/star_2.4.0j_encode --genomeLoad NoSharedMemory --outBAMcompression 10 --outFileNamePrefix clip1.genome.map/ --outFilterMultimapNmax 1 --outFilterMultimapScoreRange 1 --outFilterScoreMin 10 --outFilterType BySJout --outReadsUnmapped Fastx --outSAMattrRGline ID:foo --outSAMattributes All --outSAMmode Full --outSAMtype BAM Unsorted --outSAMunmapped None --outStd Log --readFilesIn ${x}.trim.unmap.fastq
Convert unmapped fastq to fasta: fastq_to_fasta.py $y ${x}.mask.fasta
Collapse identical reads: fasta2collapse.pl ${x}.mask.fasta ${x}.collapse.fasta
Create read database to map miRNAs: bowtie-build --offrate 2 --large-index   ${x}.collapse.fasta     ${x}.bowtie.index      2> ${x}.bowtie.index.log
Map miRNA to collapsed (unique) sequences: bowtie   -a  -e 35   -l 8   -n 1  -p 8   -x ${x}.bowtie.index  --no-unal   -f $U_to_T_fasta     ${x}.mirna.reverse.map.tsv  2> ${x}.mirna.reverse.map.log
Select the "best match" positive strand alignments with the least number of mismatches: collapse_bowtie_results.py ${x}.mirna.reverse.map.tsv ${x}.map.tsv
Takes output from "best match" alignments. For each unique sequence that also maps to a miRNA, recover reads from uncollapsed fasta and identify the mappable mRNA component of these chimeric candidates: find_candidate_chimeras   --bowtie_align  ${x}.map.tsv  --fa_file ${x}.mask.fasta  --metrics_file ${x}.candidate.chimeras.metrics.tsv   --out_file ${x}.target.fasta
Map chimeric portion to genome: STAR   --alignEndsType EndToEnd   --genomeDir $genome   --genomeLoad NoSharedMemory   --outBAMcompression 10   --outFileNamePrefix ${x}.candidate.genome.map   --outFilterMatchNminOverLread 0.66   --outFilterMultimapNmax 1   --outFilterMultimapScoreRange 1   --outFilterScoreMin 10   --outFilterScoreMinOverLread 0.66   --outFilterType BySJout   --outReadsUnmapped Fastx   --outSAMattrRGline ID:foo   --outSAMattributes All   --outSAMmode Full   --outSAMtype BAM Unsorted   --outSAMunmapped None   --outStd Log   --readFilesIn ${x}.candidate.chimeras.metrics.tsv   --runMode alignReads   --runThreadN 16
Sort mapped reads: samtools sort   -@ 8   -o ${x}.candidate.bam   *Aligned.out.bam
Index bam file: samtools index ${x}.candidate.genome.map
Remove PCR duplicate reads from bam files: umi_tools dedup   --random-seed 1   --stdin ${x}.candidate.genome.map   --method unique   --stdout ${x}.dedup.bam
Index bam file: samtools index ${x}.dedup.bam
Append miRNA to genome mapping for chimeric reads and create miR-specific output files:    make_chimeras_bam.py  ${x}.dedup.bam ${x}.chimeras.bam
Get the feature Counts for individual chimeras : featureCounts -a gencode.vM20.chr_patch_hapl_scaff.annotation.gtf -s 1 -T 8 -o WT_KRAS.feature.count CLR322.mmu-miR-21a-5p.chimeras.bam CLR322.mmu-miR-21a-3p.chimeras.bam CLR322.mmu-miR-30a-5p.chimeras.bam CLR322.mmu-miR-182-5p.chimeras.bam CLR322.mmu-miR-29a-3p.chimeras.bam CLR322.mmu-miR-16-5p.chimeras.bam CLR322.mmu-miR-92a-3p.chimeras.bam CLR322.mmu-miR-26a-5p.chimeras.bam CLR322.mmu-miR-7a-5p.chimeras.bam CLR322.mmu-miR-23a-3p.chimeras.bam CLR322.mmu-miR-27b-3p.chimeras.bam CLR322.mmu-miR-27a-3p.chimeras.bam CLR322.mmu-miR-183-5p.chimeras.bam CLR322.mmu-miR-93-5p.chimeras.bam CLR322.mmu-miR-20a-5p.chimeras.bam CLR322.mmu-miR-15b-5p.chimeras.bam CLR322.mmu-miR-200b-3p.chimeras.bam CLR322.mmu-miR-106b-5p.chimeras.bam CLR322.mmu-miR-17-5p.chimeras.bam CLR322.mmu-miR-15a-5p.chimeras.bam
Assembly: mm10/GRCm38
Supplementary files format and content: *.chimeras.bam.tsv.gz files are tab-delimited files containing miRNA and genomic chimeric mapping for identified chimeric reads *feature.count.tsv files contain the per-gene number of chimeras for a subset of miRNAs listed above

extract_protocol:
For each 10cm plate (~15 million cells), cells were washed once with cold phosphate buffered saline, and then UV crosslinked (254 nm, 400 mJ/cm2) on ice. Cells were then spun down, supernatant removed, and washed with cold phosphate buffered saline. Cell pellets were flash frozen on dry ice and stored at -80°C.
Chimeric eCLIP was performed as previously described (Manakov et al. BioRxiv 2022). Briefly, lysis was performed in eCLIP lysis buffer, followed by sonication and digestion with RNase I (Ambion). Immunoprecipitation of AGO2-RNA complexes was achieved with a primary mouse monoclonal Ago2 antibody (eIF2C2 (4F9) Santa Cruz, 4°C overnight) using magnetic beads pre-coupled to the secondary antibody (M-280 Sheep Anti-Mouse IgG Dynabeads, Thermo Fisher 11202D). Initial experiments used standard eCLIP conditions (10 ug of antibody and 125 uL of Dynabeads for 20×106 cells), but most experiments used decreased antibody and increased bead amounts based on the trend of decreased cross-species chimeras in those conditions (See Sup. Fig. 2X and Sup. Table X). Where indicated, 2% of each immunoprecipitated (IP) sample was saved as input control. For human/rat mixing experiments, cell pellets were lysed, sonicated, and RNase digested separately, and then mixed during addition of antibody and beads prior to overnight incubation. To phosphorylate the cleaved mRNA 5'-ends, beads were washed and treated with T4 polynucleotide kinase (PNK, 3' -phosphatase minus, NEB) and 1 mM ATP. Chimeric ligation was then performed on-bead at room temperature for one hour with T4 RNA Ligase I (NEB) and 1 mM ATP in a 150 µl total volume. As in seCLIP, samples were then dephosphorylated with alkaline phosphatase (FastAP, Thermo Fisher) and T4 PNK (NEB), and an RNA adapter was ligated to the 3′-ends of the mRNA fragments (T4 RNA Ligase, NEB). Input samples were then denatured with 1X NuPage buffer (Life Technologies) and DTT, run on 4%–12% Bis-Tris protein gels and transferred to nitrocellulose membranes. The region corresponding to bands at the appropriate Ago2 protein size plus 75 kDa was excised and treated with Proteinase K (NEB) to isolate RNA, which was column purified (Zymo). Ago ("no-gel") chimeric eCLIP samples were treated directly with Proteinase K (NEB) to isolate RNA and column purified (Zymo). For both IP and input, RNA was then reverse transcribed with SuperScript IV Reverse Transcriptase (Invitrogen), using customized buffer with 3 mM manganese chloride (to encourage read-through of crosslink sites), and 0.1 M DTT. Following reverse transcription, samples were treated as in seCLIP, including treatment with ExoSAP-IT (Affymetrix) to remove excess oligonucleotides, hydrolysis with sodium hydroxide (to degrade RNA) and addition of hydroden chloride (to balance pH). A 5’ Illumina DNA adapter (/5Phos/NNNNNNNNNNAGATCGGAAGAGCGTCGTGT/3SpC3) was then ligated to the 3′-end of cDNA fragments with T4 RNA Ligase (NEB), and after bead purification (Dynabeads MyOne Silane, Thermo Fisher), qPCR was performed on an aliquot of each sample to identify the proper number of PCR cycles. The remainder of the sample was PCR amplified with barcoded Illumina compatible primers (Q5, NEB) based on qPCR quantification and size selected using AMPure XP beads (Beckman). Libraries were quantified using Agilent TapeStation and sequenced on the Illumina HiSeq or NovaSeq platform.

treatment_protocol: Chimeric eCLIP

value: KRAS mutant mouse pancreatic cancer cells


title: A549 expressing FTL, rep1

value: lung

value: cancer cell

value: deprived of serum for 2h


title: not4abc_AZD_2

description:
Library name: Sample 11
E2_n4abc_azd


title: A549 expressing FTS, rep2

value: FTS


title: KrasG12D,chase5h,rep3

description: SLAM-seq

value: pancreas

value: mPDAC


title: not4abc_AZD_1

description:
Library name: Sample 10
E1_n4abc_azd


title: A549 expressing FTS, rep1


title: ASQ1B

city: Baltimore

first: Luciane

middle: Tsukamoto

last: Kagohara

state: MD

address: 1650 Orleans St - CRB1 Rm 490

department: Oncology

institute: Johns Hopkins University

description: Library name: ASQ1B

data_processing:
Sequencing alignment and demultiplexing with Space Ranger (10x Genomics).
Quality check and filtering with Seurat.
Normalization using SCTransform in Seurat.
Dimensional reduction using PCA in Seurat.
Gene expression analysis using Seurat.
Assembly: GRCh38
Supplementary files format and content: barcodes.tsv: spatial barcodes
Supplementary files format and content: features.tsv: genes
Supplementary files format and content: matrix.mtx: sequencing counts
Supplementary files format and content: aligned_fiducials.jpg: capture area fiducial frame
Supplementary files format and content: detected_tissue_image.jpg: image of tissue detected
Supplementary files format and content: scalefactors_json.json; scale factors
Supplementary files format and content: tissue_hires_image.png: high resolution tissue image
Supplementary files format and content: tissue_lowres_image.png: low resolution tissue image
Supplementary files format and content: tissue_positions.csv: coordinates
Supplementary files format and content: <sample>_histologicalannotation.csv: histology annotations for spatial spots
Library strategy: Spatial Transcriptomics

extract_protocol: Visium FFPE Spatial Gene Expression Reagent Kits (10x Genomics).

