title: H3K23ac, Kat6a-/– proliferating NSPCs, replicate 1

description: Library name: b2_Prol.H3K23ac_Kat6A_HOM-1


title: Cite with TCR, mouse 3, eGFP negative, CD8 T cells, HTO

status: Public on Apr 20 2026

type: SRA

city: New Haven

first: Yulong

last: Wei

state: CT

address: 295 Congress Avenue

department: Microbial Pathogenesis

country: USA

institute: Yale University

description:
Library name: Mouse_3_GFPneg_CD8_HTO
Hashtag Oligonucleotide
HTO_cellranger_antibody_list_with_barcode.csv

library_source: other

data_processing:
Cite-seq, TCR, and Multiome files were demultiplexed using Cellranger mkfastq.
Assembly: mm10
Supplementary files format and content: CellRanger output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz, per_barcode_metrics.csv
Supplementary files format and content: CellRanger output files: atac_cut_sites.bigwig, atac_fragments.tsv, atac_fragments.tsv.gz.tbi, atac_peak_annotation.tsv, atac_peaks.bed
Library strategy: CITE-seq

molecule: protein

organism: Mus musculus

source_name: mixed sample (kidney, spleen, renal lymph node)

extract_protocol:
Cite-seq with TCR: CD4 and CD8 T cells were isolated from kidney, spleen, and renal lymph node of lupus-prone MRL/MpJ-Faslpr/J or MRL/MpJ-Faslpr/J.Nur77-eGFP (sorted on eGFP expression). Single cells were encapsulated in Gel-Beads-in-Emulsion (GEMs) using the 10x Genomics Chromium Controller and Chromium Single Cell 5′ chemistry (5P HT v2 Next-GEM). Multiome: CD44hi T cells were isolated from  kidney, spleen and renal lymph node of lupus-prone MRL/MpJ-Faslpr/J mice. Single cells were encapsulated in Gel-Beads-in emulsion (GEMs) using the 10x Genomics Chromium Controller with the Chromium Single Cell Multiome ATAC + Gene Expression kit. Library preparation and sequencing were performed at the Yale Center for Genome Analysis (YCGA).
The 10x Single Cell 5' with Feature Barcoding and Immune Receptor Mapping protocols (CG000330 Rev F,  CG000208 Rev G) with TotalSeq™-C cell hashing (HTO) was used to generate 5′ gene expression, TCR V(D)J enrichment , and  HTO (hashtag) libraries.
The Single Cell Multiome ATAC + Gene Expression portocol described by 10X Genomics (CG000338 Rev G) was used to generate 3' gene expression library and ATAC libraries.

tag: tissue

tag: cell type

value: CD8 T cells

tag: genotype

value: Faslpr; Nur77-eGFP reporter (MRL/MpJ-Faslpr/J.Nur77-eGFP)

tag: library type

value: TCR

tag: hto


title: H3K14ac, Kat6a+/– proliferating NSPCs, replicate 4

city: Parkville

first: Waruni

last: Abeysekera

state: Victoria

address: 1G Royal Parade

department: Bioinformatics

country: Australia

institute: Walter and Eliza Hall Institute of Medical Research

description: Library name: b2_Prol.H3K14ac_Kat6A_HET-4

library_source: genomic

data_processing:
FASTQ files were output using standard Illumina protocols and software.
Reads were aligned to the Mus musculus (mm39) genome by Rsubread version 2.12.3.
Read counts were summarized by genomic regions - Promoters (TSS - 1kb to TSS + 1kb) and Gene body of protein coding genes, and H3K4me1+/H3K27ac+ enhancer enriched regions - using the Rsubread's featureCounts function and Rsubread's inbuilt mm39 NCBI annotation.
Assembly: mm39
Supplementary files format and content: Tab-delimited text files for Promoter region counts of protein coding genes; columns represent Entrez Gene ID, region length in base counts, and the number of sequence reads mapping to that region.
Supplementary files format and content: Tab-delimited text files for Gene body counts of protein coding genes; columns represent Entrez Gene ID, gene length in base counts, and the number of reads mapping to that gene.
Supplementary files format and content: Tab-delimited text files for H3K4me1+ enhancer enriched regions; columns represent region ID, region length in base counts, and the number of reads mapping to that region.
Supplementary files format and content: Tab-delimited text files for H3K27ac+ enhancer enriched regions; columns represent region ID, region length in base counts, and the number of reads mapping to that region.
Supplementary files format and content: Tab-delimited text files for overlapping H3K4me1+/H3K27ac+ enhancer enriched regions; columns represent region ID, region length in base counts, and the number of reads mapping to that region.
Supplementary files format and content: Separate supplementary files containing sequence read counts for all samples in matrix form are also provided for each genomic region.
Library strategy: CUT&Tag

molecule: genomic DNA

source_name: Proliferating Neural stem and progenitor cells

growth_protocol: Neural stem and progenitor cell isolation and culture The dorsal telencephalon of E12.5 embryos was dissected and placed in 1 ml of neural stem cell (NSC) proliferation medium  [Neurocult medium with the addition of proliferation supplement mouse or rat (STEMCELL Technologies, 05702), 4 µg/ml heparin solution (STEMCELL Technologies, 07980), 10 ng/ml bovine recombinant fibroblast growth factor 2 (FGF2), 20 ng/ml murine epidermal growth factor (EGF)] in Eppendorf tubes on ice. The tissue was mechanically dissociated and centrifuged at 200 g for 5 min and resuspended in fresh NSC proliferation medium. Cells were passed through a 70 µm cell strainer and plated onto 6 cm tissue culture Petri dishes and grown at 37oC in 5% CO2 in air. Proliferating neural stem and progenitor cell (NSPC) colonies were cultured as non-adherent neurospheres, 3x105 cells were passaged every 5-6 days into 10 cm dishes. For passaging, cells were collected in 15 ml Falcon tubes and chemically dissociated with Accutase (STEMCELL Technologies, 07920), centrifuged at 200 g for 5 min, washed and resuspended in 2 ml of NSC proliferation medium. Live cells were quantified using trypan blue (Bio-Rad, 1450021) exclusion in an automated cell counter (Luna II, Logos Biosystems). Cells were frozen in NSC freezing medium [Neurocult medium and proliferation supplement mouse or rat, 4 µg/ml heparin solution, 10 ng/ml FGF2, 20 ng/ml EGF, 10% DMSO] at either passage 3 or passage 4 after approximately 72 h of growth without prior dissociation.  Neural stem and progenitor cell differentiation To induce differentiation, NSPCs were passaged after 72 h of growth, 6x105 cells were washed and resuspended in 3 ml of NSC differentiation medium [Neurocult medium and proliferation supplement mouse or rat, 1% heat inactivated embryonic stem cell qualified foetal bovine serum (HI ES-FBS, Gibco, 30044333)] and plated onto 6 well plates precoated with 15 µg/mL poly-L-ornithine (Sigma-Aldrich, P4957) and 4.5 µg/mL laminin (Sigma-Aldrich, L2020). Differentiated cells were grown for 3 days at 37oC and 5% CO2.

extract_protocol:
Automated CUT&Tag sequencing To assess multiple histone marks and genome protein occupancies in each NSPCs preparation from an individual embryo, we followed an automated CUT&Tag protocol developed by Henikoff and colleagues (Janssens et al., 2021), which allows parallel processing of many samples detecting many targets in a 96-well plate format. We used a liquid handling robot (Bluewasher) for removing liquids and adding wash buffers to plates. For these CUT&Tag experiment, 1x106 NSPCs were cultured in a 20 cm dish for 3 days under proliferating conditions or 2x106 NSPCs were plated onto 10 cm dishes and cultured for 3 days under differentiation conditions. Both proliferating and differentiating NSPCs underwent chemical dissociation with Accutase (STEMCELL Technologies, 07920). NSPCs were collected, counted and washed. Cells were bound to concanavalin-A beads and permeabilised using 0.05% digitonin and 0.01% NP-40A. Permeabilised cells were counted, and 50,000 cells were aliquoted for each CUT&Tag reaction in a 96 well plate well. Cells were stained with primary antibody at 1:50 dilution overnight. Cells were stained with nanobody-Tn5 at 1:50 and tagmentation reactions were performed for 1 hour at 37˚C.
Tagmented fragments were isolated using SPRI beads. Optimal indexing PCR cycle number was determined by qPCR and all reactions for the same primary antibody were indexed using dual indexing primer (Mezger et al., 2018) for 9-20 PCR cycles. Sequencing libraries concentration ranged between 0.5-15 ng/µl as determined by QuantiFluor dsDNA. Indexed sequencing libraries were pooled and sequenced on NextSeq2000 using one P3 (1.2B reads) and three P2 (400M reads) kits producing 50 bp paired end reads and 8 bp dual indexing reads and standard Illumina sequencing primer. Some samples were re-amplified using the same number of indexing PCR cycles to acquire more material for sequencing. The antibodies to the following antigens were used: H3K9ac (Cell Signaling Technology, 9649), H3K14ac (Abcam, ab52946), H3K23ac (Millipore, 07355), MLL1 (Cell Signaling Technology, 14689), H3K4me3 (Active Motif, 36090), RNA polymerase II subunit A phosphorylated on Ser2 [POLR2A; also known as Phospho-Rpb1 CTD; (Cell Signaling Technology, 13499)], IgG (Epicypher, 13-0042).

value: Kat6a Heterozygous

tag: cut&tag antibody

value: H3K14ac (Abcam, ab52946)

tag: mouse id


title: Cite with TCR, mouse 2, eGFP negative, CD4 T cells, TCR

description:
Library name: Mouse_2_GFPneg_CD4_TCR
TCR V(D)J enrichment

library_source: transcriptomic

data_processing:
Cite-seq, TCR, and Multiome files were demultiplexed using Cellranger mkfastq.
Assembly: mm10
Supplementary files format and content: CellRanger output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz, per_barcode_metrics.csv
Supplementary files format and content: CellRanger output files: atac_cut_sites.bigwig, atac_fragments.tsv, atac_fragments.tsv.gz.tbi, atac_peak_annotation.tsv, atac_peaks.bed
Library strategy: TCR-Seq

molecule: polyA RNA

value: mixed sample (kidney, spleen, renal lymph node)

value: CD4 T cells


title: Cite with TCR, mouse 2, eGFP positive, CD4 T cells, HTO

description:
Library name: Mouse_2_GFPpos_CD4_HTO
Hashtag Oligonucleotide
HTO_cellranger_antibody_list_with_barcode.csv


title: H3K23ac, Kat6a+/– proliferating NSPCs, replicate 2

description: Library name: b2_Prol.H3K23ac_Kat6A_HET-2

value: Proliferating Neural stem and progenitor cells

value: H3K23ac (Millipore 07355)


title: H3K23ac, Kat6a+/– proliferating NSPCs, replicate 4

description: Library name: b2_Prol.H3K23ac_Kat6A_HET-4


title: Cite with TCR, mouse 3, eGFP negative, CD8 T cells, GEX

description:
Library name: Mouse_3_GFPneg_CD8_RNA
polyA RNA


title: Cite with TCR, mouse 2, eGFP positive, CD4 T cells, TCR

description:
Library name: Mouse_2_GFPpos_CD4_TCR
TCR V(D)J enrichment


title: Cite with TCR, mouse 3, eGFP positive, CD8 T cells, HTO

description:
Library name: Mouse_3_GFPpos_CD8_HTO
Hashtag Oligonucleotide
HTO_cellranger_antibody_list_with_barcode.csv