tag: tissue type

value: pancreatic adenosquamous tumor


title: CLR329

description: Library name: AGO_KRAS_IN2


title: not4abc_3

description:
Library name: Sample 9
B3_not4abc

value: Control


title: KrasG12D,chase5h,rep2

description: SLAM-seq


title: CLR326

description: Library name: AGO_KRAS_IP1

value: IP


title: KrasG12D,chase5h,rep1

description: SLAM-seq

value: pancreatic cancer cell


title: A549 expressing FGFR3, rep2


title: CLR339

description: Library name: AGO_KRAS_IN1


title: A549 expressing FGFR3, rep1


title: not4abc_2

description:
Library name: Sample 8
B2_not4abc


title: KrasG12D,chase1h,rep3

description: SLAM-seq

value: DMSO, s4U, doxycycline


title: CLR325

description: Library name: AGO_WT_IN2

value: WT Mouse pancreatic cancer cells

value: input


title: not4abc_1

description:
Library name: Sample 7
B1_not4abc

value: 12-day-old seedlings


title: A549 expressing TACC3, rep2


title: col-0_AZD8055_3

description:
Library name: Sample 6
D3_col_azd


title: KrasG12D,chase1h,rep2

description: SLAM-seq


title: A549 expressing TACC3, rep1


title: CLR324

description: Library name: AGO_WT_IP2


title: CLR323

description: Library name: AGO_WT_IN1


title: A549 expressing empty vector, rep2

value: control


title: KrasG12D,chase1h,rep1

description: SLAM-seq


title: KrasG12D, pulse24hchase0h, rep3

description: SLAM-seq


title: CLR322

description: Library name: AGO_WT_IP1


title: col-0_AZD8055_1

description:
Library name: Sample 4
D1_col_azd

value: TOR inhibitor (AZD8055)


title: A549 expressing empty vector, rep1


title: KrasG12D, pulse24hchase0h, rep2

description: SLAM-seq


title: NAT10 and Xrp1 RNAi ac4C-IP rep3

first: Wang

last: Lin

state: Shanghai

address: 2005 Songhu Road, Yangpu District, Shanghai, China

department: College of Life Science

description:
Library name: Da_GX_Ip_3
Column name in raw_All_Samples: Da_GX_Ip_3

data_processing:
Raw sequencing data was first filtered by fastp (v0.23.1), low-quality reads were discarded and the reads with adaptor sequences were trimmed. Clean reads were further treated with in-house scripts to eliminate duplication bias introduced in library preparation and sequencing.
Clean reads were first clustered according to the UMI sequences, in which reads with the same UMI sequence were grouped into the same cluster. Reads in the same cluster were compared to each other by pairwise alignment, and then reads with sequence identity over 95% were extracted to a new sub-cluster. After all sub-clusters were generated, multiple sequence alignment was performed to get one consensus sequence for each sub-clusters. After these steps, any errors and biases introduced by PCR amplification or sequencing were eliminated.
The de-duplicated consensus sequences were used for ac4C site analysis, which were mapped to the reference genome using STAR software (v2.7.6a). The reads mapped to the exon regions of each gene were counted by featureCounts (Subread-1.5.1; Bioconductor) and then the expression was calculated. The exomePeak software (v2.16.0) was used for peak calling.
The peaks were annotated using bedtools (v2.30.0). The deepTools (v3.5.1) was used for peak distribution analysis. The different peaks were identified using QNB (v1.1.11). Sequence motifs enriched in the peak regions were identified using HOMER (v4.9.1). Both Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for annotated genes were implemented using KOBAS software (v2.1.1) with a P-value cutoff of 0.05 and a input number cutoff of 2 to determine statistically significant enrichment.
Assembly: BDGP6.28 (Ensembl Release 102)
Supplementary files format and content: input FPKM all genes.csv and all bigwig files
Supplementary files format and content: ac4C-IP all peaks.csv, ac4C-IP NAT10-RNAi vs Control diff peaks.csv and ac4C-IP NAT10-RNAi Xrp1-RNAi vs Control diff peaks.csv

organism: Drosophila melanogaster

source_name: Whole 3rd larva

growth_protocol: All Drosophila stocks were maintained on standard cornmeal–yeast–agar–syrup food

extract_protocol:
Total RNA from 40 whole 3rd larva was extracted using Trizol reagent kit (Invitrogen, #15596026) according to the manufacturer’s protocol.
Total treated with DNase I (NEB,#M0303L) to remove genomic DNA contamination. RNA purity and integrity were assessed using Nanodrop, Qubit 3.0 fluorometer, and LabChip GX Touch. Polyadenylated RNA was enriched from total RNA using VAHTS mRNA Capture Beads (Vazyme,#N401), according to the manufacturer’s instructions. For ac4C-RIP, purified poly(A)+ RNA was chemically fragmented (~100 nt) by 20 mM ZnCl₂ at 95°C for 5–10 minutes. Ten percent of the fragmented RNA was reserved as input, while the remainder was incubated with ac4C antibody (Abcam, #ab252215) and RNasin (Promega,#N2615) at 4°C for 2 hours. RNA–antibody complexes were pulled down using Protein G magnetic beads (Thermo Scientific,#88848), washed extensively, and recovered by TRIzol extraction. RNA-seq libraries were prepared from both input and immunoprecipitated RNA using the KCTM Digital mRNA Library Prep Kit (Seqhealth Tech. Co., Ltd., Wuhan, China), which incorporates a 12-base unique molecular identifier (UID) to reduce PCR and sequencing bias.The library preparation included the enrichment of PCR products corresponding to fragments ranging from 200 to 500 base pairs. The enriched libraries were quantified and sequenced on a NovaSeq X Plus (Illumina) platform using the PE150 sequencing mode to generate paired-end reads.

treatment_protocol: Whole-body knockdown of NAT10 using da-GAL4, tub-GAL80ts resulted in severe developmental delay. Thus, embryos were collected for 24 h at 18 °C, then aged to first-instar larvae and shifted to 30 °C. Control larvae were harvested after 72 h, and NAT10 knockdown larvae after 120 h.

value: Whole 3rd larva

value: Dats-GAL4 > UAS-NAT10-RNAi Xrp1-RNAi


title: col-0_3

description:
Library name: Sample 3
A3_col


title: NAT10 and Xrp1 RNAi ac4C-IP rep2

description:
Library name: Da_GX_Ip_2
Column name in raw_All_Samples: Da_GX_Ip_2


title: KrasG12D, pulse24hchase0h, rep1

description: SLAM-seq


title: hMOs_NR_replicate1

city: beijing

first: xinyu

last: She

address: shuangqing road, haidian district

department: school of life sciences

institute: Tsinghua university

library_source: transcriptomic single cell

data_processing:
The demultiplexing, barcoded processing, gene counting were made using the Cell Ranger software v6.0.2 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger).
Assembly: GRCh38-2020-A genome
Supplementary files format and content: Tab-separated values files and matrix files

source_name: midbrain organoid

growth_protocol: The hMOs were maintained in the incubator at 37°C and 5% CO2, using different kinds of medium according to different developmental stages.

extract_protocol:
The hMOs were dissociated into single-cell suspensions using the Neural Tissue Dissociation Kit (Miltenyi Biotec, #130-092-628), and cell particles were resuspended in an ice-cold buffer (0.04% BSA in PBS).
Single-cell gene expression libraries were generated with theChromium™ Single Cell 3’ Library and Gel Bead Kit v3.1 (10X Genomics, Pleasanton, California, USA) according to the manufacturer's instruction.

value: midbrain organoid

value: the cells associated with the midbrain


title: hMOs_LR_replicate1


title: NAT10 and Xrp1 RNAi ac4C-IP rep1

description:
Library name: Da_GX_Ip_1
Column name in raw_All_Samples: Da_GX_Ip_1


title: col-0_1

description:
Library name: Sample 1
A1_col


title: hMOs_HC_replicate1


title: KrasG12D, no pulse, rep2

description: SLAM-seq

value: DMSO, doxycycline


title: KrasG12D, no pulse, rep1

description: SLAM-seq

value: pancreas


title: NAT10 RNAi ac4C-IP rep2

description:
Library name: Da_GD_Ip_2
Column name in raw_All_Samples: Da_GD_Ip_2

value: Dats-GAL4 > UAS-NAT10-RNAi


title: MMTV-PyMT3 DRG RNA

city: Medford

first: Madeleine

last: Oudin

address: 200 College Ave

institute: Tufts University

description: Library name: Sample5

data_processing:
Trimming using trimmomatic, Mapping with STAR, featurecounts for counts, Deseq2
Assembly: Mus_musculus.GRCm39.dna.primary_assembly
Supplementary files format and content: Comma delimited, deseq output genes with p<0.05.

source_name: Dorsal root ganglia

extract_protocol:
RNA was extracted using Direct-zol RNA Miniprep Plus Kits (R2071-A)
Illumina Stranded mRNA

value: Dorsal root ganglia

value: MMTV-PyMT


title: NAT10 RNAi ac4C-IP rep3

description:
Library name: Da_GD_Ip_3
Column name in raw_All_Samples: Da_GD_Ip_3


title: KrasG12D, no pulse, rep3

description: SLAM-seq

value: mPDAC


title: col-0_AZD8055_2

description:
Library name: Sample 5
D2_col_azd


title: col-0_2

description:
Library name: Sample 2
A2_col

value: Control


title: Midbody rep4

city: Aurora

first: Matthew

last: Taliaferro

state: CO

department: Biochemistry and Molecular Genetics

institute: University of Colorado Anschutz Medical Campus

description: Library name: Midbody rep4

data_processing:
Read pairs contained both the sequence of the reporter-embedded MPRA oligo as well as a unique molecular identifier (UMI) installed during reverse transcription. Custom python scripts extracted the number of unique UMIs observed for each MPRA oligo in the data.
Assembly: hg38
Supplementary files format and content: tab-delimited text file that contains the number of unique UMIs observed for each MPRA oligo
Library strategy: MPRA

source_name: HeLa

growth_protocol: Cells grown in DMEM supplemented with 10% FBS and 1% pen-strep

extract_protocol:
Midbodies were isolated as in Farmer et al 2023 (PMID 37922419). Midbody and whole cell RNA samples were extracted using a Zymo Quick RNA Microprep kit
Targeted RNAseq libraries were constructed as in Arora et al 2022 (PMID 36107770)

value: HeLa

value: Midbody RNA


title: Pooled snRNAseq samples

city: Nashville

first: Valentina

last: Cigliola

state: TN

address: 2220 Pierce Avenue

institute: Vanderbilt University

description: Library name: Sample 1

data_processing:
Demultiplexing, barcode, counting, and matrix construction were done in Trailmaker software. Raw-counts matrices were read into Seurat (v.5) and filtered to cells containing over 500 unique detected genes. Standard normalization, feature selection, and scaling procedures were employed. After principal component analysis and dimension reduction via uniform manifold approximation and projection, cells were annotated by cluster based on canonical genes.
Assembly: Genome assembly GRCz11 (zebrafish)
Supplementary files format and content: Comma seperated metadata and matrix files per sample, and pooled R Seurat object