title: H3K14ac, Kat6a+/+ proliferating NSPCs, replicate 2

description: Library name: b2_Prol.H3K14ac_Kat6A_WT-2

value: Kat6a Wildype


title: H3K14ac, Kat6a+/+ proliferating NSPCs, replicate 1

description: Library name: b2_Prol.H3K14ac_Kat6A_WT-1


title: Cite with TCR, mouse 2, eGFP positive, CD4 T cells, GEX

description:
Library name: Mouse_2_GFPpos_CD4_RNA
polyA RNA


title: Cite with TCR, mouse 2, eGFP negative, CD8 T cells, TCR

description:
Library name: Mouse_2_GFPneg_CD8_TCR
TCR V(D)J enrichment


title: H3K23ac, Kat6a+/+ proliferating NSPCs, replicate 3

description: Library name: b2_Prol.H3K23ac_Kat6A_WT-3


title: Cite with TCR, mouse 4, eGFP negative, CD8 T cells, HTO

description:
Library name: Mouse_4_GFPneg_CD8_HTO
Hashtag Oligonucleotide
HTO_cellranger_antibody_list_with_barcode.csv


title: H3K23ac, Kat6a+/+ proliferating NSPCs, replicate 1

description: Library name: b2_Prol.H3K23ac_Kat6A_WT-1


title: Cite with TCR, mouse 2, eGFP negative, CD8 T cells, HTO

description:
Library name: Mouse_2_GFPneg_CD8_HTO
Hashtag Oligonucleotide
HTO_cellranger_antibody_list_with_barcode.csv

value: HTO


title: Cite with TCR, mouse 2, eGFP positive, CD8 T cells, HTO

description:
Library name: Mouse_2_GFPpos_CD8_HTO
Hashtag Oligonucleotide
HTO_cellranger_antibody_list_with_barcode.csv


title: male_aged_NSF_4

city: Sai Kung

first: Yukinori

last: Hirano

address: Clear water bay

department: LIFS

country: Hong Kong

institute: The Hong Kong University of Science and Technology

description:
Library name: S014
S014_NSF

data_processing:
The adaptors were trimmed via Trimommatic, followed by classification into ASV.
Assembly: ASV indicated in tax_table.txt
Supplementary files format and content: Matrix table with raw gene counts for every ASV and every sample
Supplementary files format and content: tab-delimited text files (ASV_result.txt) include counts of ASV for each fly.

organism: Drosophila melanogaster

source_name: Body

growth_protocol: Flies were raised under a 12 h:12 h, light:dark cycle, at 25℃ and 60% humidity.

extract_protocol:
Bodies were individually isolated and subjected to DNA extraction.
DNA was amplified by PCR targetting 250 bp of 16s rDNA. After 1st PCR, P7 and P5 primers carrying indices were used to further tag the amplified fragment, followed by analysis using NovaSeq according to the manufacturer’s instructions.

treatment_protocol: Flies at 25-d-old (aged) were collected after sleep assay to determine individuals showing sleep fragmentation.

value: Body

tag: cell line

value: adult

value: male

tag: strain

value: Cantonized w1118

tag: treatment

value: 25-d-old (aged)

tag: batch

tag: group

value: normal sleeping fly


title: Cite with TCR, mouse 4, eGFP positive, CD8 T cells, HTO

description:
Library name: Mouse_4_GFPpos_CD8_HTO
Hashtag Oligonucleotide
HTO_cellranger_antibody_list_with_barcode.csv

value: CD8 T cells


title: H3K14ac, Kat6a-/– proliferating NSPCs, replicate 2

description: Library name: b2_Prol.H3K14ac_Kat6A_HOM-2

value: Kat6a Homozygous


title: Cite with TCR, mouse 4, eGFP positive, CD8 T cells, TCR

description:
Library name: Mouse_4_GFPpos_CD8_TCR
TCR V(D)J enrichment

value: Faslpr; Nur77-eGFP reporter (MRL/MpJ-Faslpr/J.Nur77-eGFP)

value: TCR


title: Cite with TCR, mouse 1 CD8 T cells, HTO

description:
Library name: Mouse_1_CD8_HTO
Hashtag Oligonucleotide
HTO_cellranger_antibody_list_with_barcode.csv

value: Faslpr (MRL/MpJ-Faslpr/J)


title: male_aged_NSF_3

description:
Library name: S013
S013_NSF


title: H3K9ac, Kat6a+/– proliferating NSPCs, replicate 4

description: Library name: b2_Prol.H3K9ac_Kat6A_HET-4

value: Kat6a Heterozygous

value: H3K9ac (Cell Signaling Technology 9649)


title: H3K23ac, Kat6a+/+ proliferating NSPCs, replicate 2

description: Library name: b2_Prol.H3K23ac_Kat6A_WT-2

value: Proliferating Neural stem and progenitor cells


title: Cite with TCR, mouse 1 CD8 T cells, TCR

description:
Library name: Mouse_1_CD8_TCR
TCR V(D)J enrichment

value: mixed sample (kidney, spleen, renal lymph node)


title: male_aged_NSF_2

description:
Library name: S012
S012_NSF


title: H3K14ac, Kat6a+/– proliferating NSPCs, replicate 3

description: Library name: b2_Prol.H3K14ac_Kat6A_HET-3

value: H3K14ac (Abcam, ab52946)


title: Cite with TCR, mouse 3, eGFP negative, CD8 T cells, TCR

description:
Library name: Mouse_3_GFPneg_CD8_TCR
TCR V(D)J enrichment


title: H3K9ac, Kat6a+/– proliferating NSPCs, replicate 3

description: Library name: b2_Prol.H3K9ac_Kat6A_HET-3


title: Cite with TCR, mouse 1 CD8 T cells, GEX

description:
Library name: Mouse_1_CD8_RNA
polyA RNA

value: GEX


title: H3K14ac, Kat6a+/– proliferating NSPCs, replicate 2

description: Library name: b2_Prol.H3K14ac_Kat6A_HET-2


title: H3K14ac, Kat6a+/– proliferating NSPCs, replicate 1

description: Library name: b2_Prol.H3K14ac_Kat6A_HET-1


title: Uncompressed 3

city: Notre Dame

first: Julian

last: Najera

state: IN

address: 2031 McCourtney Hall East

institute: University of Notre Dame

description:
Library name: P2-C3
NIH3T3 fibroblasts grown under standard uncompressed conditions

data_processing:
Reads were aligned using the STAR alignment tool (v2.7.2)
Assembly: GRCm39
Supplementary files format and content: tab-delimited text file includes counts matrix for all samples
Supplementary files format and content: tab-delimited file includes raw counts for each individual sample

source_name: embryonic

growth_protocol: NIH3T3 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1X P/S in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
DirectZol RNA Miniprep
RNA libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit

treatment_protocol: NIH3T3 fibroblasts were subjected to 0.14 kPa of compression or 0 kPa of compression (uncompressed).

value: embryonic

value: fibroblast

value: WT

value: uncompressed


title: Compressed 3

description:
Library name: P2-C2
NIH3T3 fibroblasts subjected to in vitro mechanical compression

value: compressed


title: Uncompressed 2

description:
Library name: P2-C1
NIH3T3 fibroblasts grown under standard uncompressed conditions


title: H3K14ac, Kat6a+/+ proliferating NSPCs, replicate 3

description: Library name: b2_Prol.H3K14ac_Kat6A_WT-3

value: Kat6a Wildype


title: male_aged_SF_5

description:
Library name: S010
S010_SF

value: Body

value: adult

value: male

value: Cantonized w1118

value: 25-d-old (aged)

value: sleep-fragmented fly


title: Uncompressed 1

description:
Library name: P1-C2
NIH3T3 fibroblasts grown under standard uncompressed conditions


title: Cite with TCR, mouse 3, eGFP positive, CD8 T cells, GEX

description:
Library name: Mouse_3_GFPpos_CD8_RNA
polyA RNA

value: CD8 T cells

value: Faslpr; Nur77-eGFP reporter (MRL/MpJ-Faslpr/J.Nur77-eGFP)


title: male_aged_SF_4

description:
Library name: S009
S009_SF


title: Cite with TCR, mouse 3, eGFP positive, CD8 T cells, TCR

description:
Library name: Mouse_3_GFPpos_CD8_TCR
TCR V(D)J enrichment

value: mixed sample (kidney, spleen, renal lymph node)

value: TCR


title: H3K9ac, Kat6a+/+ proliferating NSPCs, replicate 3

description: Library name: b2_Prol.H3K9ac_Kat6A_WT-3

value: Proliferating Neural stem and progenitor cells

value: H3K9ac (Cell Signaling Technology 9649)


title: H3K9ac, Kat6a-/– proliferating NSPCs, replicate 3

description: Library name: b2_Prol.H3K9ac_Kat6A_HOM-3

value: Kat6a Homozygous


title: H3K9ac, Kat6a+/+ proliferating NSPCs, replicate 2

description: Library name: b2_Prol.H3K9ac_Kat6A_WT-2


title: male_aged_NSF_5

description:
Library name: S015
S015_NSF

value: normal sleeping fly


title: H3K9ac, Kat6a-/– proliferating NSPCs, replicate 2

description: Library name: b2_Prol.H3K9ac_Kat6A_HOM-2


title: Cite with TCR, mouse 2, eGFP positive, CD8 T cells, TCR

description:
Library name: Mouse_2_GFPpos_CD8_TCR
TCR V(D)J enrichment


title: UF1hypo_shUHRF1

type: genomic

city: Guangzhou

first: Jiongliang

last: Wang

state: Guangdong

address: 190 Kaiyuan Avenue

country: China

institute: GUANGZHOU INSTITUTES OF BIOMEDICINE AND HEALTH,CHINESE ACADEMY OF SCIENCES

hyb_protocol: illumina

scan_protocol: illumina

data_processing:
Chip Analysis Methylation Pipeline (ChAMP) package v2.26.0
Normalized beta

label: Cy3 and Cy5

organism: Homo sapiens

source_name: colon

label_protocol: illumina

extract_protocol: cell-free DNA

value: colon


title: male_aged_SF_2

description:
Library name: S007
S007_SF


title: Cite with TCR, mouse 2, eGFP negative, CD8 T cells, GEX

description:
Library name: Mouse_2_GFPneg_CD8_RNA
polyA RNA

value: GEX


title: H3K9ac, Kat6a-/– proliferating NSPCs, replicate 1

description: Library name: b2_Prol.H3K9ac_Kat6A_HOM-1


title: male_aged_SF_1

description:
Library name: S006
S006_SF


title: male_aged_NSF_1

description:
Library name: S011
S011_NSF


title: UF1hypo_shScr


title: Id2 KO, Term, ATAC, rep1

city: washington dc

first: Shaoqi

last: Zhu

state: WASHINGTON

address: 725 21st st nw

institute: the george washington university

description:
Library name: Day-8-sgID2-Term-1
Day-8-sgID2-Term_scaled.bw
Day-8-sgID2-Term_peaks.narrowPeak

data_processing:
The sequencing quality of ATAC-seq libraries was assessed by FastQC (v0.11.8)(https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)
ATAC-seq reads were trimmed using Trim Galore (0.6.7) (-q 20 --phred33 --stringency 3 --fastqc --length 30 -e 0.1) to trim low quality sequences and remove adapters.
Bowtie2 v2.3.4.1 was used to align the sequencing reads to the mm10 mouse genome, and only uniquely mapped reads were retained with PCR duplicates removal using MarkDuplicate from Picard(picard-2.25.5-0)
MACS2( V2.1.1.20160309) as used for calling ATAC peaks, with FDR < 0.00001 and  --fe-cutoff 4 .
Assembly: mm10
Supplementary files format and content: bigWig

source_name: spleen

growth_protocol: Naive P14 TCR transgenic CD8+ T cells were nucleofected with sgRNAs targeting Thy1 (as a negative control) and those targeting Id2. The nucleofected P14 cells were adoptively transferred into CD45-disparate recipients and subsequently infected with LCMV-Clone 13 to induce chronic viral infection. On day 8 post-infection,  Tim3+Slamf6- P14 cells were sorted and purified as effector exhausted CD8+ T (Tex-eff) cells, while Tim3−Slamf6+ P14 cells were sorted and purified as precursors to stem-like exhausted CD8+ T (Tex-prec) cells, and both subsets were used for ATAC-seq analysis. WT or CD4-Cre+ Id3 fl/fl mice were directed infected with LCMV-Clone 13 to induce chronic viral infection. On day 8 post-infection, antigen-specific CD8+ T cells were detected with GP33- MHC-I tetramer. Among tetramer+ cells,  Tim3+Slamf6- P14 cells were sorted and purified as effector exhausted CD8+ T (Tex-eff) cells, while Tim3−Slamf6+ P14 cells were sorted and purified as precursors to stem-like exhausted CD8+ T (Tex-prec) cells, and both subsets were used for ATAC-seq analysis.