organism: Danio rerio

source_name: Spinal cord

extract_protocol:
Spinal cord segments (2.0 mm anterior and posterior to the injury site) were dissected from gata4wcm6/wcm6 and wild-type fish at 2 wpi, flash-frozen, and stored at −80 °C. Nuclei isolation was adapted from a previously established protocol. All solutions, reagents, and tools were pre-chilled and maintained on ice throughout the procedure. Nuclei Isolation Buffer 1 (NIM1) consisted of 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, and 10 mM Tris prepared in RNase-free water. Nuclei Isolation Buffer 2 (NIM2) was prepared by supplementing NIM1 with 1 µM DTT, 0.4 U/µL RNase inhibitor, and 0.1% Triton X-100. A swinging-bucket centrifuge was pre-cooled to 4 °C prior to sample processing. Tissue samples were incubated in cold NIM2 buffer and gently dissociated by trituration on ice, followed by centrifugation at 200 × g for 10 minutes at 4 °C. The resulting pellet was resuspended in Nuclei Buffer containing 0.75% bovine serum albumin and passed through a 40 µm cell strainer into a 15-mL conical tube coated with 1% bovine serum albumin. Following filtration, Hoechst dye (1 µg/mL) and Nuclei Fixation Solution were added, and samples were incubated on ice for 10 minutes. Nuclei were fluorescence-activated cell sorted (FACS) in 1× phosphate buffer (approximately 1 × 10⁶ nuclei per sample), transferred to a new 1% bovine serum albumin–coated 15-mL conical tube, and centrifuged at 200 × g for 10 minutes at 4 °C. The supernatant was carefully removed, leaving approximately 1.0 mL above the pellet. Nuclei were permeabilized by adding Nuclei Permeabilization Solution and incubating on ice for 3 minutes, followed by neutralization with Nuclei Neutralization Buffer and centrifugation at 200 × g for 10 minutes at 4 °C. The final pellet was fully resuspended in Nuclei Buffer supplemented with DMSO and stored at −80 °C until library preparation.
Single-nucleus RNA-sequencing libraries were generated using the PARSE Evercode Whole Transcriptome Mini v2 kit according to the manufacturer’s instructions.

value: Spinal cord

value: gata4wcm6/wcm6 and wildtype


title: MMTV-PyMT2 DRG RNA

description: Library name: Sample4


title: KrasWT,chase12h,rep3

description: SLAM-seq

value: pancreatic cancer cell

value: KrasWT

value: DMSO, s4U


title: MMTV-PyMT1 DRG RNA

description: Library name: Sample3


title: NAT10 RNAi ac4C-IP rep1

description:
Library name: Da_GD_Ip_1
Column name in raw_All_Samples: Da_GD_Ip_1

value: Whole 3rd larva


title: Midbody rep3

description: Library name: Midbody rep3


title: KrasWT,chase12h,rep2

description: SLAM-seq


title: Control ac4C-IP rep3

description:
Library name: Da_WT_Ip_3
Column name in raw_All_Samples: Da_WT_Ip_3


title: Midbody rep2

description: Library name: Midbody rep2


title: KrasWT,chase12h,rep1

description: SLAM-seq


title: FVB3 control DRG RNA

description: Library name: Sample2

value: FvB/NJ


title: Control ac4C-IP rep2

description:
Library name: Da_WT_Ip_2
Column name in raw_All_Samples: Da_WT_Ip_2


title: FVB2 control DRG RNA

description: Library name: Sample1


title: Midbody rep1

description: Library name: Midbody rep1


title: KrasWT,chase5h,rep3

description: SLAM-seq


title: NAT10 and Xrp1 RNAi input rep3

description:
Library name: Da_GX_In_3
Column name in raw_All_Samples: Da_GX_In_3

value: Dats-GAL4 > UAS-NAT10-RNAi Xrp1-RNAi


title: WT4_ICELL8 cx whole heart tissue

city: South San Francisco

first: Chi-Ming

last: Li

state: CA

address: 750 Gateway Blvd

department: Target Discovery & Biology

institute: Amgen

description:
Library name: WT4_ICELL8 cx
Takara ICELL8 cx

data_processing:
Initial processing of ICELL8 cx full-length snRNA-seq data was done with Takara’s Cogent™ NGS Analysis Pipeline (https://www.takarabio.com/learning-centers/next-generation-sequencing/bioinformatics-resources/cogent-ngs-analysis-pipeline, v.2.0.1). Cogent demux was used to demultiplex fastq files followed by running cogent analyze with Takara’s pre-built Cogent reference mm10 for mouse and hg38 for human. Analyze_incl_introns_genematrix.csv files produced by Cogent pipeline[16] were read into R (v.4.2.1) and used to create the Seurat (v.5.1.0) object[13]. For mouse data, nuclei barcodes were filtered based on the number of genes per nucleus (3000 < number of genes < 20,000), number of reads per nucleus (10,000 < number of reads < 1,400,000), and percentage of mitochondrial genes per nucleus (< 20%). For human data, nuclei barcodes were filtered using the following thresholds: 10,000 < number of counts < 100,000 and less than 30% mitochondrial reads per nucleus. The threshold for mitochondrial reads was less stringent than for 10X as applying those same criteria to the ICELL8 data resulted in dramatic losses of nuclei.
Assembly: Mouse mm10
Supplementary files format and content: csv for icell8 gene expression matrix

molecule: nuclear RNA

source_name: Whole heart

extract_protocol:
Fresh heart tissue was harvested and quickly transferred into a 2 ml nuclease-free Eppendorf tube containing 1 ml ice-cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2 in nuclease-free water) containing freshly added 0.1% NP-40 and 0.2 U/µl RiboLock RNase inhibitor (ThermoFisher Scientific, Waltham, MA). Tissue samples were cut into smaller pieces using scissors and transferred to a 7 ml Dounce homogenizer containing 1 ml lysis buffer on ice. Tissue was homogenized with 10 strokes each of the loose and tight pestle followed by incubation on ice for 15 minutes. Homogenized tissue was filtered through a 40 µm cell strainer and cell strainer was rinsed with 0.5 ml nuclei resuspension buffer (2% bovine serum albumin (BSA) in 1X phosphate buffered saline (PBS)) with 0.2 U µl-1 RiboLock RNase inhibitor. Samples were centrifuged at 700 rcf for 5 minutes at 4°C. Resulting pellet nuclei was washed with 1 ml nuclei resuspension buffer followed by centrifugation. Pellet containing nuclei was resuspended in 1 ml nuclei resuspension buffer and stained with 3 µl DRAQ5 (ThermoFisher Scientific). DRAQ5+ nuclei were sorted on a BD FACSAria Fusion (BD Biosciences, Franklin Lakes, NJ) using a 100 µm nozzle into 100 µl nuclei collection buffer (1X PBS, 2% BSA) with 0.6 U µl-1 RiboLock RNase inhibitor. Following flow cytometry, sorted DRAQ5+ nuclei were centrifuged at 2,000 rcf for 5 minutes at 4°C, and nuclei were resuspended in 100 µl 1X PBS, prior to loading into the Chromium or ICELL8 cx systems as described below.
ICELL8 cx platform: After manually counting nuclei using Trypan Blue, samples were loaded at a concentration of 40,000 nuclei ml-1. Libraries were prepared using the manufacturers’ instructions- SMART-Seq Pro (ICELL8 cx Single-Cell System User Manual kit (Takara Bio, San Jose, CA)-and sequenced on a NovaSeqX (Illumina) instrument following manufacturers’ guidelines: 151 X 8 X 8 X 151 [read1 X i7 index X i5 index X read2].

value: Whole heart

tag: strain

value: Male

value: wildtype

tag: age

value: 8-10 weeks


title: Control ac4C-IP rep1

description:
Library name: Da_WT_Ip_1
Column name in raw_All_Samples: Da_WT_Ip_1


title: WT3_ICELL8 cx whole heart tissue

description:
Library name: WT3_ICELL8 cx
Takara ICELL8 cx


title: KrasWT,chase5h,rep2

description: SLAM-seq

value: pancreas


title: Whole cell rep4

description: Library name: Whole cell rep4

value: Whole cell RNA


title: KrasWT,chase5h,rep1

description: SLAM-seq

value: mPDAC


title: Whole cell rep3

description: Library name: Whole cell rep3

value: HeLa


title: NAT10 and Xrp1 RNAi input rep2

description:
Library name: Da_GX_In_2
Column name in raw_All_Samples: Da_GX_In_2


title: Whole cell rep2

description: Library name: Whole cell rep2


title: WT2_ICELL8 cx whole heart tissue

description:
Library name: WT2_ICELL8 cx
Takara ICELL8 cx


title: NAT10 and Xrp1 RNAi input rep1

description:
Library name: Da_GX_In_1
Column name in raw_All_Samples: Da_GX_In_1


title: KrasWT,chase1h,rep3

description: SLAM-seq


title: WT1_ICELL8 cx whole heart tissue

description:
Library name: WT1_ICELL8 cx
Takara ICELL8 cx


title: Whole cell rep1

description: Library name: Whole cell rep1


title: KrasWT,chase1h,rep2

description: SLAM-seq


title: NAT10 RNAi input rep3

description:
Library name: Da_GD_In_3
Column name in raw_All_Samples: Da_GD_In_3

value: Dats-GAL4 > UAS-NAT10-RNAi


title: Th2 cells, Sample 4, mid IL13

city: Chicago

first: Isabella

middle: M.

last: Salamone

state: IL

address: 920 E 58TH ST

department: Human Genetics

institute: University of Chicago

description:
Library name: Sample4mid
sgRNA read counts

library_source: other

data_processing:
sgRNA enrichment analysis was performed using MAGeCK, comparing the IL-13-high population counts to counts averaged across IL-13-high, IL-13-low, and middle from the same donor using mageck test --paired.
Supplementary files format and content: tab-delimited text file includes raw counts per sgRNA
Library strategy: CRISPR Screen

molecule: other

source_name: primary Th2 cells

growth_protocol: Naïve CD4+ T cells were isolated from freshly thawed PBMCs using the EasySep Human Naïve CD4+ T Cell Isolation Kit (STEMCELL Technologies) and polarized to Th2 using ImmunoCult-XF T Cell Expansion Medium supplemented with ImmunoCult Th2 Differentiation Supplement (STEMCELL Technologies). Immediately following isolation, naïve CD4+ T cells were diluted to 1x106 cells/mL, then activated with ImmunoCult Human CD3/CD28 T Cell Activator (STEMCELL Technologies). CD4+ T cells were counted every 3-4 days and diluted to 1x106 cells/mL with fresh medium as needed; at day ten, cells were pelleted, resuspended in completely fresh medium, and re-activated.

extract_protocol:
Cells were lysed in NK lysis buffer (50 mM Tris, 50 mM EDTA, 1% SDS) with proteinase K overnight at 55C to reverse crosslinking, then DNA was precipitated with isopropanol.
sgRNA sequences were amplified and sequencing primers ligated as described in Joung et al. 2017

treatment_protocol: Four samples of CD4+ T cells from three donors were transduced with lentivirus one day after thawing at MOI = 0.2. CD4+ T cells were transduced in a 24-well plate as 6 wells of 9.2x105 cells each. The original media was removed from each well and stored at 37C, then replaced with 1 mL fresh media + 8 μg/mL polybrene + lentivirus. Cells were spinfected at 1000 x g for 90 min at room temperature, then the media was removed in its entirety and replaced with the original media. Fourteen days after spinfection, CD4+ T cells were stimulated with 5ng/mL PMA (Sigma-Aldrich) and 500ng/mL ionomycin (Sigma-Aldrich) for two hours, then 5 μg/mL brefeldin A (Biolegend) and 2 μM monensin (Biolegend) for an additional two hours.

value: primary Th2 cells

value: Th2 polarized, transduced with lentiCRISPR library


title: WT4_10X 5' whole heart tissue

description:
Library name: WT4_10X 5_
10X Genomics

data_processing:
Initial processing of Chromium snRNA-seq data was done with Cell Ranger Pipeline (https://www.10xgenomics.com/support/software/cell-ranger/latest, v.8.0.0). Cellranger mkfastq was used to demultiplex bcl files followed by cellranger count with 10X Genomics’ pre-built Cell Ranger reference refdata-gex-mm10-2020-A (https://cf.10xgenomics.com/supp/cell-exp/refdata-gex-mm10-2020-A.tar.gz for mouse, http://cf.10xgenomics.com/supp/cell-exp/refdata-cellranger-GRCh38-3.0.0.tar.gz for human). Filtered_feature_bc_matrix was read into R (v.4.2.1) with Seurat (v.5.1.0) function Read10X [13]. Doublets were detected by scDblFinder (v.1.2.0) for each sample separately and removed from the downstream analyses[14]. Nuclei from all samples were merged into a single Seurat object and analyzed separately for each dataset.  Initial processing of ICELL8 cx full-length snRNA-seq data was done with Takara’s Cogent™ NGS Analysis Pipeline (https://www.takarabio.com/learning-centers/next-generation-sequencing/bioinformatics-resources/cogent-ngs-analysis-pipeline, v.2.0.1). Cogent demux was used to demultiplex fastq files followed by running cogent analyze with Takara’s pre-built Cogent reference mm10 for mouse and hg38 for human. Analyze_incl_introns_genematrix.csv files produced by Cogent pipeline[16] were read into R (v.4.2.1) and used to create the Seurat (v.5.1.0) object[13]. For mouse data, nuclei barcodes were filtered based on the number of genes per nucleus (3000 < number of genes < 20,000), number of reads per nucleus (10,000 < number of reads < 1,400,000), and percentage of mitochondrial genes per nucleus (< 20%). For human data, nuclei barcodes were filtered using the following thresholds: 10,000 < number of counts < 100,000 and less than 30% mitochondrial reads per nucleus. The threshold for mitochondrial reads was less stringent than for 10X as applying those same criteria to the ICELL8 data resulted in dramatic losses of nuclei.
Assembly: Mouse mm10
Supplementary files format and content: hd for 10x gene expression matrix and tsv for icell8 gene expression matrix

extract_protocol:
Fresh heart tissue was harvested and quickly transferred into a 2 ml nuclease-free Eppendorf tube containing 1 ml ice-cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2 in nuclease-free water) containing freshly added 0.1% NP-40 and 0.2 U/µl RiboLock RNase inhibitor (ThermoFisher Scientific, Waltham, MA). Tissue samples were cut into smaller pieces using scissors and transferred to a 7 ml Dounce homogenizer containing 1 ml lysis buffer on ice. Tissue was homogenized with 10 strokes each of the loose and tight pestle followed by incubation on ice for 15 minutes. Homogenized tissue was filtered through a 40 µm cell strainer and cell strainer was rinsed with 0.5 ml nuclei resuspension buffer (2% bovine serum albumin (BSA) in 1X phosphate buffered saline (PBS)) with 0.2 U µl-1 RiboLock RNase inhibitor. Samples were centrifuged at 700 rcf for 5 minutes at 4°C. Resulting pellet nuclei was washed with 1 ml nuclei resuspension buffer followed by centrifugation. Pellet containing nuclei was resuspended in 1 ml nuclei resuspension buffer and stained with 3 µl DRAQ5 (ThermoFisher Scientific). DRAQ5+ nuclei were sorted on a BD FACSAria Fusion (BD Biosciences, Franklin Lakes, NJ) using a 100 µm nozzle into 100 µl nuclei collection buffer (1X PBS, 2% BSA) with 0.6 U µl-1 RiboLock RNase inhibitor. Following flow cytometry, sorted DRAQ5+ nuclei were centrifuged at 2,000 rcf for 5 minutes at 4°C, and nuclei were resuspended in 100 µl 1X PBS, prior to loading into the Chromium or ICELL8 cx systems as described below.
Chromium platform:After manually counting nuclei using Trypan Blue, based on the concentration, samples were loaded at a volume to target 20,000 nuclei. Libraries were prepared using the manufacturers’ instructions-10X 3’ v4 GEM-X (Chromium GEM-X Single Cell 3’ Reagent Kits v4 Document Number CG000731 Rev B August 2024) or 10X 5’ v3 GEM-X (Chromium GEM-X Single Cell 5’ Reagent Kits v3 Document Number CG000733 Rev A March 2024) kits (10X Genomics)- and sequenced on a NovaSeqX (Illumina, San Diego, CA) instrument following manufacturers’ guidelines: 28 X 10 X 10 X 90 [read1 X i7 index X i5 index X read2]. ICELL8 cx platform: After manually counting nuclei using Trypan Blue, samples were loaded at a concentration of 40,000 nuclei ml-1. Libraries were prepared using the manufacturers’ instructions- SMART-Seq Pro (ICELL8 cx Single-Cell System User Manual kit (Takara Bio, San Jose, CA)-and sequenced on a NovaSeqX (Illumina) instrument following manufacturers’ guidelines: 151 X 8 X 8 X 151 [read1 X i7 index X i5 index X read2].


title: KrasWT,chase1h,rep1

description: SLAM-seq

value: pancreatic cancer cell


title: NAT10 RNAi input rep2

description:
Library name: Da_GD_In_2
Column name in raw_All_Samples: Da_GD_In_2


title: WT3_10X 5' whole heart tissue

description:
Library name: WT3_10X 5_
10X Genomics


title: Th2 cells, Sample 4, low IL13

description:
Library name: Sample4low
sgRNA read counts


title: KrasWT, pulse24hchase0h, rep3

description: SLAM-seq

value: KrasWT


title: NAT10 RNAi input rep1

description:
Library name: Da_GD_In_1
Column name in raw_All_Samples: Da_GD_In_1


title: Th2 cells, Sample 4, high IL13

description:
Library name: Sample4high
sgRNA read counts


title: WT2_10X 5' whole heart tissue

description:
Library name: WT2_10X 5_
10X Genomics


title: KrasWT, pulse24hchase0h, rep2

description: SLAM-seq

value: DMSO, s4U


title: Control input rep3

description:
Library name: Da_WT_In_3
Column name in raw_All_Samples: Da_WT_In_3

value: Whole 3rd larva


title: Th2 cells, Sample 3, mid IL13

description:
Library name: Sample3mid
sgRNA read counts


title: WT1_10X 5' whole heart tissue

description:
Library name: WT1_10X 5_
10X Genomics


title: KrasWT, pulse24hchase0h, rep1

description: SLAM-seq


title: Control input rep2

description:
Library name: Da_WT_In_2
Column name in raw_All_Samples: Da_WT_In_2


title: Th2 cells, Sample 3, low IL13

description:
Library name: Sample3low
sgRNA read counts


title: WT4_10X 3' whole heart tissue

description:
Library name: WT4_10X 3_
10X Genomics


title: KrasWT, no pulse, rep3

description: SLAM-seq

value: DMSO


title: WT3_10X 3' whole heart tissue

description:
Library name: WT3_10X 3_
10X Genomics


title: Control input rep1

description:
Library name: Da_WT_In_1
Column name in raw_All_Samples: Da_WT_In_1


title: Th2 cells, Sample 3, high IL13

description:
Library name: Sample3high
sgRNA read counts


title: KrasWT, no pulse, rep2

description: SLAM-seq


title: NAT10 KD rep3

description:
Library name: en_NAT10-RNAi_3
Column name in processed files:en_GD-3

data_processing:
The resulting cDNA library was sequenced using Illumina Novaseq6000
Reads were further filtered by fastp (version 0.18.0)
paired-end clean reads were mapped to the reference genome using HISAT2 2.1.0 and other parameters set as a default.
The mapped reads of each sample were assembled by using StringTie v1.3.1 in a reference-based approach. For each transcription region, a FPKM value was calculated to quantify its expression abundance and variations, using RSEM software.
Assembly: Ensembl_release109
Supplementary files format and content: csv files include FPKM for each sample
Supplementary files format and content: csv files include diff gene expression(NAT10-RNAi vs Control)
Supplementary files format and content: csv files include diff gene expression(NAT10-RNAi Xrp1-RNAi vs Control)

source_name: Wing disc

growth_protocol: All Drosophila stocks were maintained on standard cornmeal–yeast–agar–syrup food at 25 °C.

extract_protocol:
Total RNA from 100 dissected wing disc was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol.
RNA quality was assessed on an Agilent 2100 Bioanalyzer(Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beadsThen the enriched mRNA was fragmented into short fragments using fragmentation buffer and reversly transcribed into cDNA by using NEBNext Ultra RNA Library Prep Kit for Illumina(NEB#7530,New England Biolabs, Ipswich, MA, USA).

treatment_protocol: Wing discs were dissected from third instar larvae of the indicated genotypes in ice-cold PBS.