extract_protocol:
ATAC-seq was performed on WT and ID3KO GP33-tetramer+ CD8+ T cells, or CD45.2+ sgThy1 and sgId2 P14 CD8+ T cells sorted on day 8 post-infection with LCMV- clone 13.
Sort-purified cells were treated with TN5 transposase to generate tagged DNA fragments. TN5-tagged DNA fragements were purified, PCR amplified to build ATAC-seq libraries. The resulting libraries were seqeunced with Illumina NovaSeq 6000 platform.

treatment_protocol: Sorted cells were used directly for downstream analyses without further treatment.

value: spleen

value: CD45.2+ CD8+ T cells

value: sgRNA targeting Thy1 and Id2

value: day 8 post-infection with LCMV Cl13


title: Id2 KO, Stem, ATAC, rep3

description:
Library name: Day-8-sgID2-Stem-3
Day-8-sgID2-Stem_scaled.bw
Day-8-sgID2-Stem_peaks.narrowPeak


title: HCT116_shUHRF1


title: aged_SF_head_5

description:
Library name: male_aged_SF_head_5
Sleep fragmented fly

data_processing:
The adaptors were trimmed via Trimommatic, followed by mapping to the Drosophila reference genome, dm6 from UCSC using STAR.
The reads with low mapping quality below 8 and the non-primary mapped reads were eliminated.
The filtered reads were analyzed with HTSeq-count to obtain numbers of the reads mapped on exons, which was further analyzed on R using DESeq2
Assembly: dm6
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample
Supplementary files format and content: tab-delimited text files include statistical results and fold enrichment comparing between groups

source_name: Head

extract_protocol:
Heads and bodies were individually isolated and subjected to total RNA extraction for RNA-seq.
The library for nuclear RNA-seq was prepared by Oligo d(T)25 Magnetic Beads (New England Biolabs inc. Ipswich, MA, USA), and NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs inc. Ipswich, MA, USA), according to the manufacturer’s instructions.

treatment_protocol: Flies at 5-d-old (young) or 25-d-old (aged) were collected after sleep assay to determine individuals showing sleep fragmentation.

value: Head

value: adult

value: male

value: Cantonized w1118


title: H3K9ac, Kat6a+/– proliferating NSPCs, replicate 1

description: Library name: b2_Prol.H3K9ac_Kat6A_HET-1

value: Kat6a Heterozygous


title: Compressed 2

description:
Library name: P1-C3
NIH3T3 fibroblasts subjected to in vitro mechanical compression

value: embryonic

value: fibroblast

value: WT

value: compressed


title: Id2 KO, Stem, ATAC, rep1

description:
Library name: Day-8-sgID2-Stem-1
Day-8-sgID2-Stem_scaled.bw
Day-8-sgID2-Stem_peaks.narrowPeak


title: H3K9ac, Kat6a+/– proliferating NSPCs, replicate 2

description: Library name: b2_Prol.H3K9ac_Kat6A_HET-2

value: Proliferating Neural stem and progenitor cells

value: H3K9ac (Cell Signaling Technology 9649)


title: aged_SF_head_4

description:
Library name: male_aged_SF_head_4
Sleep fragmented fly

value: 25-d-old (aged)


title: HCT116_shScr

value: colon


title: Compressed 1

description:
Library name: P1-C1
NIH3T3 fibroblasts subjected to in vitro mechanical compression


title: Id2 KO, Term, ATAC, rep3

description:
Library name: Day-8-sgID2-Term-3
Day-8-sgID2-Term_scaled.bw
Day-8-sgID2-Term_peaks.narrowPeak

value: spleen


title: male_aged_SF_3

description:
Library name: S008
S008_SF

value: Body

value: sleep-fragmented fly


title: mSI_org_Lkb1HomBud_KrasG12D_3

city: Utrecht

first: Jelte

last: van der Vaart

address: Universiteitsweg 100

department: Center for Molecular Medicine

country: Netherlands

institute: University Medical Center Utrecht

description: Library name: C6K2.3

data_processing:
Single-end RNASeq reads were processed using the UMCU RNASeq pipeline (https://github.com/UMCUGenetics/RNASeq, v2.3.0) with default settings. Read quality was assessed by FastQC (0.11.4) followed by splice-aware alignment against the mouse reference genome (GRCm38) with STAR (2.4.2a). RNA expression quantification was performed with htseq-count (0.6.0) in reverse-stranded mode. Multiple sequence runs were integrated using standardized integration parameters.
Assembly: GRCm38
Supplementary files format and content: tab-delimited text file with raw mapped counts for samples sequenced in run MER3145
Supplementary files format and content: tab-delimited text file with raw mapped counts for samples sequenced in run PLU6264
Supplementary files format and content: tab-delimited text file with raw mapped counts for samples sequenced in run BUG7424

molecule: total RNA

source_name: Small Intestine

growth_protocol: Small intestinal organoids were grown under standard conditions. This includes ENR growth medium: advanced DMEM/F12 (Gibco) supplemented with 1% Penicillin-Streptomycin (Sigma-Aldrich), 10 mM HEPES (Gibco), 1% Ultraglutamine 1 (Lonza), 1x B27 (Sigma-Aldrich), 1x N2 (Sigma-Aldrich) and 1 mM N-acetylcysteine (Sigma- Aldrich). Basal culture medium was further supplemented with 50 ng/mL murine recombinant Egf (Peprotech), 2.5% R-spondin1 (RSPO) conditioned medium (homemade) and 1% Noggin conditioned medium (homemade)

extract_protocol:
Organoids were lysed in RLT lysis buffer (Qiagen) 3-5 days after passaging. Biological triplicates were employed. To isolate RNA from samples, the “miRNA CT 400” protocol was run on a QIAsymphony isolation robot using the QIAsymphony RNA Kit (931636) on each sample in 400ul of the RLT Plus lysis buffer (1053393). RNA quality was checked with the Agilent Fragment Analyzer 5300 system using the RNA Kit (15nt) (Cat. DNF-471-1000). And RNA quantity was measured with the Invitrogen™ Qubit ™ Fluorometer using the Qubit RNA HS Assay Kit (Cat. Q32855).
100ng of total RNA was used to prepare TruSeq Stranded mRNA libraries (Cat. 20020594) following the manufacturers protocol, with full (WT, Lkb1+/- and Lkb1-/- organoids) or half (Lkb1mut + Apc-/- and Lkb1mut + KrasG12D organoids) the reagents volume. After the library preparation libraries were checked with the Fragment Analyzer system dsDNA 910 Reagent Kit (35-1500bp) (Cat. DNF-910-K1000) and with Qubit dsDNA HS Assay Kit (Cat. Q32854). Sample libraries were pooled equimolar.

value: Small Intestine

value: mSI organoids

value: Lkb1 -/- and KrasG12D


title: Id2 WT, Term, ATAC, rep2

description:
Library name: Day-8-sgTHY1.2-Term-2
Day-8-sgTHY1.2-Term_scaled.bw
Day-8-sgTHY1.2-Term_peaks.narrowPeak

value: CD45.2+ CD8+ T cells

value: sgRNA targeting Thy1 and Id2

value: day 8 post-infection with LCMV Cl13


title: H3K9ac, Kat6a+/+ proliferating NSPCs, replicate 1

description: Library name: b2_Prol.H3K9ac_Kat6A_WT-1

value: Kat6a Wildype


title: UF1hypo_UHRF1+-7


title: Id2 WT, Term, ATAC, rep3

description:
Library name: Day-8-sgTHY1.2-Term-3
Day-8-sgTHY1.2-Term_scaled.bw
Day-8-sgTHY1.2-Term_peaks.narrowPeak


title: Id2 WT, Term, ATAC, rep1

description:
Library name: Day-8-sgTHY1.2-Term-1
Day-8-sgTHY1.2-Term_scaled.bw
Day-8-sgTHY1.2-Term_peaks.narrowPeak


title: Id2 KO, Stem, ATAC, rep2

description:
Library name: Day-8-sgID2-Stem-2
Day-8-sgID2-Stem_scaled.bw
Day-8-sgID2-Stem_peaks.narrowPeak


title: HCT116


title: Id2 KO, Term, ATAC, rep2

description:
Library name: Day-8-sgID2-Term-2
Day-8-sgID2-Term_scaled.bw
Day-8-sgID2-Term_peaks.narrowPeak


title: mSI_org_Lkb1HomBud_KrasG12D_2

description: Library name: C6K2.2


title: mSI_org_Lkb1HomBud_KrasG12D_1

description: Library name: C6K2.1


title: mSI_org_Lkb1HomCys_KrasG12D_2

description: Library name: C20K3.2


title: Cite with TCR, mouse 2, eGFP positive, CD8 T cells, GEX

description:
Library name: Mouse_2_GFPpos_CD8_RNA
polyA RNA

value: mixed sample (kidney, spleen, renal lymph node)

value: CD8 T cells

value: Faslpr; Nur77-eGFP reporter (MRL/MpJ-Faslpr/J.Nur77-eGFP)

value: GEX


title: mSI_org_Lkb1HomCys_KrasG12D_3

description: Library name: C20K3.3


title: aged_SF_head_1

description:
Library name: male_aged_SF_head_1
Sleep fragmented fly

value: Head

value: adult

value: male

value: Cantonized w1118


title: UHRF1+-19

value: colon


title: aged_NSF_head_4

description:
Library name: male_aged_NSF_head_4
normal sleeping fly

value: 25-d-old (aged)


title: MT1hypo_GSK862_50nM


title: mSI_org_Lkb1HomCys_KrasG12D_1

description: Library name: C20K3.1

value: Small Intestine


title: mSI_org_Lkb1Het_KrasG12D_3

description: Library name: C9K3.3

value: mSI organoids

value: Lkb1 +/- and KrasG12D


title: aged_NSF_head_3

description:
Library name: male_aged_NSF_head_3
normal sleeping fly


title: mSI_org_Lkb1Het_KrasG12D_1

description: Library name: C9K3.1


title: mSI_org_WT_KrasG12D_3

description: Library name: K4.3


title: aged_NSF_head_1

description:
Library name: male_aged_NSF_head_1
normal sleeping fly


title: aged_SF_head_2

description:
Library name: male_aged_SF_head_2
Sleep fragmented fly


title: MT1hypo_DMSO


title: mSI_org_WT_KrasG12D_2

description: Library name: K4.2


title: Id2 WT, Stem, ATAC, rep3

description:
Library name: Day-8-sgTHY1.2-Stem-3
Day-8-sgTHY1.2-Stem_scaled.bw
Day-8-sgTHY1.2-Stem_peaks.narrowPeak

value: spleen

value: CD45.2+ CD8+ T cells


title: MT1hypo_DAC_10nM


title: aged_NSF_head_5

description:
Library name: male_aged_NSF_head_5
normal sleeping fly


title: MT1hypo_GSK862_500nM


title: HCT116_GSK862_500nM


title: Id2 WT, Stem, ATAC, rep2

description:
Library name: Day-8-sgTHY1.2-Stem-2
Day-8-sgTHY1.2-Stem_scaled.bw
Day-8-sgTHY1.2-Stem_peaks.narrowPeak

value: sgRNA targeting Thy1 and Id2

value: day 8 post-infection with LCMV Cl13


title: HCT116_GSK862_50nM


title: Id2 WT, Stem, ATAC, rep1

description:
Library name: Day-8-sgTHY1.2-Stem-1
Day-8-sgTHY1.2-Stem_scaled.bw
Day-8-sgTHY1.2-Stem_peaks.narrowPeak


title: mSI_org_Lkb1Het_KrasG12D_2

description: Library name: C9K3.2


title: MT1hypo_DAC_100nM


title: HCT116_DAC_100nM


title: mSI_org_Lkb1HomCys_APCKO_3

description: Library name: C20A3.3

value: Lkb1 -/- and Apc -/-


title: Id3 KO, Term, ATAC, rep3

description:
Library name: Day-8-ID3-KO-tet-Term-3
Day-8-ID3-KO-tet-Term_scaled.bw
Day-8-ID3-KO-tet-Term_peaks.narrowPeak

value: Cd11a high,  GP33-tetramer+, CD8+ T cells

value: WT and Id3 KO with Cd4-Cre


title: aged_NSF_head_2

description:
Library name: male_aged_NSF_head_2
normal sleeping fly


title: Id3 KO, Stem, ATAC, rep1

description:
Library name: Day-8-ID3-KO-tet-Stem-1
Day-8-ID3-KO-tet-Stem_scaled.bw
Day-8-ID3-KO-tet-Stem_peaks.narrowPeak


title: Id3 KO, Term, ATAC, rep2

description:
Library name: Day-8-ID3-KO-tet-Term-2
Day-8-ID3-KO-tet-Term_scaled.bw
Day-8-ID3-KO-tet-Term_peaks.narrowPeak


title: Id3 KO, Term, ATAC, rep1

description:
Library name: Day-8-ID3-KO-tet-Term-1
Day-8-ID3-KO-tet-Term_scaled.bw
Day-8-ID3-KO-tet-Term_peaks.narrowPeak


title: HCT116_DMSO


title: mSI_org_Lkb1HomCys_APCKO_1

description: Library name: C20A3.1


title: Id3 WT, Term, ATAC, rep2

description:
Library name: Day-8-WT-tet-Term-2
Day-8-WT-tet-Term_scaled.bw
Day-8-WT-tet-Term_peaks.narrowPeak


title: mSI_org_WT_KrasG12D_1

description: Library name: K4.1


title: young_head_5

description:
Library name: male_young_head_5
normal sleeping fly

value: 5-d-old (young)


title: aged_SF_body_5

description:
Library name: male_aged_SF_body_5
Sleep fragmented fly

value: Body


title: Id3 KO, Stem, ATAC, rep3

description:
Library name: Day-8-ID3-KO-tet-Stem-3
Day-8-ID3-KO-tet-Stem_scaled.bw
Day-8-ID3-KO-tet-Stem_peaks.narrowPeak


title: NMIBC tumor biopsy 1192302F

city: Bothell

first: Joseph

middle: D

last: Dekker

state: Washington

institute: Pfizer

description: Library name: 1192302F

data_processing:
Reads were aligned to the human GENCODE v28 transcriptome using STAR aligner (v2.6.0c)
Gene expression was quantified using RSEM (v1.3.0)
QC metrics were computed using picard (v2.18.7) and RNA-SeQC (v1.1.8)
Data4Cure was utilized for analysis and visualization (https://www.data4cure.com)
Assembly: hg38
Supplementary files format and content: Tab-delimited text file with log2-transformed RSEM TPM values per sample

source_name: bladder tumor

growth_protocol: Not applicable (clinical tumor specimens)

extract_protocol:
Qiagen RNeasy Plus Universal Mini kit, quantified by Qubit 2.0, integrity by TapeStation
NEBNext Ultra II RNA Library Prep Kit for Illumina, polyA selection with Oligo d(T) beads, ~500 ng input, ~10 PCR cycles, validated by TapeStation/Qubit/qPCR, sequenced by GENEWIZ/Azenta

treatment_protocol: Not applicable (untreated surgical specimens)

value: bladder tumor

tag: biobank

value: BioIVT


title: mSI_org_Lkb1HomBud_APCKO_2

description: Library name: C6A.2


title: young_head_1

description:
Library name: male_young_head_1
normal sleeping fly

value: Head

value: adult

value: male

value: Cantonized w1118


title: young_head_3

description:
Library name: male_young_head_3
normal sleeping fly


title: Id3 WT, Term, ATAC, rep1

description:
Library name: Day-8-WT-tet-Term-1
Day-8-WT-tet-Term_scaled.bw
Day-8-WT-tet-Term_peaks.narrowPeak


title: aged_SF_head_3

description:
Library name: male_aged_SF_head_3
Sleep fragmented fly

value: 25-d-old (aged)


title: young_head_4

description:
Library name: male_young_head_4
normal sleeping fly


title: mSI_org_Lkb1HomBud_APCKO_1

description: Library name: C6A.1

value: Small Intestine

value: mSI organoids


title: Id3 WT, Stem, ATAC, rep3

description:
Library name: Day-8-WT-tet-Stem-3
Day-8-WT-tet-Stem_scaled.bw
Day-8-WT-tet-Stem_peaks.narrowPeak

value: spleen


title: NMIBC tumor biopsy 1239506F

description: Library name: 1239506F


title: mSI_org_Lkb1HomCys_APCKO_2

description: Library name: C20A3.2


title: aged_SF_body_2

description:
Library name: male_aged_SF_body_2
Sleep fragmented fly


title: NMIBC tumor biopsy 1209437F

description: Library name: 1209437F


title: Id3 KO, Stem, ATAC, rep2

description:
Library name: Day-8-ID3-KO-tet-Stem-2
Day-8-ID3-KO-tet-Stem_scaled.bw
Day-8-ID3-KO-tet-Stem_peaks.narrowPeak


title: mSI_org_Lkb1Het_APCKO_2

description: Library name: C9A2.2

value: Lkb1 +/- and Apc -/-


title: Id3 WT, Stem, ATAC, rep1

description:
Library name: Day-8-WT-tet-Stem-1
Day-8-WT-tet-Stem_scaled.bw
Day-8-WT-tet-Stem_peaks.narrowPeak

value: day 8 post-infection with LCMV Cl13


title: mSI_org_Lkb1Het_APCKO_3

description: Library name: C9A2.3


title: NMIBC tumor biopsy 1240255F

description: Library name: 1240255F


title: HCT116_DAC_10nM

value: colon


title: aged_SF_body_1

description:
Library name: male_aged_SF_body_1
Sleep fragmented fly


title: Id3 WT, Term, ATAC, rep3

description:
Library name: Day-8-WT-tet-Term-3
Day-8-WT-tet-Term_scaled.bw
Day-8-WT-tet-Term_peaks.narrowPeak

value: Cd11a high,  GP33-tetramer+, CD8+ T cells


title: NMIBC tumor biopsy 1192680F

description: Library name: 1192680F


title: young_head_2

description:
Library name: male_young_head_2
normal sleeping fly


title: TN_CD_female_17w_187_heart

city: LAUSANNE

first: Johan

last: Auwerx

address: Station 15

department: SV-IBI

country: Switzerland

institute: Ecole Polytechnique Fédérale de Lausanne EPFL

description: Library name: HDP-018755_heart

data_processing:
Bulk RNA barcoding and sequencing (BRB-seq) libraries were generated and sequenced by Alithea Genomics (Switzerland). The raw data was demultiplexed, cleaned, and aligned to the mouse reference genome GRCm39/mm39 using R (version 4.1.a) and START (version 2.7.9a). Count matrices were used for downstream analyses
Assembly: genome GRCm39/mm39
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample

source_name: heart

extract_protocol:
RNA was extracted from liver and heart tissues using Direct-zol-96 RNA kit (Zymo research)
The generation of bulk RNA Barcoding and sequencing (BRB-seq) libraries was performed using the MERCURIUSTM BRB-seq library preparation kit for Illumina and following the manufacturer’s manual

value: heart


title: RT_CD_male_17w_186_heart

description: Library name: HDP-018880_heart


title: mSI_org_WT_APCKO_2

description: Library name: A2.2

value: Apc -/-


title: mSI_org_Lkb1HomBud_APCKO_3

description: Library name: C6A.3

value: Lkb1 -/- and Apc -/-


title: aged_SF_body_3

description:
Library name: male_aged_SF_body_3
Sleep fragmented fly

value: Body


title: aged_SF_body_4

description:
Library name: male_aged_SF_body_4
Sleep fragmented fly


title: aged_NSF_body_4

description:
Library name: male_aged_NSF_body_4
normal sleeping fly


title: TN_WD_male_17w_185_heart

description: Library name: HDP-018703_heart


title: aged_NSF_body_3

description:
Library name: male_aged_NSF_body_3
normal sleeping fly


title: Id3 WT, Stem, ATAC, rep2

description:
Library name: Day-8-WT-tet-Stem-2
Day-8-WT-tet-Stem_scaled.bw
Day-8-WT-tet-Stem_peaks.narrowPeak

value: WT and Id3 KO with Cd4-Cre


title: NMIBC tumor biopsy 1240530F

description: Library name: 1240530F

value: bladder tumor

value: BioIVT


title: RT_CD_male_17w_184_heart

description: Library name: HDP-018952_heart


title: mSI_org_WT_APCKO_3

description: Library name: A2.3


title: aged_NSF_body_5

description:
Library name: male_aged_NSF_body_5
normal sleeping fly

value: adult

value: male


title: mSI_org_Lkb1Het_APCKO_1

description: Library name: C9A2.1


title: RT_WD_male_17w_182_heart

description: Library name: HDP-018961_heart


title: NMIBC tumor biopsy 1240507F

description: Library name: 1240507F


title: young_body_5

description:
Library name: male_young_body_5
normal sleeping fly

value: Cantonized w1118

value: 5-d-old (young)


title: mSI_org_WT_APCKO_1

description: Library name: A2.1

value: Small Intestine

value: mSI organoids


title: NMIBC tumor biopsy 1210237F

description: Library name: 1210237F


title: TN_WD_female_3d_181_heart

description: Library name: HDP-019011_heart


title: Liver Ercc1 d/- AL 16wks #4

first: Wilbert

last: Vermeij

address: Heidelberglaan 25

institute: Princess Máxima Center

description: Library name: IRL23

data_processing:
Processing of raw sequencing reads was performed on an in house-generated data analysis pipeline. Removal of sequence adaptors, tRNA, and rRNA from both mRNA- and Ribo-seq and truncation of mRNA reads was performed using Trimmomatic (version 0.39). Trimmed reads were aligned to the coding region (CDS) of the mouse reference genome (annotation: gencode.vM20.annotation.gtf; genome: GRCm38.p6.genome.fa; http://gencodegenes.org/mouse/release_M20.html) using STAR (version 2.7.0f). Read counts for each gene were obtained using FeatureCounts (as part of SubRead version 1.6.4).
For ORF quantification, ribosome-profiling adapters were clipped from reads and filtered for the standard quality threshold used by CutAdapt v3.4. Fragments shorter than 25 nucleotides were discarded. Next, reads were filtered for mitochondrial, rRNA and tRNA sequence contaminations using Bowtie2 v2.4.2. All reads not mapped to the custom contaminant annotation were seen as ribosome-protected fragments (RPF) and kept. The RPFs were mapped to the Ensembl mouse genome (GRCm38 - Ensembl release 102) with STAR v2.7.8a and mapped RPFs were analyzed with ORFquant using default settings. All CDS regions from the ORFquant output were analyzed for potential P-sites across all three frames by a custom Python script. In-frame P-sites were retained, yielding one nucleotide (NT) position per codon. An intersection operation was performed on the resulting BED file with the actual P-sites per sample calculated by ORFquant, providing the number of P-sites present per transcript per sample.
Assembly: GRCm38.p6.genome.fa gencode.vM20.annotation.gtf
Supplementary files format and content: ribo_liver_counts_file.txt; Tab-delimited count files produced by featureCounts
Supplementary files format and content: ORF_count_file_psites.txt; Tab-delimited txt file based on BED files produced by ORFquant
Library strategy: Ribo-Seq

molecule: cytoplasmic RNA

source_name: Liver

growth_protocol: Animals were housed individually in ventilated cages under specific pathogen-free conditions, in carefully controlled environments (20–22 °C, 12h light:12h dark cycle). All animals were bred and maintained on AIN93G synthetic pellets (Research Diet Services B.V., Wijk bij Duurstede, The Netherlands; gross energy content 4.9 kcal/g dry mass, digestible energy 3.97 kcal/g). On average, 6-16 week old Ercc1Δ/− mice ate 2.3 g food per day. The animals that were given dietary restriction (DR) were so from 7 weeks of age, when development was largely completed, starting with 10% restriction. DR was gradually increased weekly with 10%, until 30% DR (1.6 g/day), which was provided from 9 weeks of age onward as previously published. Adult WT mice ate on average 3.0 g food per day, resulting in 2.1 g/day for 30% DR. To avoid alterations in the biological clock, food was provided to the animals just before the start of the dark (active) period, Zeitgeber Time (ZT) 12:00, except for the day of sacrifice, when no food was given prior to collect.