value: Wing disc

value: epithelial cells

value: en-GAL4 > UAS-NAT10-RNAi


title: Muscle, 3BNC117 plus PD-L1

city: Atlanta

first: Gregory

middle: K

last: Tharp

state: GA

address: 954 Gatewood Dr

department: Developmental and Cognitive Neuroscience

institute: Yerkes National Primate Research Center

description: Library name: p25128-s002_RMc21

data_processing:
Space Ranger v4.0.1 pipeline was used to map FASTQ files to the human GRCh38-2020-A reference and Visium Human Transcriptome Probe Set v2.0
Assembly: GRCh38-2020-A
Supplementary files format and content: barcodes.tsv.gz: list of spatial barcodes
Supplementary files format and content: features.tsv.gz: list of gene IDs
Supplementary files format and content: matrix.mtx.gz: gene expression count data in  Matrix Market Exchange Format
Supplementary files format and content: aligned_fiducials.jpg: aligned fiducials of the tissue image
Supplementary files format and content: detected_tissue_image.jpg: image of tissue and spots
Supplementary files format and content: scalefactors_json.json: scalefactors in json format
Supplementary files format and content: tissue_hires_imagge.png: hi-res images of tissue
Supplementary files format and content: tissue_lowres_image.png: low-res image of tissue
Supplementary files format and content: tissue_positions.parquet: spatial barcodes and coordinates specifying spots in parquet format
Library strategy: Spatial Transcriptomics

organism: Macaca mulatta

source_name: muscle

extract_protocol:
Muscle samples from rhesus macaques were fixed in 10% neutral buffered formalin (NBF), processed, and blocked in paraffin. RNA was extracted from scrolls collected from the formalin-fixed, paraffin-embedded (FFPE) tissue blocks with the RNeasy FFPE kit (Qiagen), and subjected to RNA quality assessment, where samples were confirmed to have DV200 scores >30%. Tissue sections with 5 μm thickness were prepared from the FFPE blocks and placed onto glass slides.
The 6.5 mm x 6.5 mm area of interest (AOI) for each tissue section was then chosen, followed by decrosslinking and hybridization of the Visium Human Transcriptome Probes Set v2.0 with the tissue in these AOIs. The hybridized probes are released and captured by the Visium HD slide oligos using the Visium CytAssist instrument (10x Genomics), amplified, and the final sequencing libraries were generated. Libraries were validated by capillary electrophoresis on a TapeStation 4200 (Agilent).

treatment_protocol: Macaques were administered AAV9.PD-L1 and/or AAV9.3BNC117 at a dose of 2.5 × 10¹² vector genomes (vg) kg⁻¹ of each vector in PBS at a volume less than 1 mL and delivered intramuscularly across eight sites: two injections in each of the lower and upper quadriceps, biceps, and deltoid muscles.

value: muscle

value: 3BNC117 plus PD-L1


title: Activated MuSC H4K20me1 biol rep3

city: Madison

first: Jeonghye

middle: Hannah

last: Hwang

state: Wisconsin

address: 1111 Highland ave.

department: Department of Cell and Regenerative Biology

institute: University of Wisconsin-Madison

description: Library name: Act3_H4K20me1

library_source: genomic

data_processing:
Paired-end raw FASTQ reads were trimmed for adapter sequences and low-quality bases using Trim Galore (v0.6.10) with default parameters.
Trimmed reads were aligned to the mouse reference genome using Bowtie2 (v2.5.4) with the --very-sensitive preset. Alignment parameters were set to --no-mixed --no-discordant -X 1000 for high-confidence paired-end mapping and a maximum fragment length of 1000 bp.
Initial alignments were converted to BAM format and sorted using Samtools (v1.22.1).  Duplicates were identified and removed using Picard Tools (v3.4.0) MarkDuplicates.
Reads overlapping with ENCODE blacklist regions (ENCFF547MET) were removed using bedtools (v2.31.1) intersect. Only propery paired (-f 2) reads with a mapping quiality (MAPQ) score greater than 30 were kept. Reads mapping to the mitochondrial genome were excluded.
The filtered BAM files were converted to bigWig format using deepTools (v3.5.6) bamCoverage. The genome was binned into 10 bp windows, and signal intensity was RPKM normalized. A smoothing window of 30 bp was applied (--smoothLength 30) to reduce local noise.
Assembly: mm10
Supplementary files format and content: bigWig,  RPKM-normalized signal tracks for genome browser visualization
Library strategy: CUT&Tag

molecule: genomic DNA

source_name: skeletal muscle

extract_protocol:
Freshly isolated MuSCs were bound to Concanavalin A magnetic beads and permeabilized. Cells were incubated overnight at 4 °C with primary antibodies, followed by secondary antibody incubation and binding of protein A–Tn5 transposase preloaded with sequencing adapters. Targeted tagmentation was performed at 37 °C in the presence of MgCl₂. DNA fragments were released by SDS and proteinase K treatment and purified by phenol–chloroform extraction followed by ethanol precipitation.
CUT&Tag DNA fragments were PCR-amplified using indexed i5 and i7 primers and NEBNext HiFi 2× PCR Master Mix. The number of amplification cycles was determined by qPCR to minimize PCR duplicates. Libraries were quantified using the Qubit dsDNA High Sensitivity Assay and fragment size distribution was assessed using an Agilent Bioanalyzer High Sensitivity DNA kit.

treatment_protocol: Muscle stem cells (MuSCs) were isolated from mouse skeletal muscle under quiescent or activated conditions. Quiescent MuSCs were freshly isolated from non-injured muscle, whereas activated MuSCs were isolated 2 days post-injury (2 dpi) following BaCl₂ injection into the tibialis anterior (TA) muscle. For conditional knockout samples, Kmt5a deletion was induced in adult mice using a tamoxifen-inducible Cre recombinase system prior to MuSC isolation. Cells were immediately processed for CUT&Tag.

value: skeletal muscle

value: muscle stem cell

value: WT

value: BaCl₂ injury


title: Th2 cells, Sample 2, mid IL13

description:
Library name: Sample2mid
sgRNA read counts


title: WT2_10X 3' whole heart tissue

description:
Library name: WT2_10X 3_
10X Genomics

value: Whole heart

value: Male

value: wildtype

value: 8-10 weeks


title: KrasWT, no pulse, rep1

description: SLAM-seq

value: pancreas

value: mPDAC

value: pancreatic cancer cell

value: KrasWT


title: WT1_10X 3' whole heart tissue

description:
Library name: WT1_10X 3_
10X Genomics


title: Th2 cells, Sample 2, low IL13

description:
Library name: Sample2low
sgRNA read counts

value: primary Th2 cells

value: Th2 polarized, transduced with lentiCRISPR library


title: Salmonella ΔintΔrdf::cat rep 3

city: Santiago

first: Susan

last: Bueno

state: Región Metropolitana

address: Avda. Libertador Bernardo O’Higgins #340

department: Departamento de Genetica Molecular y Microbiología

country: Chile

institute: Pontificia Universidad Católica de Chile

description:
Library name: Sample 6
Column name in All_samples_raw_counts.xlsx: dInt_dRDF_3

data_processing:
Quality control of raw reads was carried out using FastQC v0.11.9. Adapters and low-quality sequences were trimmed from raw reads using Trim Galore! v0.6.6. and the trimmed reads were aligned to the reference genome using STAR v2.6.1d. BAM file filtering and indexing was carried out using SAMtools. RNAseq library quality control was implemented using RSeQC v4.0.0 and QualiMap v2.2.2-dev. Duplicate reads were marked using Picard tools v2.23.9. Library complexity was estimated using Preseq v2.0.3. Duplication rate quality control was performed using dupRadar v1.18.0. Reads overlapping with coding sequences were assigned to genes using featureCounts v2.0.1. Classification of rRNA genes and their reads were based on annotations and RepeatMasker rRNA tracks from the UCSC genome browser when applicable. Differential gene transcription analysis was completed using DESeq2 v1.28.0. Functional enrichment analysis was achieved using g:Profiler python API v1.0.0. Quality control and analysis results plots were visualized using MultiQC v1.9. BAM indexing and read coverage plots were done with IGV 2.16.0.
Assembly: Reference sequence used for read alignment: ASM950v1
Supplementary files format and content: xlsx file contains raw counts for each sample.

organism: Salmonella enterica subsp. enterica serovar Enteritidis

source_name: strain: P125109

growth_protocol: Overnight cultures were diluted in LB broth to OD600 = 0.01 and grown at 37°C and shaking until OD600 = 0.6.

extract_protocol:
The cultures were centrifugated at 8,000g for 8 min, the supernatant was discarded, and RNA was isolated using TRizol and chloroform. Samples were further purified from salts using drop dialysis in MF-Millipore™ membranes (0.025µm pore diameter) in nuclease-free water for 4 h on ice.
Libraries were prepared using the Zymo-Seq RiboFree Total RNA Library Prep Kit and sequenced on an Illumina NovaSeq platform to a sequencing depth of at least 10 million read pairs (150 bp paired-end sequencing) per sample.

value: genotype:deltaInt deltaRDF::cat


title: Muscle, 3BNC117-only

description: Library name: p25128-s001_RNp21

value: muscle


title: 3D human dental pulp stem cell spheroids treated with spermidine and TGF-β3, biological replicate 3

city: Harbin

first: WenXuan

last: Zhang

address: No. 300 Xuefu Road

institute: The Second Affiliated Hospital of Harbin Medical University

description: Library name: DPSC_3ST_3

data_processing:
Raw sequencing reads were subjected to quality control, including removal of adaptor sequences and low-quality reads.
Clean reads were aligned to the human reference genome (Homo_sapiens: NCBI_GCF_000001405.40_GRCh38.p14) using a standard RNA-seq alignment pipeline.
Gene expression levels were quantified based on read counts generated from aligned sequencing reads.
Expression normalization and differential expression analysis were performed between experimental groups using bioinformatics analysis pipelines.
Functional enrichment analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, were performed to identify biological pathways associated with differentially expressed genes.
Assembly: Homo_sapiens (human): NCBI_GCF_000001405.40_GRCh38.p14
Supplementary files format and content: Tab-delimited text file containing gene-level expression matrix, including gene identifiers, gene symbols, FPKM, TPM, raw read counts, and normalized read counts for each RNA-seq sample.

source_name: Dental pulp

growth_protocol: Human dental pulp stem cells (DPSCs) were isolated from extracted healthy human third molars and cultured in α-MEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37℃ in a humidified atmosphere containing 5% CO2. Cells at passages 3–6 were used for subsequent experiments.

extract_protocol:
Total RNA was extracted from each sample using TRIzol reagent according to the manufacturer's instructions. RNA quantity and quality were assessed using standard quality control procedures. Qualified RNA samples were used for subsequent RNA sequencing library preparation.
RNA sequencing libraries were constructed from qualified total RNA samples using the standard mRNA-seq library preparation workflow according to the manufacturer's protocol. The libraries were sequenced on the DNBSEQ-T7 platform using paired-end sequencing.

treatment_protocol: For two-dimensional (2D) culture, DPSCs were seeded and maintained under conventional adherent culture conditions. For three-dimensional (3D) culture, DPSCs were assembled into spheroids using a spheroid formation method and cultured under 3D conditions.ST-GH treatment was performed by culturing DPSCs or DPSC spheroids with a GelMA/HAMA-based hydrogel system containing spermidine (SPD) and transforming growth factor-β3 (TGF-β3). Control groups received no SPD/TGF-β3 stimulation. Samples were collected for RNA extraction after the indicated culture period.

value: Dental pulp

value: Human dental pulp stem cells

value: ST-GH treatment under 3D spheroid culture


title: No Pregnancy (Virgin) 24 month old C57Bl/6J 8

city: Melbourne

first: Bing H

last: Wang

state: VIC

address: 75 Commercial Road

country: Australia

institute: Baker Heart and Diabetes Institute

description: Library name: AA50_H

data_processing:
raw reads were demultiplexed by bcl2fastq version 2.20
and fastq files resulted were subjected to preprocessing by TrimGalore suite v0.6.10, involving Illumina adapter and priming site removal using Cutadapt version 4.8, followed by quality control assessments with FastQC v0.12.1 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc)
Before further demultiplexed the splits of fastq files and concatenated back into 23 samples for further data-processing, Multi-QC version 1.21  was performed to validate the sequencing-quality and -consistency for each split.
The filtered reads were mapped to the mouse reference genome (GRCm38.p6.vM25) using STAR aligner version 2.7.11b
BAM files were processed and indexed using Samtools version 1.19.2  and Bamtools version 2.5.2 to provide mapping statistics.
HTSeq v0.11.1 was employed for constructing counting matrices, and an aggregated quality control summary was compiled with Multi-QC.
Assembly: GRCm38.p6.vM25
Supplementary files format and content: read count normalized by HTSeq (in .txt format)

source_name: Heart_LV mid section

extract_protocol:
Hearts were perfused with ice cold phosphate buffered saline (PBS) before excision from chest cavity. The left ventricle was further dissected into 3 sections (base, mid section and apex) on a moist absorbent pad, and flash frozen in liquid nitrogen.
RNA was harvested using RNEasy Mini plus Kit (Qiagen)
cDNA was generated using Illumina Stranded Total RNA Prep kit with Ribo-Zero Plus depletion
NovaSeq X with 150 paired-end reads and sequenced to a depth of a minimum of 50M reads per sample

treatment_protocol: No treatments or interventions given

value: Heart_LV mid section


title: Salmonella ΔintΔrdf::cat rep 2

description:
Library name: Sample 5
Column name in All_samples_raw_counts.xlsx: dInt_dRDF_2


title: Th2 cells, Sample 2, high IL13

description:
Library name: Sample2high
sgRNA read counts


title: 3D human dental pulp stem cell spheroids treated with spermidine and TGF-β3, biological replicate 2

description: Library name: DPSC_3ST_2


title: No Pregnancy (Virgin) 24 month old C57Bl/6J 7

description: Library name: AA49_H


title: Th2 cells, Sample 1, mid IL13

description:
Library name: Sample1mid
sgRNA read counts


title: Salmonella ΔintΔrdf::cat rep 1

description:
Library name: Sample 4
Column name in All_samples_raw_counts.xlsx: dInt_dRDF_1


title: 3D human dental pulp stem cell spheroids treated with spermidine and TGF-β3, biological replicate 1

description: Library name: DPSC_3ST_1


title: No Pregnancy (Virgin) 24 month old C57Bl/6J 6

description: Library name: AA47_H


title: Th2 cells, Sample 1, low IL13

description:
Library name: Sample1low
sgRNA read counts


title: Human dental pulp stem cell spheroids cultured under 3D conditions, biological replicate 3

description: Library name: DPSC_3D_3

value: 3D spheroid culture


title: Th2 cells, Sample 1, high IL13

description:
Library name: Sample1high
sgRNA read counts


title: Salmonella ΔROD21::frt rep 2

description:
Library name: Sample 2
Column name in All_samples_raw_counts.xlsx: dROD21_2


title: No Pregnancy (Virgin) 24 month old C57Bl/6J 5

description: Library name: AA46_H


title: Human dental pulp stem cell spheroids cultured under 3D conditions, biological replicate 2

description: Library name: DPSC_3D_2


title: No Pregnancy (Virgin) 24 month old C57Bl/6J 4

description: Library name: AA45_H


title: primary Th2 cells, KO, donor 3

description:
Library name: Th2KO3
MN-IMS7-RS20-KO-Donor3

data_processing:
Reads were aligned using STAR, read counts generated with HTSeq, and differential gene expression determined using edgeR with a model including donor
Assembly: hg19
Supplementary files format and content: tab-delimited text file includes raw counts per gene

extract_protocol:
RNA was isolated from cells using the RNeasy Micro Kit (Qiagen).
RNA-seq libraries were prepared using NEBNext Poly(A) mRNA Isolation Module and NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs).

treatment_protocol: Primary T cells from three independent donors were transduced with lentivirus containing a GFP reporter, the Cas9 ORF, and sgRNAs targeting SLC27A3 or a non-targeting control as described above. The next day, GFP+ cells were isolated using a FACSymphony B6 (BD Biosciences), then polarized to the Th2 lineage. Fourteen days after spinfection, cells were stimulated with 5ng/mL PMA (Sigma-Aldrich) and 500ng/mL ionomycin (Sigma-Aldrich) for four hours, then pelleted.

value: SLC27A3 knockdown

value: Th2 polarization


title: Salmonella ΔROD21::frt rep 3

description:
Library name: Sample 3
Column name in All_samples_raw_counts.xlsx: dROD21_3


title: Salmonella ΔROD21::frt rep 1

description:
Library name: Sample 1
Column name in All_samples_raw_counts.xlsx: dROD21_1


title: Human dental pulp stem cell spheroids cultured under 3D conditions, biological replicate 1

description: Library name: DPSC_3D_1


title: No Pregnancy (Virgin) 24 month old C57Bl/6J 3

description: Library name: AA44_H


title: 2D cultured human dental pulp stem cells treated with spermidine and TGF-β3, biological replicate 3

description: Library name: DPSC_2ST_3

value: ST-GH treatment under 2D culture


title: No Pregnancy (Virgin) 24 month old C57Bl/6J 2

description: Library name: AA42_H


title: V5_ph_KO_1-3hr_exp3_chrom1

city: Martinsried

first: Jürg

last: Müller

state: Bavaria

address: Am Klopferspitz

country: Germany

institute: Max Planck Institute of Biochemistry

description: Library name: sample 34

data_processing:
Reads from fastq files were aligned to the dm6 genome (and to the D. pseudoobscura dp3 genome for subsequent normalization) using STAR (Dobin et al., 2013). Only sequences that mapped uniquely to the genome with a maximum of two mismatches were considered for further analyses.
Bam files were used as inputs to run Bioconductor packages.
The proportion of D. pseudoobscura reads as compared to D. melanogaster reads in input and in samples was used to normalize the ChIP-seq datasets for histone marks in embryos. Reference samples for normalization with D. pseudoobscura chromatin are indicated. Other samples were normalized based on total read number per million reads. Normalization factor for each sample is indicated.
Assembly: dm6
Supplementary files format and content: Normalized bigwig files (bin size = 1) were generated with deepTools (Ramirez et al., 2016).

source_name: 1-3hr old embryos

growth_protocol: Drosophila embryos were grown on standard medium at 25˚C

extract_protocol:
Lysates were clarified from sonicated fixed nuclei. Histone/Protein-DNA complexes were isolated with antibody. D. pseudoobscura chromatin was spiked in before the addition of the antibody.
Libraries were prepared according to manufacturer's instructions.
ChIP-Seq

value: 1-3hr old embryos

tag: antibodies

value: V5 (Cell Signalling (#D3H8Q))

tag: normalization factor


title: 2D cultured human dental pulp stem cells treated with spermidine and TGF-β3, biological replicate 2

description: Library name: DPSC_2ST_2


title: primary Th2 cells, KO, donor 2

description:
Library name: Th2KO2
MN-IMS7-RS20-KO-Donor2


title: primary Th2 cells, NT, donor 3

description:
Library name: Th2NT3
MN-IMS7-RS20-NT-Donor3

value: non-targeting control


title: No Pregnancy (Virgin) 24 month old C57Bl/6J 1

description: Library name: AA41_H


title: primary Th2 cells, NT, donor 2

description:
Library name: Th2NT2
MN-IMS7-RS20-NT-Donor2


title: Quiescent MuSC H4K20me1 biol rep2

description: Library name: Q2_H4K20me1

value: skeletal muscle

value: muscle stem cell

value: WT


title: PL12 cell line rep 1

city: New York

first: Yuan

last: Hao

address: Laura and Isaac Perlmutter Cancer Center

institute: NYU Langone Health

description: Library name: PL12A

data_processing:
Raw read quality was assessed using FASTQC. Reads were aligned to the Mus musculus reference genome (GRCm38, GENCODE M25) using the STAR aligner with default settings. Gene-level quantification (raw counts, transcripts per million [TPM], and fragments per kilobase million [FPKM]) was performed using the RSEM algorithm. Differential gene expression analysis was conducted using the DESeq2.
Assembly: GRCm38, Genome Reference Consortium Mouse Build 38 Organism
Supplementary files format and content: text file contains trancripts per million for each sample

source_name: mouse LUSC

extract_protocol:
Total RNA was extracted from PL and PLA murine cell lines using the RNeasy Plus Universal Mini Kit (QIAGEN) according to the manufacturer’s instructions.
cDNA synthesis was performed using the SMARTer PCR cDNA Synthesis Kit (Clontech), followed by library preparation with the Nextera XT DNA Library Preparation Kit (Illumina), following standard protocols

value: mouse LUSC


title: 2D cultured human dental pulp stem cells treated with spermidine and TGF-β3, biological replicate 1

description: Library name: DPSC_2ST_1


title: 16HBE, SLC27A3 KD, rep3

description:
Library name: 16HBEKD3
MN-IS-1s-SLC27A3-03_S39_L008

data_processing:
Read counts per gene were obtained with Salmon version 1.5.1 on transcripts from human Gencode release 19. Estimated counts were used in exploratory analysis (transformed with DESeq2’s rlog function) and in DESeq2 version 1.32.0.
Raw counts table was generated using STAR (aligned to hg19) and Htseq
Assembly: hg19
Supplementary files format and content: tab-delimited text file includes raw counts per gene

growth_protocol: Human immortalized 16HBE14o- bronchial epithelial cells (16HBEs, Sigma-Aldrich) were grown in DMEM supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin on tissue culture-treated plastic coated with 10 μg/mL fibronectin, 100 μg/mL bovine serum albumin fraction V, and 30 μg/mL bovine collagen.