extract_protocol:
Snap frozen tissue samples from the same livers as used for mRNA sequencing were used to perform ribosome profiling with minimal modifications as previously described. Samples were powdered with a BioSpec Pulverizer (#59012MS) on dry ice. Pulverized material was lysed with 500ul ice-cold lysis buffer (20 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 0.1% IGEPAL CA-630 (Sigma-Aldrich, USA), 1 mM DTT, 10 U/ml RNase-free DNase 1 (Lucigen, USA), 100 µg/ml cycloheximide (Sigma-Aldrich, USA)) for 15 minutes on ice, triturated ten times through a 23G needle, and clarified through centrifugation at 20000 g at 4 °C for 10 min. Total RNA content of sample lysates was estimated using the Qubit™ RNA broad range (BR) Assay Kit (Thermo Fisher Scientific, USA) on an Invitrogen™ Qubit™ 4 fluorometer (Thermo Fisher Scientific, USA). Lysates were then digested per 200 µl aliquots with RNAse I (10 units per 20 µg of RNA; Lucigen, USA) for generation of ribosome-protected fragments (RPFs) at 20 °C for 45 min while shaking at 400 RPM on a thermomixer. The digestion reaction was stopped by adding 5 µl (5U) of SUPERase*In RNAse inhibitor (Thermo Fisher Scientific, USA) and placing the samples on ice. Digested lysates were transferred to Microspin S-400 HR sephacryl columns (Sigma-Aldrich, USA) equilibrated with 3 ml of cold RNase-free polysome buffer (20 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM MgCl2) and centrifuged at 600g at room temperature for 2 min. 10 µl 10% SDS was added to the digested lysates and RPFs were extracted using 3 volumes (660 µl) of Trizol LS (Fisher Scientific, USA), followed by addition of 1 volume (880 µl) of ethanol and RNA purification using the Zymo Direct-zol RNA micro prep kit (Zymo Research, USA) with slight modifications to the manufacturer’s instructions: Columns were spun dry for 1 min at 12000 g and isolated RPFs were eluted in 20 µl nuclease-free water (Sigma-Aldrich, USA). Depletion of rRNA was performed using RiboPOOL technology (siTOOLs Biotech; riboPOOL ribo-seq cat# dp-K012-000052, Germany) with a slight modification to the manufacturer’s instructions: 200 pmol of RiboPOOL and 100 µl of beads were used per sample. Next, RNA was purified using the Zymo RNA Clean and Concentrator-5 kit (Zymo Research, USA) and size-selected through denaturing PAGE using 15% TBE-Urea gels (Thermo Fisher Scientific, USA). RNA fragments corresponding to 26–34 nucleotides were excised and recovered from gel slices by crushing the gel and rocking at 20 °C, 700 RPM for 2 hours on a thermomixer. RNA solutions were transferred to Costar Spin-X filter tubes (Thermo Fisher Scientific, USA) and filtered through centrifugation at 2350 g for 6 min. 2 µl of GlycoBlue (Thermo Fisher Scientific, USA) and 700 µl of isopropanol were added per aliquot and RNA was left to precipitate overnight at −80 °C.
Following precipitation, RNA fragments were pelleted by centrifugation at 21130 g at 4 °C for 45 min. Pellets were washed with 1ml of 80% ice-cold ethanol and centrifuged at 21130 g at 4 °C for 10 min. RNA pellets were air-dried for 3-4 min and dissolved in 60.75 µl nuclease-free water on ice. RNA fragments were dephosphorylated using 30 U of T4 PNK (Lucigen, USA) for 1 h at 37 °C and purified using Zymo RNA Clean and Concentrator-5 kit (Zymo Research, USA), where isolated RNA fragments were eluted in 9.5 µl nuclease-free water. Purified fragments were ligated to a pre-adenylated 3’oligonucleotide linker using 100 U of T4 RNA ligase 2 Deletion Mutant (Lucigen, USA) and 5 U T4 RNA ligase 1 (Thermo Fisher Scientific, USA) at 23 °C for 3 h. Leftover linker was removed using 5’Deadenylase (New England Biolabs, USA) and Rec J Exonuclease (Lucigen, USA). Linker-ligated RNA fragments were reverse-transcribed into cDNA using EpiScript reverse transcriptase (Lucigen, USA). cDNA was treated with Exonuclease I (Lucigen, USA) for 30 min at 37 °C, followed by 15 min at 80 °C with a reduction to 4 °C; and further treated with 5 U of RNAse I (Lucigen, USA) and 2.5 U of Hybridase Thermostable RNase H (Lucigen, USA) at 55 °C for 5 min followed by an incubation step at 4 °C to stop the reaction. Treated cDNA was purified using the Zymo Oligo Clean and Concentrator Kit (Zymo Research, USA) with modifications to the manufacturer’s instructions: Columns were spun dry for 1 min at 21130 g and isolated RPFs were eluted in 9.5 µl nuclease-free water (Sigma-Aldrich, USA).  Size-selection of cDNA containing ligated linkers was performed through denaturing PAGE using 10% TBE-Urea gels (Thermo Fisher Scientific, USA). cDNA fragments corresponding to 70–80 nucleotides were excised and extracted with ammonium acetate and SDS, followed by overnight precipitation with isopropanol as described above. Size-selected cDNA was circularized for 3 h at 60 °C using 100 U of circLigase I (Lucigen, USA) followed by heat inactivation for 10 min at 80 °C and amplified using Phusion high-fidelity polymerase (New England Biolabs, USA) with reverse primers containing unique barcode sequences for 10 cycles of: 30 sec at 98 °C, 15 sec at 94 °C, 5 sec at 55 °C, 10 sec at 65 °C. Following amplification, 5 µl of 3 M NaCl (Thermo Fisher Scientific, USA), 1 ml of ethanol and 2 µl of GlycoBlue (Thermo Fisher Scientific, USA) were added to each aliquot of cDNA and left to precipitate overnight at −80 °C as described above.  Amplified cDNA libraries were size-selected using 8% non-denaturing TBE-Urea gels (Thermo Fisher Scientific, USA). cDNA libraries corresponding to 150 nucleotides were excised and recovered from gel slices by crushing and subsequent rocking at 37 °C at 700 RPM on a thermomixer for 2 hours. cDNA solutions were transferred to Costar Spin-X filter tubes (Thermo Fisher Scientific, USA) and filtered through centrifugation at 2350g for 6 min. cDNA libraries were purified using the Zymo DNA Clean and Concentrator-5 kit (Zymo Research, USA) with modifications to the manufacturer’s instructions: Columns were spun dry for 2 min at 12000 g and isolated RPFs were eluted in 13 µl nuclease-free water (Sigma-Aldrich, USA).  cDNA libraries were quantified using Qubit™ DNA high sensitivity (HS) Assay Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions on an Invitrogen™ Qubit™ 4 fluorometer (Thermo Fisher Scientific, USA) and Bioanalyzer 2100 (Agilent) using the High Sensitivity DNA kit and pooled in equimolar ratios. Sequencing was performed on a NextSeq2000 (Illumina; 1x50bp) at the Utrecht Sequencing Facility (USEQ) to a depth varying between 37831148 and 70879824 total reads per sample.

treatment_protocol: See growth protocol.

value: F1 Hybrid C57BL6J/FVBN

value: Liver

tag: Sex

value: Male

value: Ercc1 d/-


title: young_body_4

description:
Library name: male_young_body_4
normal sleeping fly


title: young_body_3

description:
Library name: male_young_body_3
normal sleeping fly


title: NMIBC tumor biopsy 1147248F

description: Library name: 1147248F


title: TN_WD_female_27d_179_heart

description: Library name: HDP-018998_heart

value: heart


title: young_body_2

description:
Library name: male_young_body_2
normal sleeping fly


title: NMIBC tumor biopsy 1240279F

description: Library name: 1240279F


title: TN_WD_male_3d_180_heart

description: Library name: HDP-019022_heart


title: NMIBC tumor biopsy 1193848F

description: Library name: 1193848F


title: Liver Ercc1 d/- 30% DR 16wks #3

description: Library name: IRL20


title: Liver Ercc1 d/- AL 7wks #4

description: Library name: IRL24


title: aged_NSF_body_1

description:
Library name: male_aged_NSF_body_1
normal sleeping fly

value: 25-d-old (aged)


title: NMIBC tumor biopsy 1298446F

description: Library name: 1298446F


title: RT_CD_male_17w_183_heart

description: Library name: HDP-018881_heart


title: Bacillus subtilis subsp. subtilis str. 168 Delta-samI mutant, biofilm, replicate 3

city: Bielefeld

first: Christian

last: Ruckert-Reed

address: Sequenz 1

department: Medical School OWL

country: Germany

institute: Bielefeld University

description: Library name: DsamI_3

data_processing:
Basecalls were performed using RTA v 1.18.66.3
Read trimming/filtering was performed using trimmomatic v0.39 with parameters PE SLIDINGWINDOW:4:15 MINLEN:25 CROP:50
Sequenced reads were mapped to  NC_000964.3 using bowtie2 v2.3.2 with parameters -X 600
Raw read counts per gene were calculated using featureCounts v2.0.0 with parameters -O -M -t gene -g ID -s 2
Read count normalization and data analysis were performed using DESeq2
Assembly: NC_000964.3
Supplementary files format and content: tab-delimited matrix table with raw read counts for every gene and every sample
Supplementary files format and content: tab-delimited matrix table with normalized read counts derived via DESeq2 for each gene with at least 10 raw counts in at least 3 samples

source_name: grown as biofilms on agar plate

extract_protocol:
Biofilms were transferred into a tube and submerged in 800 µL DNA/RNA Shield supplemented with 10 mg/mL lysozyme. To homogenize biofilms were first passed through a 23g1 needle 10-15 times and then sonicated for 12 bursts (duty cycle 20 %, output control 2). 30 min incubation at 37 °C under shaking conditions and subsequent sonification for 4x20 bursts lysed the cells, and debris were pelleted by centrifugation at max. speed for 2 min. To isolate total RNA the protocol of the Quick RNA Miniprep Plus Kit (Zymo Research) was followed. Concentration of isolated RNA was measured with a NanoDrop and integrity was confirmed in a denaturing agarose gel (Aranda et al., 2012).
High-quality total RNA aliquots of the biological replicates were rRNA-depleted using Pan-Bacteria riboPOOL (siTOOLs BIOTECH). Successful rRNA depletion was confirmed using Agilent RNA Pico 6000 kit on an Agilent 2100 Bioanalyzer (Agilent Technologies). The TruSeq Stranded mRNA Library Prep Kit from Illumina, starting with fragment, prime, and finish was used to prepare the cDNA libraries. The cDNA libraries were then sequenced in paired-end mode on an Illumina NextSeq2000 system with 50 nt read length and a minimum of 5 million clusters per sample.

tag: strain background

value: B. subtilis subsp. subtilis str. 168

value: grown as biofilms on agar plate

value: Delta samI


title: aged_NSF_body_2

description:
Library name: male_aged_NSF_body_2
normal sleeping fly

value: Body


title: TN_CD_male_17w_178_heart

description: Library name: HDP-018700_heart


title: young_body_1

description:
Library name: male_young_body_1
normal sleeping fly


title: Liver Ercc1 +/+ AL 104wks #4

description: Library name: IRL22

value: Ercc1 +/+

value: AL/104 wks


title: Liver Ercc1 d/- 30% DR 16wks #4

description: Library name: IRL21


title: Bacillus subtilis subsp. subtilis str. 168 Delta-samI mutant, biofilm, replicate 2

description: Library name: DsamI_2


title: RT_WD_male_17w_176_heart

description: Library name: HDP-018963_heart


title: Shocked_E_Low8

description: Library name: E8

extract_protocol:
Heads were individually isolated and subjected to total RNA extraction for RNA-seq.
The library for nuclear RNA-seq was prepared by Oligo d(T)25 Magnetic Beads (New England Biolabs inc. Ipswich, MA, USA), and NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs inc. Ipswich, MA, USA), according to the manufacturer’s instructions.

treatment_protocol: Flies were subjected to electric shocks at 60 V for 5 min, analyzed by behavioral assay to determine a place preference at 1.5 h and frozen at 2.5 h.

value: Head

value: adult

value: male

tag: treatment group


title: Liver Ercc1 d/- AL 7wks #3

description: Library name: IRL15


title: NMIBC tumor biopsy 1239504F

description: Library name: 1239504F

value: bladder tumor

value: BioIVT


title: Liver Ercc1 +/+ AL 104wks #3

description: Library name: IRL19

value: F1 Hybrid C57BL6J/FVBN


title: Shocked_E_Low9

description: Library name: E9

value: Cantonized w1118


title: Bacillus subtilis subsp. subtilis str. 168 Delta-samI mutant, biofilm, replicate 1

description: Library name: DsamI_1


title: Liver Ercc1 +/+ AL 16wks #4

description: Library name: IRL18

value: Liver

value: Male


title: Liver Ercc1 +/+ 30% DR 16wks #4

description: Library name: IRL16


title: TN_WD_female_17w_173_heart

description: Library name: HDP-018697_heart

value: heart


title: Liver Ercc1 +/+ AL 16wks #3

description: Library name: IRL13


title: Liver Ercc1 d/- 30% DR 16wks #2

description: Library name: IRL17

value: Ercc1 d/-


title: Bacillus subtilis subsp. subtilis str. 168 wildtype, biofilm, replicate 3

description: Library name: WT168_3

value: B. subtilis subsp. subtilis str. 168

value: wildtype


title: Spa_Root_Ctrl_Bulk_R3

city: Stanford

first: Guannan

state: CA

address: 371 Jane Stanford Way

department: Biology

institute: Stanford University

description: Roots from 4-6-day-old seedlings for bulk RNA-seq

data_processing:
The demultiplexing, barcoded processing, gene counting were performed using Cell Ranger (v5.0.1, 10X Genomics)
Assembly: Arabidopsis (TAIR10) from The Arabidopsis Information Resource (TAIR; https://www.arabidopsis.org/), C. sativa (v2) from Brassicaceae Database (BRAD; http://brassicadb.cn), E. salsugineum (v1.0) and S. parvula (v2.2) from Phytozome 12 (https://phytozome-next.jgi.doe.gov/) (Phytozome ID 574 and 173, respectively), reference genome for S. irio (v1.0) from Brassicaceae Database (BRAD; http://brassicadb.cn) with the updated gene model annotation (Sun et al., 2022) from the CoGe database (https://genomevolution.org/) with the Genome ID 57216.
Supplementary files format and content: tab-separated value files and matrix files; rds files

organism: Schrenkiella parvula

source_name: Root

extract_protocol:
Primary root tips of seedlings from different species were cut and placed into a 35 mm petri dish containing a 70 um strainer and 4 mL enzyme solution (1.25% [w/v] Cellulase [“ONOZUKA” R-10, Yakult], 0.1%[w/v] Pectolyase [P-3026, Sigma-Aldrich], 0.4 M Mannitol, 20 mM MES [pH 5.7], 20 mM KCl, 10 mM CaCl2, 0.1% [w/v] bovine serum albumin). The sample dish was rotated at 85 rpm for 50 min at 25°C. The root tips were then transferred to a slide glass and gently pressed with a cover glass using a pencil rubber to release protoplasts. The tissues were transferred back to the enzyme solution and incubated for another 10 minutes. Then, the protoplast solution was filtered through a 70 μm filter, and then through a 40 μm filter twice using 50 mL conical tubes. The filtered solution was centrifuged at 500g for 10 min at 22°C using a swinging bucket rotor and the supernatant was discarded. Protoplasts were resuspended using 500 μL Solution A (0.4 M Mannitol, 20 mM MES [pH 5.7], 20 mM KCl, 10 mM CaCl2, 0.1% [w/v] bovine serum albumin) and transferred to a 2 mL round bottom tube. After centrifugation at 200g for 6 min, the pelleted protoplasts were resuspended with 30–50 μL Solution A to achieve the desired cell concentration (~1,000 protoplasts/μL).
The protoplast suspension was loaded into Chromium microfluidic chips (Next GEM Single Cell 3’ kit) and barcoded with a 10X Chromium Controller (10X Genomics). mRNA from the barcoded cells were subsequently reverse-transcribed and sequencing libraries constructed with reagents from a Chromium Single Cell 3’ reagent kit v3.1 chemistry dual index (10X Genomics) according to the manufacturer’s instructions.

value: Root

tag: age

value: 4-6-day-old seedlings

value: Control


title: Bacillus subtilis subsp. subtilis str. 168 wildtype, biofilm, replicate 1

description: Library name: WT168_1

value: grown as biofilms on agar plate


title: TN_WD_male_27d_172_heart

description: Library name: HDP-018985_heart

tag: tissue


title: Shocked_E_Low4

description: Library name: E4

value: Head

tag: cell type


title: TN_WD_male_27d_175_heart

description: Library name: HDP-019016_heart


title: Shocked_E_Low7

description: Library name: E7

value: adult

tag: genotype


title: Bacillus subtilis subsp. subtilis str. 168 wildtype, biofilm, replicate 2

description: Library name: WT168_2


title: TN_WD_female_27d_171_heart

description: Library name: HDP-018996_heart


title: RT_WD_male_17w_177_heart

description: Library name: HDP-018875_heart


title: Shocked_E_Low6

description: Library name: E6

value: male


title: Shocked_E_Low5

description: Library name: E5


title: Spa_Root_Ctrl_Bulk_R1


title: Liver Ercc1 +/+ 30% DR 16wks #3

description: Library name: IRL14

value: Ercc1 +/+


title: Liver Ercc1 d/- AL 7wks #2

description: Library name: IRL11


title: TN_WD_female_9d_174_heart

description: Library name: HDP-019076_heart


title: RT_CD_female_17w_169_heart

description: Library name: HDP-018894_heart


title: Spa_Root_NaCl_scRNAseq_R2

description: Roots from 4-6-day-old seedlings for scRNA-seq

library_source: transcriptomic single cell

value: 100 mM NaCl treatment


title: Spa_Root_Ctrl_Bulk_R2


title: Liver Ercc1 +/+ AL 16wks #2

description: Library name: IRL9

value: F1 Hybrid C57BL6J/FVBN


title: TN_WD_female_17w_168_heart

description: Library name: HDP-018738_heart


title: Liver Ercc1 +/+ AL 104wks #2

description: Library name: IRL12

value: AL/104 wks


title: Shocked_E_Low3

description: Library name: E2

value: Cantonized w1118


title: Liver Ercc1 +/+ 30% DR 16wks #2

description: Library name: IRL10

value: Liver

value: Male


title: Shocked_E_Low2

description: Library name: E10


title: RT_WD_male_17w_170_heart

description: Library name: HDP-018886_heart

value: heart


title: Shocked_E_Low1

description: Library name: E1


title: TN_WD_female_17w_166_heart

description: Library name: HDP-018704_heart


title: Spa_Root_Ctrl_scRNAseq_R2


title: Liver Ercc1 d/- AL 16wks #2

description: Library name: IRL7

value: Ercc1 d/-


title: Spa_Root_NaCl_scRNAseq_R1


title: TN_CD_male_9w_164_heart

description: Library name: HDP-018788_heart


title: Liver Ercc1 +/+ 30% DR 16wks #1

description: Library name: IRL6


title: Liver Ercc1 +/+ AL 104wks #1

description: Library name: IRL5


title: Shocked_D_Mid8

description: Library name: D8


title: Shocked_D_Mid4

description: Library name: D4


title: Spa_Root_ABA_scRNAseq_R2

value: 5 M ABA treatment


title: Esa_Root_Ctrl_Bulk_R2

organism: Eutrema salsugineum

value: Root


title: TN_WD_male_9d_167_heart

description: Library name: HDP-019082_heart


title: Liver Ercc1 d/- AL 16wks #3

description: Library name: IRL8


title: Shocked_D_Mid3

description: Library name: D3


title: Spa_Root_ABA_scRNAseq_R1

value: 4-6-day-old seedlings


title: Shocked_D_Mid7

description: Library name: D7


title: Shocked_D_Mid6

description: Library name: D6


title: Liver Ercc1 d/- AL 16wks #1

description: Library name: IRL3


title: Liver Ercc1 +/+ AL 16wks #1

description: Library name: IRL2


title: TN_WD_female_17w_165_heart

description: Library name: HDP-018718_heart


title: Esa_Root_Ctrl_Bulk_R1

value: Control


title: Shocked_D_Mid5

description: Library name: D5


title: Spa_Root_Ctrl_scRNAseq_R1


title: Liver Ercc1 d/- 30% DR 16wks #1

description: Library name: IRL1


title: TN_WD_male_27d_159_heart

description: Library name: HDP-019009_heart


title: Esa_Root_Ctrl_Bulk_R3


title: TN_WD_male_3d_163_heart

description: Library name: HDP-019004_heart


title: Transcriptome of the wildtype Pseudomonas putida KT2440 in the glucose M9 with 4 g/L isoprenol (replicate 2)

city: Incheon

first: Hyungyu

last: Lim

address: Inha ro 100

country: South Korea

institute: Inha University

data_processing:
Raw RNA-Seq reads were mapped to the original reference genome using Bowtie2 and transcript abundance was analyzed using summarizeOverlaps (Lawrence et al., 2013). Calculation of transcripts per million (TPM) and prediction of differentially expressed genes were conducted using DESeq2 (Love et al., 2014).
Assembly: NC_002947.4
Supplementary files format and content: csv

organism: Pseudomonas putida KT2440

source_name: Pseudomonas putida KT2440

growth_protocol: All microbial cultures were conducted at 30 ˚C using either the modified minimal M9 medium (4 g/L glucose, 2 g/L (NH4)2SO4, 6.8 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 2 mM MgSO4, 0.1 mM CaCl2, 500 μL/L 2000X trace element solution). The composition of the trace element solution is 4.5 g/L ZnSO4⋅7H2O, 0.7 g/L MnCl2⋅4H2O, 0.3 g/L CoCl2⋅6H2O, 0.2 g/L CuSO4⋅2H2O, 0.4 g/L Na2MoO4⋅2H2O, 4.5 g/L CaCl2⋅2H2O, 3.0 g/L FeSO4⋅7H2O, 1.0 g/L H3BO3, 0.1 g/L KI, 15 g/L disodium ethylenediaminetetraacetate (Mohamed et al., 2017).