extract_protocol:
RNA was isolated from 16HBEs treated with non-targeting (NT) or SLC27A3-targeting sgRNAs using the RNeasy Mini Kit (Qiagen).
RNA-seq libraries were prepared using NEBNext Poly(A) mRNA Isolation Module and NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs).

treatment_protocol: Genes of interest were knocked down in 16HBE cells using Cas9 ribonucleoproteins (RNPs). Briefly, guides targeting expressed exons of interest were designed with GUIDES using GTEx data from lung, skin, and blood72. To form RNPs, crRNAs were duplexed with a universal tracrRNA (Integrated DNA Technologies) at equimolar concentrations, then mixed with Alt-R Cas9 enzyme (Integrated DNA Technologies) at a ratio of 1.15:1 with equal volume PBS. 16HBE cells were nucleofected with RNPs using the 4D-Nucleofector X Unit program CM-158 (Lonza). Nucleofection was performed on 2.5x105 cells per knockdown using SG Cell Line 4D-Nucleofector X Kit S (Lonza). Immediately after nucleofection, cells were transferred to a 6-well plate to recover for one week.

value: immortalized bronchial epithelial cell


title: Multiple Pregnancy  24 month old C57Bl/6J  8

description: Library name: 9MP_H


title: Psc_ph_wt_1-3hr_exp3_chrom1

description: Library name: sample 29

value: DSHB (6E8)


title: Human dental pulp stem cells cultured under 2D conditions, biological replicate 3

description: Library name: DPSC_2D_3

value: Dental pulp

value: 2D culture


title: Multiple Pregnancy  24 month old C57Bl/6J  7

description: Library name: 8P_H


title: 16HBE, SLC27A3 KD, rep2

description:
Library name: 16HBEKD2
MN-IS-1s-SLC27A3-02_S25_L008


title: V5_ph_wt_1-3hr_exp3_chrom1

description: Library name: sample 35


title: Psc_ph_KO_1-3hr_exp3_chrom1

description: Library name: sample 28


title: Multiple Pregnancy  24 month old C57Bl/6J  6

description: Library name: 8MP_H


title: Human dental pulp stem cells cultured under 2D conditions, biological replicate 2

description: Library name: DPSC_2D_2

value: Human dental pulp stem cells


title: 16HBE, SLC27A3 KD, rep1

description:
Library name: 16HBEKD1
MN-IS-1s-SLC27A3-01_S11_L008


title: Psc_ph_deltaSAM_1-3hr_exp3_chrom1

description: Library name: sample 26


title: Human dental pulp stem cells cultured under 2D conditions, biological replicate 1

description: Library name: DPSC_2D_1


title: Multiple Pregnancy  24 month old C57Bl/6J  5

description: Library name: 7P_H

value: Heart_LV mid section


title: D. subpalustris, mixed male and female adult flies, RNA rep2

city: Houghton

first: Paul

middle: Donald

last: Goetsch

state: MI

address: 1400 Townsend Drive

department: Biological Sciences

institute: Michigan Technological University

description: Library name: D.subpalustris RNAseq rep2

data_processing:
RNAseq reads were trimmed using fastp
Trimmed RNA-seq reads were aligned to the draft softmasked genome assembly GCA_054883435.1 using STAR v2.7.11b with the --outSamstrandField intronMotif option enabled
Preliminary genome annotations were generated using BRAKER v3.0.6 on the softmasked genome assembly
RNAseq reads were remapped to the resulting genome annotation using STAR v2.7.11b to assess the percentage of uniquely mapped reads between replicates
Assembly: PDG_Dspa_wgs, GCA_054883435.1
Supplementary files format and content: tab deliminated text file (.tab) containing unnormalized gene-level raw read counts output by STAR

organism: Drosophila subpalustris

source_name: Whole body

growth_protocol: Fly stocks were maintained on cormeal-sucrose-yeast medium at room temperature.

extract_protocol:
RNA was extracted using a standard Trizol-chloroform phase separation followed by isopropanol precipitation from 10-20 mixed male and female adult flies
RNAseq libraries were generated from a total of 1 µg purified RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBext Ultra II Directional Library Prep Kit for Illumina (NEB). Samples were multiplexed using the NEBNext Multiplex Oligos for Illumina kit.

value: Whole body


title: Pho_ph_wt_1-3hr_exp3_chrom1

description: Library name: sample 24

value: Pho (Papp and Mueller 2006)


title: 16HBE, NT, rep4

description:
Library name: 16HBENT4
MN-IS-1s-NT-04_S43_L008


title: Multiple Pregnancy  24 month old C57Bl/6J  4

description: Library name: 4MP_H


title: 16HBE, NT, rep3

description:
Library name: 16HBENT3
MN-IS-1s-NT-03_S29_L008


title: D. subpalustris, mixed male and female adult flies, RNA rep1

description: Library name: D.subpalustris RNAseq rep1


title: Multiple Pregnancy  24 month old C57Bl/6J  3

description: Library name: 3MP_H


title: 16HBE, NT, rep2

description:
Library name: 16HBENT2
MN-IS-1s-NT-02_S15_L008


title: input_ph_KO_1-3hr_exp3_chrom1

description: Library name: sample 21


title: Multiple Pregnancy  24 month old C57Bl/6J  2

description: Library name: 11P_H


title: D. palustris, mixed male and female adult flies, RNA rep2

description: Library name: D.palustris RNAseq rep2

data_processing:
RNAseq reads were trimmed using fastp
Trimmed RNA-seq reads were aligned to the draft softmasked genome assembly GCA_051621885.1 using STAR v2.7.11b with the --outSamstrandField intronMotif option enabled
Preliminary genome annotations were generated using BRAKER v3.0.6 on the softmasked genome assembly
RNAseq reads were remapped to the resulting genome annotation using STAR v2.7.11b to assess the percentage of uniquely mapped reads between replicates
Assembly: PDG_Dpal_wgs, GCA_051621885.1
Supplementary files format and content: tab deliminated text file (.tab) containing unnormalized gene-level raw read counts output by STAR

organism: Drosophila palustris


title: 16HBE, NT, rep1

description:
Library name: 16HBENT1
MN-IS-1s-NT-01_S1_L008


title: Multiple Pregnancy  24 month old C57Bl/6J  1

description: Library name: 10P_H


title: PFOA 700 ng/L from 8 hpf, 24 hpf, biological replicate 4

status: Public on Aug 10 2026

city: Durham

first: Zainab

last: Afzal

state: NC

address: 700 George Street, BBRI/Kumar Lab

institute: North Carolina Central University

description: Library name: Z20

data_processing:
Raw sequencing reads underwent quality-control assessment.
Reads were aligned to the Danio rerio reference genome assembly GRCz11.
Gene-level read counts were generated from aligned reads.
Differential expression analysis was performed using the edgeR algorithm in the Novomagic analysis platform.
Assembly: GRCz11
Supplementary files format and content: csv file contains raw counts for each sample
Supplementary files format and content: csv file contains FPKM values for each sample

source_name: whole embryo

growth_protocol: Wild-type Danio rerio adults were maintained under standard zebrafish husbandry conditions. Embryos were generated from independent spawning events, staged by hours post-fertilization, and maintained in embryo medium until collection at 24 hpf.

extract_protocol:
Total RNA was extracted from pooled whole embryos using the Direct-zol RNA Miniprep Kit (Zymo Research, catalog R2073-A), including on-column DNase treatment to remove genomic DNA.
RNA-seq libraries were prepared using the Illumina TruSeq Stranded mRNA Library Prep protocol. Libraries were sequenced on an Illumina NovaSeq platform to generate 150-bp paired-end reads.

treatment_protocol: Embryos were left untreated or exposed to 700 ng/L PFOA at different developmental timepoints begining at the 1-cell, 3.5 hpf, 5 hpf, or 8 hpf stage. All treatment groups were collected at 24 hpf for RNA extraction.

value: whole embryo

value: Embryos were exposed to 700 ng/L perfluorooctanoic acid (PFOA) beginning at 8 hpf and collected at 24 hpf.

tag: group

value: PFOA treated

tag: batch

value: total RNA


title: input_ph_deltaSAM_1-3hr_exp3_chrom1

description: Library name: sample 19

value: 1-3hr old embryos


title: D. palustris, mixed male and female adult flies, RNA rep1

description: Library name: D.palustris RNAseq rep1


title: PFOA 700 ng/L from 5 hpf, 24 hpf, biological replicate 3

description: Library name: Z15

value: Embryos were exposed to 700 ng/L perfluorooctanoic acid (PFOA) beginning at 5 hpf and collected at 24 hpf.


title: PFOA 700 ng/L from 5 hpf, 24 hpf, biological replicate 4

description: Library name: Z16


title: PFOA 700 ng/L from 5 hpf, 24 hpf, biological replicate 2

description: Library name: Z14


title: PFOA 700 ng/L from 8 hpf, 24 hpf, biological replicate 1

description: Library name: Z17


title: PFOA 700 ng/L from 8 hpf, 24 hpf, biological replicate 3

description: Library name: Z19


title: PFOA 700 ng/L from 5 hpf, 24 hpf, biological replicate 1

description: Library name: Z13


title: PFOA 700 ng/L from 3.5 hpf, 24 hpf, biological replicate 4

description: Library name: Z12

value: Embryos were exposed to 700 ng/L perfluorooctanoic acid (PFOA) beginning at 3.5 hpf and collected at 24 hpf.


title: PFOA 700 ng/L from 3.5 hpf, 24 hpf, biological replicate 3

description: Library name: Z11


title: TET2KO heart, AngII, rep 3

city: London

first: Hashum

last: Sum

address: 125 Coldharbour Lane

institute: King's College London

description:
Library name: H6
BAM: H6.bam

data_processing:
Raw sequence reads were trimmed to remove adapter sequences and low-quality bases using Trimmomatic v.0.36. Paired-end reads were aligned to the Mus musculus GRCm38 (mm10) reference genome using the STAR aligner v.2.5.2b, yielding alignment files (sample.bam).
Gene-level hit counts were extracted using featureCounts from the Subread package v.1.5.2 (hit-counts/raw_counts.csv).
Gene expression levels were quantified and normalized as TPM values (hit-counts/TPM_values.csv).
Differential gene expression analysis was performed using DESeq2, and Gene Ontology (GO) enrichment analyses were conducted using GeneSCF (Significant-DEGs.csv, Differential_expression_analysis_table.csv, GO_analysis).
Assembly: mm10
Supplementary files format and content: Comma-delimited CSV file containing raw gene read counts (hit-counts/raw_counts.csv).
Supplementary files format and content: Comma-delimited CSV file containing TPM expression values (hit-counts/TPM_values.csv).

source_name: heart

growth_protocol: Mice were maintained under standard housing conditions with ad libitum access to food and water.