extract_protocol:
For transcriptomic analysis, cell cultures at the exponential phase (OD600 0.4-0.6) were mixed with two volumes of RNA protect Bacteria Reagent (Qiagen, Hilden, Germany) for stabilization and their total RNA was extracted by using Quick-RNA Fungal/Bacterial Miniprep Kit (Zymo Research). Residual genomic DNA was removed by incubating the samples with DNase I (RNase-free, NEB). Ribosomal RNA was removed by hybridizing with 30-bp oligonucleotides designed to bind the ribosomal RNA of KT2440 specifically and treating with Thermostable RNase H purchased from Lucigen (Middleton, WI, USA). The remaining oligonucleotides were removed by incubating the resulting samples with DNase I (NEB). The samples were purified by using an RNA Clean & Concentrator Kit (Zymo Research).
Paired-end RNA-Seq libraries were constructed using a KAPA RNA HyperPrep kit from Kapa Biosystems (Wilmington, MA, USA).

treatment_protocol: 4 g/L of glucose or/and isoprenol was added to the treatment samples.

value: Pseudomonas putida KT2440

value: Bacteria

value: WT

value: glucose and isoprenol


title: Liver Ercc1 d/- AL 7wks #1

description: Library name: IRL4


title: TN_CD_male_9w_162_heart

description: Library name: HDP-018814_heart


title: Esa_Root_ABA_scRNAseq_R2


title: Shocked_C_High8

description: Library name: C8

value: Head

value: adult

value: male


title: Esa_Root_ABA_scRNAseq_R1


title: Shocked_D_Mid2

description: Library name: D2


title: Shocked_C_High6

description: Library name: C6


title: TN_CD_female_17w_157_heart

description: Library name: HDP-018707_heart


title: Transcriptome of the wildtype Pseudomonas putida KT2440 in the glucose M9 with 4 g/L isoprenol (replicate 1)


title: Shocked_D_Mid1

description: Library name: D1


title: Shocked_C_High4

description: Library name: C4


title: Esa_Root_NaCl_scRNAseq_R2

value: 100 mM NaCl treatment


title: Esa_Root_NaCl_scRNAseq_R1


title: Shocked_C_High7

description: Library name: C7

value: Cantonized w1118


title: Esa_Root_Ctrl_scRNAseq_R1


title: Transcriptome of the wildtype Pseudomonas putida KT2440 in the glucose M9 (replicate 2)

value: glucose


title: TN_WD_female_9d_154_heart

description: Library name: HDP-019085_heart


title: Ribo_miRFP-D-EGFP

city: Ithaca

first: Saori

last: Uematsu

state: New York

address: 526 Campus Road, Cornell University

institute: Cornell University

description: Library name: Sample_87

data_processing:
The sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq (v2.20.0.422) software.
The 5’ and 3’ adapters of the reads were trimmed by Cutadapt (version 2.8) using the following parameters:  -a “A{10}” -m 15 –max-n=0.1. The trimmed reads with length shorter than 15 nucleotides were excluded from the analysis.
The trimmed reads were first aligned to rRNAs using Bowtie (version 1.2.3) with the following parameters: -v 0 –norc.
The reads unaligned to rRNAs were then mapped to the custom mouse transcriptome using STAR (version 2.7.10a) with default parameters. To avoid ambiguity, reads mapped to multiple positions or with >2 mismatches were disregarded for further analysis.
Assembly: GRCh39.p13
Supplementary files format and content: tab-delimited text files including normalized read counts per codon on individual mRNAs for Ribo-seq.
Supplementary files format and content: comma-delimited text files including read counts mapped to each position on reporter mRNA Ribo-seq.
Library strategy: Ribo-Seq

extract_protocol:
For mRNA reporters, 8 μg of mRNA was transfected into HEK293-Kb cells (~80% confluence) in 10 cm dishes for 8 h. For plasmid reporters, 1 μg plasmid was transfected into 35 mm dishes for 24 h. Cells were washed with cold PBS and lysed in polysome lysis buffer (10 mM HEPES, pH 7.4; 100 mM KCl; 5 mM MgCl₂; 100 μg/mL cycloheximide; 1% Triton X-100). Lysates were cleared by centrifugation at 20,000 × g for 10 min at 4 °C and digested with RNase I (Ambion, 750 U per 100 A₂₆₀ units) at 4 °C for 1 h. Total RNA was extracted using TRIzol LS (Invitrogen) for cDNA library construction.
Ribosome-protected fragments (25–35 nt) or disome footprints (40-60 nt) were resolved on a 15% polyacrylamide TBE–urea gel (Invitrogen) and visualized with SYBR Gold. Excised RNA fragments were eluted overnight in RNA elution buffer (300 mM sodium acetate, pH 5.2; 1 mM EDTA; 0.1 U/μL SUPERase·In), cleared through Spin-X columns, and ethanol-precipitated. cDNA libraries were constructed using T4 PNK, Poly(A) polymerase, and custom Ezra enzyme, followed by ligation of 5' adapters, streptavidin bead-based purification, and reverse transcription following the previous study [28]. Final cDNA was amplified with barcoded primers, size-selected (180 bp), quantified, pooled, and sequenced on an Illumina MiniSeq or NextSeq 500.


title: K562 dCas9-KRAB cells, hCRISPRi-v2 library, NPC1-targeting sgRNA (sgNPC1), LLOMe-treated, biological replicate 2

city: Berkeley

first: Roberto

last: Zoncu

address: Li Ka Shing Center

institute: University of California, Berkeley

description:
Library name: K562_sgNPC1_LLOMe_rep2
220520_K562_sgNPC1_LLOMe_Rep_2_S14

data_processing:
Lane 1 of the 220526 NextSeq run (flowcell AHWKFVBGXL) produced empty FASTQ files and was excluded from the submission; sgRNA counts were derived from the remaining three lanes of that run and all four lanes of the 220520 run (flowcell AHWGVLBGXL).
Assembly: hg19
Supplementary files format and content: Basecalls were performed using bclconvert or bcl2fastq. Count tables were generated using the ScreenProcessing pipeline (https://github.com/ucsf-lgr/ScreenProcessing) .
Library strategy: CRISPR Screen

growth_protocol: sgNT K562 dCas9-KRAB and sgNPC1 K562 dCas9-KRAB cells were infected with genome wide sgRNA library lentivirus to target 1000x coverage of all guides at a low MOI to ensure roughly one guide per cell. 550 million cells from each cell line were pelleted and resuspended in pure virus supplemented with 1 μl/ml polybrene (Millipore Sigma, TR-1003-G). Cells in virus were plated into 6 well plates and spinfected at 1,000g for 1 hour. Cells were then supplemented with complete media and cultured in flasks in shaking incubators at 100xg. Cells were maintained in shaking conditions throughout the screen. After ~24 hours, virus was removed and cells were resuspended in fresh media. The next day BFP infection percentage was assessed (34% for NT, 45% for NPC1) and selection was started with 1 ug/mL puromycin (Gibco, A1113803). Cells were selected with 1 μg/mL puromycin for 6 days until population reached ~80-90% BFP+. Cells were grown for an additional 3 days to reach numbers necessary for the screen. On day T0 of the screen, samples were split for 850X coverage of the library to each replicate. 2 replicates from each cell line were frozen in media with 10% DMSO as backup. 2 replicates from each cell line were pelleted and stored at -80C for T0 gDNA samples. 2 replicates from each cell line were seeded at 0.25e6 cells/mL and treated with 0.5 mM LLOMe Hydrochloride (Cayman Chemicals, 16008) or 0.01% DMSO mock treatment for another arm of the screen (not shown) and cultured for 2 days. Drug treatment was then removed from cells and replaced with fresh media for two days. This cycle of drug treatment and recovery was repeated after which cells were expanded for 6 days until cell numbers were sufficient to end the screen. Multiple cell pellets representing 850X coverage of the library were stored at -80C for each sample for gDNA processing.

extract_protocol:
Cell pellets from the screen were processed using the NucleoSpin Blood XL Kit (Macherey Nagel, 740950) according to manufacturer’s directions and extracted DNA was quantified with DeNovix Nanodrop. After DNA extraction, samples were processed for NGS sequencing using the protocol described in Heo, SJ., Enriquez, L.D, Federman, S. et al51. DNA samples were amplified by PCR using NEBNext Ultra II Q5 MasterMix (NEB, M0544) with a common reverse primer (CAAGCAGAAGACGGCATACGAGATATGCTGTTTCCAGCTTAGCTCTT) and a unique forward primer per sample containing a 6 bp barcode (AATGATACGGCGACCACCGAGATCTACACGATCGGAAGAGCACACGTCTGAACTCCAGTCACnnnnnnGCACAAAAGGAAACTCACCCT) (sequences listed in supplemental table). For each sample total gDNA was amplified in multiple PCRs with an input of 10 μg per reaction. PCRs were run with the following cycling conditions: 98C for 30 s, followed by 24 cycles of 98C for 10 s and 62.6C for 75 s, and a final elongation at 65C for 5 mins. Reactions were pooled and purified with double-sided SPRI beads (Beckman Coulter, A63882). Samples were quantified on the Agilent TapeStation and pooled to generate a library with equal representation of each sample. Library was sequenced on the Illumina NextSeq 550 using a NextSeq High Output kit (75 cycles) (Illumina, 20024906). A custom sequencing primer (GTGTGTTTTGAGACTATAAGTATCCCTTGGAGAACCACCTTGTTGG) was added to the standard Illumina sequencing primer. Library runs were supplemented with PhiX DNA to increase sequence diversity.
The lentiviral expression vector used for CRISPR guide expression is available on Addgene under the following name: pLGR1002 (188320). The sgRNA sequences as well as the sgRNA library cloning procedure performed is previously (PMID 38243310, PMID 27661255). sgRNA library representation was confirmed by NGS sequencing. Guide library virus was prepared using the Mega Lentivirus protocol from the Weissman Lab with modifications (https://weissman.wi.mit.edu/resources/Large_scale_lentivirus_production.pdf). 18 million LentiX 293T cells (Takara Bio, 632180) were plated on a 15 cm plate. The next day, transfection mixture was prepared using 125 μl Mirus LT1 transfection reagent (Mirus Bio, MIR2304), 1.5 ml DMEM (Gibco, 11965-092), 18 μg psPax2 (packaging vector), 4 μg MD2G (env expression plasmid, VSVG), and 18 μg of lentiviral plasmid (sgRNA library). Transfection mixture was incubated for 15-20 mins at room temperature. Transfection mix was added to the 293T cells and incubated for 6 hours. After 6 hours the media was replaced with fresh 293T media supplemented with 2 μg/mL ViralBoost (ALSTEM, VB100). Lentiviral supernatant was collected 48 hours after transfection and filtered (0.45um). Lentivirus was stored at -80C until use. Lentivirus titering was performed in K562 cells prior to the screen. Multiplicity of infection (MOI) was determined by percent infection shown by BFP signal via flow cytometry. 2e5 K562 sgNT and K562 sgNPC1 cells were transduced with guide library lentivirus in a range of 5-250 μL in K562 media supplemented with 1μl/mL Polybrene (Millipore Sigma, TR-1003-G). Cells were left in virus for 24 hours and then changed to regular media. 48 hours post infection cells were analyzed on the Attune NXT flow cytometer to determine BFP fluorescence and MOI. Lentivirus for individual guide RNA experiments was produced in 6 well plates with adjusted protocol. 0.8e6 Lenti-X 293T cells per well were seeded the day before transfection. Transfection mix consists of 7.5 μL Mirus transfection reagent (MIR 2700), 100 μL DMEM (Gibco, 11965-092), 1.2 μg psPAX2, 0.3 μg MD2G, and 1.2 μg guide plasmid. Protocol follows as above.