extract_protocol:
Total RNA was extracted from mid-ventricular heart tissue samples using standard RNA isolation protocols.
Sequencing libraries were prepared from mouse total RNA using poly(A) selection for mRNA enrichment, followed by Illumina-compatible library preparation kits for paired-end sequencing (PE 2x150) on the Illumina NovaSeq 6000 platform.

treatment_protocol: Adult wild-type and endothelial-specific TET2 knockout mice were subjected to chronic infusion of Angiotensin II (1.0 mg/kg/day) via subcutaneous osmotic minipumps for 28 days to induce cardiac hypertrophy and hypertensive remodeling.

value: heart

value: cardiac muscle tissue

value: Angiotensin II


title: PFOA 700 ng/L from 3.5 hpf, 24 hpf, biological replicate 1

description: Library name: Z9


title: PFOA 700 ng/L from 1-cell, 24 hpf, biological replicate 4

description: Library name: Z8

value: Embryos were exposed to 700 ng/L perfluorooctanoic acid (PFOA) beginning at 1-cell and collected at 24 hpf.


title: TET2KO heart, AngII, rep 2

description:
Library name: H5
BAM: H5.bam


title: TET2KO heart, AngII, rep 1

description:
Library name: H4
BAM: H4.bam


title: PFOA 700 ng/L from 8 hpf, 24 hpf, biological replicate 2

description: Library name: Z18


title: PFOA 700 ng/L from 1-cell, 24 hpf, biological replicate 3

description: Library name: Z7


title: WT heart, AngII, rep 2

description:
Library name: H2
BAM: H2.bam

value: WT


title: Untreated control, 24 hpf, biological replicate 4

description: Library name: Z4

value: Untreated control; maintained in embryo medium and collected at 24 hpf.

value: Untreated


title: Untreated control, 24 hpf, biological replicate 3

description: Library name: Z3


title: TET2KO lung endothelial cells, AngII, rep 1

description:
Library name: EC4
BAM: EC4.bam

data_processing:
Sequence reads were trimmed to remove adapter sequences and low-quality bases using Trimmomatic v.0.36.
Trimmed reads were mapped to the Mus musculus GRCm38 reference genome using the STAR aligner v.2.5.2b.
Unique gene hit counts were quantified using featureCounts from the Subread package v.1.5.2.
Differential gene expression analysis between wild-type and TET2KO groups was performed using DESeq2.
Assembly: Mus_musculus/GRCm38
Supplementary files format and content: raw_counts.csv: Comma-separated text file containing raw gene hit counts for all samples.
Supplementary files format and content: TPM_values.csv: Comma-separated text file containing Transcripts Per Million (TPM) normalized expression values.

source_name: lung

growth_protocol: Mice were subjected to 28 days of chronic Angiotensin II infusion, after which lung endothelial cells were isolated.

extract_protocol:
Total RNA was extracted using standard procedures, followed by poly(A)-selected mRNA library preparation.
Libraries were prepared using poly(A) selection and sequenced as paired-end (2x150 bp) on the Illumina NovaSeq platform.

treatment_protocol: Chronic infusion of Angiotensin II for 28 days in wild-type and endothelial-specific TET2 knockout mice.

value: lung

value: lung endothelial cells


title: WT heart, AngII, rep 1

description:
Library name: H1
BAM: H1.bam


title: PFOA 700 ng/L from 1-cell, 24 hpf, biological replicate 1

description: Library name: Z5


title: Untreated control, 24 hpf, biological replicate 2

description: Library name: Z2


title: WT lung endothelial cells, AngII, rep 3

description:
Library name: EC3
BAM: EC3.bam


title: Untreated control, 24 hpf, biological replicate 1

description: Library name: Z1


title: WT lung endothelial cells, AngII, rep 2

description:
Library name: EC2
BAM: EC2.bam


title: WT lung endothelial cells, AngII, rep 1

description:
Library name: EC1
BAM: EC1.bam


title: GSK126 treated group

city: Bethesda

first: Jianyi

last: Ding

address: 9000 Rockville Pike

department: CMTB

institute: NHLBI, NIH

description:
Library name: sample2
10x Genomics

data_processing:
The libraries were mapped to the 10X GRCh38-2020-A human reference genome using CellRanger version 7.2.0. Data were further processed using Seurat version 4.1.1 running in R v4.2.0.
Assembly: hg38
Supplementary files format and content: Tab-separated values files and matrix files

extract_protocol:
Single-cell suspensions were prepared and loaded into the Chromium Next GEM Chip G alongside reverse transcription master mix. Single cells were partitioned into nanoliter-scale Gel Bead-in-Emulsion (GEMs) using the Chromium Controller (10x Genomics), where cells were lysed and poly-adenylated mRNA molecules were captured by olge-dT primers containing a 10x cell barcode and a Unique Molecular Identifier (UMI). Full-length barcoded complementary DNA (cDNA) was synthesized via reverse transcription within the intact GEMs. Following emulsion breakage, cDNA was recovered and purified using Dynabeads / magnetic Silane beads.
Purified cDNA underwent targeted PCR amplification. The resulting product was enzymatically fragmented, end-repaired, A-tailed, and size-selected using SPRIselect beads. Standard Illumina P5 and P7 adapters along with sample index sequences (dual index sets) were ligated to the fragments. Final sample index PCR amplification was performed to yield the final Illumina-ready single-cell 3' gene expression library, which was sequenced using paired-end reads on an Illumina platform.

value: human induced pluripotent stem cells


title: Mouse lung tissue, cold exposure (4 °C, 6 h), biological replicate 3

city: Shanghai

first: Junkun

last: Jiang

address: 500 Zhennan Road, Putuo District

institute: Tongji University

description: Library name: Cold_3

data_processing:
RNA-seq data were processed using CLC Genomics Workbench.
Sequencing reads were quality assessed and adapter sequences were removed.
Clean reads were aligned to the mouse reference genome (mm10) using CLC Genomics Workbench.
Gene-level read counts were extracted using CLC Genomics Workbench to generate the raw count matrix.
Assembly: mm10
Supplementary files format and content: Excel file containing gene-level raw read count matrix for all samples.

source_name: Lung

growth_protocol: Male C57BL/6J mice were maintained under standard laboratory conditions with free access to food and water.

extract_protocol:
Total RNA was extracted from mouse lung tissues using standard RNA extraction methods.
RNA-seq libraries were constructed using the NEBNext Ultra II RNA Library Prep Kit following the manufacturer's instructions.

treatment_protocol: Mice were exposed to cold temperature (4 °C) for 6 h or maintained at room temperature as control. Lung tissues were collected after treatment.

value: Lung

value: wild type

value: cold exposure 6 h


title: DMSO treated group

description:
Library name: sample1
10x Genomics

value: DMSO


title: Mouse lung tissue, cold exposure (4 °C, 6 h), biological replicate 2

description: Library name: Cold_2


title: Mouse lung tissue, cold exposure (4 °C, 6 h), biological replicate 1

description: Library name: Cold_1


title: HeLa cells, Coxiella infected, PBS-treated

city: Glendale

first: Dhritiman

last: Samanta

state: Arizona

institute: Midwestern University

description:
Library name: HeLa-Coxiella-infected-PBS
ZCXBY5_3_3_WT_DPBS

data_processing:
Quality of the fastq files was assessed using FastQC v0.12.1. Reads were then quality filtered using fastp v0.24.0 with poly-X tail trimming, 3' quality-based tail trimming, a minimum Phred quality score of 15, and a minimum length requirement of 50 bp. Quality-filtered reads were aligned to the reference genome using STAR aligner v2.7.11 with non-canonical splice junction removal and output of unmapped reads, followed by coordinate sorting using samtools v1.22.1. PCR and optical duplicates were removed using UMI-based deduplication with UMIcollapse v1.1.0. Alignment quality metrics, strand specificity, and read distribution across genomic features were assessed using RSeQC v5.0.4 and Qualimap v2.3, with results aggregated into a comprehensive quality control report using MultiQC v1.32. Gene-level expression quantification was performed using featureCounts (subread package v2.1.1) with strand-specific counting, multi-mapping read fractional assignment, exons and three prime UTR as the feature identifiers, and grouped by gene_id.
Final gene counts were annotated with gene biotype and other metadata extracted from the reference GTF file.  Sample-sample correlations for sample-sample heatmap and PCA were calculated on normalized counts (TMM, trimmed mean of M-values) using Pearson correlation. Differential expression was done with edgeR v4.0.16 using standard practice including filtering for low-expressed genes with edgeR::filterByExprwith default values. Functional enrichment, when available, is performed using gene set enrichment analysis with gseapy v0.12 using the MSigDB Hallmark gene set.
Assembly: GRCh38_114
Supplementary files format and content: comma separated values, raw counts for all samples
Supplementary files format and content: tab-separated values, gene expression matrix

source_name: HeLa

growth_protocol: HeLa cells were plated in a 6-well plate (5x10^4 per well) and either mock-infected or infected with WT C. burnetii for 2 h. After washing extensively with PBS, cells were incubated in 10% RPMI.

extract_protocol:
Cells were then centrifuged at 400xg for 5 min, and the pellet was washed once with PBS and centrifuged again. Pellets were then resuspended in 50 µL Zymo DNA/RNA shieldTM (Zymo Research, Irvine, CA, USA) and vortexed thoroughly to lyse the cells and shear the chromosomal DNA. A total of four samples, named Mock-PBS, Mock-acetate, WT-PBS, and WT-acetate, were then sent for RNAseq and analysis to Plasmidsaurus (South San Francisco, CA, USA). The authors did not perform the extraction in-house. Therefore we quote the following from Plasidsaurus website - " We extract total RNA using a bead-based extraction approach from cells preserved in Zymo DNA/RNA Shield". Also from Plasmidsaurus website - "We assess post-extraction (or received purified RNA) concentration using a fluorescence based plate reader assay and normalize sample concentrations before proceeding. Please note we do not assess an RNA Integrity Number (RIN) before proceeding, nor do we hold samples if the extracted RNA concentration is low. Making sure you send an adequate number of intact cells is key to getting optimal results."
From Plasmidsaurus website - "we convert mRNA into complementary DNA (cDNA) via reverse transcription and second-strand synthesis, followed by tagmentation, library indexing, and amplification."

treatment_protocol: At 2 dpi, cells were treated with either PBS or 50 mM acetate and incubated in the treatment conditions for 24 h. At 3 dpi, cells were harvested, counted, and diluted to 2x10^5 cells/mL.

value: HeLa

value: Cervical epithelial

value: Coxiella infection + PBS (control)


title: Mouse lung tissue, room temperature control, biological replicate 2

description: Library name: Control_2

value: room temperature control


title: Mouse lung tissue, room temperature control, biological replicate 1

description: Library name: Control_1