value: lymphoblast (chronic myelogenous leukemia)

value: dCas9-KRAB + sgNPC1 (NPC1 knockdown)

value: 1 mM LLOMe (L-leucyl-L-leucine methyl ester), 12 h

value: replicate 2


title: RT_CD_female_17w_153_heart

description: Library name: HDP-018884_heart

value: heart


title: RT_CD_female_17w_158_heart

description: Library name: HDP-018920_heart


title: TN_WD_male_17w_152_heart

description: Library name: HDP-018733_heart


title: K562 dCas9-KRAB cells, hCRISPRi-v2 library, NPC1-targeting sgRNA (sgNPC1), LLOMe-treated, biological replicate 1

description:
Library name: K562_sgNPC1_LLOMe_rep1
220520_K562_sgNPC1_LLOMe_Rep_1_S13

value: replicate 1


title: RT_CD_female_17w_160_heart

description: Library name: HDP-018897_heart


title: Transcriptome of the wildtype Pseudomonas putida KT2440 in the isoprenol M9 (replicate 2)

value: isoprenol


title: Shocked_C_High5

description: Library name: C5


title: Naïve_B_High7

description: Library name: B9


title: Disome_Asp_GAT-GAC

description: Library name: Sample_84


title: TN_WD_female_27d_151_heart

description: Library name: HDP-018986_heart


title: K562 dCas9-KRAB cells, hCRISPRi-v2 library, NPC1-targeting sgRNA (sgNPC1), DMSO vehicle, biological replicate 2

description:
Library name: K562_sgNPC1_DMSO_rep2
220520_K562_sgNPC1_DMSO_Rep_2_S12

value: DMSO (vehicle control)


title: Esa_Root_Ctrl_scRNAseq_R2

value: Root

value: 4-6-day-old seedlings

value: Control


title: TN_CD_male_3d_156_heart

description: Library name: HDP-019001_heart


title: TN_CD_male_3d_147_heart

description: Library name: HDP-019000_heart


title: K562 dCas9-KRAB cells, hCRISPRi-v2 library, non-targeting sgRNA (sgNT), LLOMe-treated, biological replicate 2

description:
Library name: K562_sgNT_LLOMe_rep2
220520_K562_sgNT_LLOMe_Rep_2_S2

value: dCas9-KRAB + non-targeting sgRNA


title: Transcriptome of the wildtype Pseudomonas putida KT2440 in the glucose M9 (replicate 1)

value: Pseudomonas putida KT2440

value: Bacteria

value: WT


title: Transcriptome of the wildtype Pseudomonas putida KT2440 in the isoprenol M9 (replicate 1)


title: Shocked_C_High3

description: Library name: C3

value: Head

value: adult

value: male

value: Cantonized w1118


title: K562 dCas9-KRAB cells, hCRISPRi-v2 library, NPC1-targeting sgRNA (sgNPC1), pre-treatment (T0), biological replicate 1

description:
Library name: K562_sgNPC1_T0_rep1
220520_K562_sgNPC1_T0_1_S15

value: lymphoblast (chronic myelogenous leukemia)

value: dCas9-KRAB + sgNPC1 (NPC1 knockdown)

value: none (pre-treatment baseline)


title: K562 dCas9-KRAB cells, hCRISPRi-v2 library, NPC1-targeting sgRNA (sgNPC1), pre-treatment (T0), biological replicate 2

description:
Library name: K562_sgNPC1_T0_rep2
220520_K562_sgNPC1_T0_2_S16

value: replicate 2


title: Disome_Asp_GAC

description: Library name: Sample_80


title: Shocked_C_High2

description: Library name: C2


title: TN_WD_male_17w_161_heart

description: Library name: HDP-018753_heart

value: heart


title: TN_WD_male_27d_148_heart

description: Library name: HDP-019015_heart


title: TN_CD_female_27d_155_heart

description: Library name: HDP-019018_heart


title: Naïve_B_High2

description: Library name: B2


title: Ribo_miRFP-P-EGFP

description: Library name: Sample_86


title: Disome_Q-P_mix

description: Library name: Sample_85


title: Shocked_C_High1

description: Library name: C1


title: Naïve_B_High4

description: Library name: B6


title: Disome_K-A

description: Library name: Sample_83


title: Naïve_B_High6

description: Library name: B8


title: K562 dCas9-KRAB cells, hCRISPRi-v2 library, NPC1-targeting sgRNA (sgNPC1), DMSO vehicle, biological replicate 1

description:
Library name: K562_sgNPC1_DMSO_rep1
220520_K562_sgNPC1_DMSO_Rep_1_S11

value: DMSO (vehicle control)

value: replicate 1


title: Naïve_B_High5

description: Library name: B7


title: Disome_A-K

description: Library name: Sample_82


title: RT_WD_female_17w_150_heart

description: Library name: HDP-018849_heart


title: TN_CD_female_9w_145_heart

description: Library name: HDP-018822_heart


title: K562 dCas9-KRAB cells, hCRISPRi-v2 library, non-targeting sgRNA (sgNT), DMSO vehicle, biological replicate 2

description:
Library name: K562_sgNT_DMSO_rep2
220520_K562_sgNT_DMSO_Rep_2_S6


title: Disome_Arg_IV

description: Library name: Sample_78


title: RT_CD_female_17w_144_heart

description: Library name: HDP-018922_heart


title: Naïve_A_Mid5

description: Library name: A5


title: TN_CD_male_9w_149_heart

description: Library name: HDP-018836_heart


title: Disome_Arg_CGCCGC-CGGCGG

description: Library name: Sample_81


title: K562 dCas9-KRAB cells, hCRISPRi-v2 library, non-targeting sgRNA (sgNT), pre-treatment (T0), biological replicate 2

description:
Library name: K562_sgNT_T0_rep2
220520_K562_sgNT_T0_2_S8


title: Disome_Arg_AGC

description: Library name: Sample_79


title: Disome_Tyr

description: Library name: Sample_76


title: Naïve_B_High3

description: Library name: B5


title: Naïve_B_High1

description: Library name: B1


title: Naïve_A_Mid7

description: Library name: A7


title: Naïve_A_Mid6

description: Library name: A6


title: TN_WD_male_9d_146_heart

description: Library name: HDP-019074_heart


title: K562 dCas9-KRAB cells, hCRISPRi-v2 library, non-targeting sgRNA (sgNT), LLOMe-treated, biological replicate 1

description:
Library name: K562_sgNT_LLOMe_rep1
220520_K562_sgNT_LLOMe_Rep_1_S1

value: 1 mM LLOMe (L-leucyl-L-leucine methyl ester), 12 h


title: Naïve_A_Mid4

description: Library name: A4


title: K562 dCas9-KRAB cells, hCRISPRi-v2 library, non-targeting sgRNA (sgNT), pre-treatment (T0), biological replicate 1

description:
Library name: K562_sgNT_T0_rep1
220520_K562_sgNT_T0_1_S7


title: TN_CD_male_17w_143_heart

description: Library name: HDP-018713_heart


title: Disome_Ser

description: Library name: Sample_73


title: Disome_Thr

description: Library name: Sample_74


title: Ribo_HEK293_Arg_-Arg

description: Library name: Sample_7


title: Disome_Val

description: Library name: Sample_77


title: Disome_Trp

description: Library name: Sample_75


title: K562 dCas9-KRAB cells, hCRISPRi-v2 library, non-targeting sgRNA (sgNT), DMSO vehicle, biological replicate 1

description:
Library name: K562_sgNT_DMSO_rep1
220520_K562_sgNT_DMSO_Rep_1_S5


title: TN_CD_female_27d_141_heart

description: Library name: HDP-019019_heart


title: RT_WD_male_17w_142_heart

description: Library name: HDP-018847_heart


title: RT_WD_male_17w_140_heart

description: Library name: HDP-018876_heart


title: Naïve_A_Mid3

description: Library name: A3


title: Ribo_SAMD9_Con

description: Library name: Sample_4


title: Ribo_HEK293_Arg

description: Library name: Sample_6


title: Disome_Met

description: Library name: Sample_71


title: Disome_Phe

description: Library name: Sample_72


title: WT.BLEO_5

city: Ann Arbor

first: Bioinformatics

last: Core

state: MI

address: BRCF Bioinformatics Core, 2800 Plymouth Rd

institute: University of Michigan

description: Library name: 15157-PV-9

data_processing:
Reads were trimmed using Cutadapt, mapped to the reference genome GRCm38 using STAR, and gene-quantified with RSEM. (Alignment options followed ENCODE standards for RNA-seq.)
Assembly: GRCm38
Supplementary files format and content: Sample counts via RSEM-STAR

source_name: Lung

extract_protocol:
Lung total RNA was isolated from lung bipoeis using Qiagen RNeasy Fibrous Tissue Mini Kit (Cat no: 74704).
Sequencing was performed by the UM Advanced Genomics Core, with libraries constructed and subsequently subjected to 150 paired-end cycles on the NovaSeq-6000 platform (Illumina). Data were pre-filtered to remove genes

treatment_protocol: Female C57BL/6J mice (8–12 weeks old) and FMO3 knockout mice (C57BL/6J-Fmo3^em1Bdgr/J; strain #037364) were obtained from The Jackson Laboratory.  Mice received daily subcutaneous (s.c.) injections of bleomycin (10 mg/kg/day) or phosphate-buffered saline (PBS) for 10 days (5 days per week). Animals were euthanized on day 22, and lung were used for RNA isolation.

value: Lung

value: BLEO


title: Ribo_SAMD9_Dox

description: Library name: Sample_5


title: RT_CD_female_17w_139_heart

description: Library name: HDP-018921_heart


title: Naïve_A_Mid2

description: Library name: A2


title: Naïve_A_Mid1

description: Library name: A1


title: Disome_Lys

description: Library name: Sample_70


title: TN_CD_male_9d_136_heart

description: Library name: HDP-019056_heart


title: TN_CD_female_9w_138_heart

description: Library name: HDP-018809_heart


title: TN_CD_female_9w_135_heart

description: Library name: HDP-018816_heart


title: WT.BLEO_3

description: Library name: 15157-PV-7


title: WT.BLEO_4

description: Library name: 15157-PV-8


title: Disome_Ile

description: Library name: Sample_68


title: Ribo_HEK293_control_rep1

description: Library name: Sample_1


title: TN_CD_female_17w_134_heart

description: Library name: HDP-018717_heart


title: Ribo_HEK293_control_rep2

description: Library name: Sample_2


title: Disome_His

description: Library name: Sample_67


title: SeV2 CL-7 Dox-OFF day 4 rep2

city: Kyoto

first: Koji

last: Eto

address: 53 Kawahara-cho, Shogoin Kawahara-cho, Sakyo-ku

department: CiRA

country: Japan

institute: Kyoto University

description: Library name: con_d4_42

data_processing:
Sequencing was performed on an Illumina NextSeq500. FASTQ files were generated using bcl2fastq-2.20.
Adapter sequences and low-quality bases were trimmed from the raw sequenced reads using cutadapt ver v3.4. Trimmed reads were mapped to human reference genome sequences (hg38) using STAR ver 2.7.9a with GENCODE (release 32, GRCh38.p13) GTF file.
Uniquely mapped reads were summarized at the gene level using htseq-count ver. 0.13.5 with the GENCODE GTF file.
Assembly: hg38
Supplementary files format and content: tab-delimited text file includes raw counts for each sample

source_name: imMKCL

growth_protocol: The maturation of imMKCLs was conducted in a humidified incubator at 37°C with 5% CO2. Cells were maintained at a density of 1.0 × 105 cells/mL for six days. The base medium was supplemented with 15% FBS and Cocktail B. Cocktail B comprised 50 ng/mL recombinant human kit ligand protein, 200 ng/mL TA-316, 15 µM KP-457 (Kaken Pharmaceutical), 0.75 µM SR-1 (Calbiochem), and 10 µM Y-27632 (Wako).

extract_protocol:
Total RNA was isolated using an RNeasy mini kit (Qiagen) following the manufacturer’s protocol.
RNA Library preparation was performed using a TruSeq Stranded mRNA Sample Prep Kit (Illumina) according to the manufacturer’s instructions.

treatment_protocol: imMKCLs culture under Dox-OFF condition for 4 days, and samples were prepared every 24 h from Dox-OFF day 0 to day 4.

value: imMKCL

value: megakaryocyte

value: DMSO


title: Disome_Leu

description: Library name: Sample_69

value: WT


title: TN_WD_male_9d_137_heart

description: Library name: HDP-019073_heart


title: WT.BLEO_2

description: Library name: 15157-PV-6


title: Ribo_HEK293_control_rep3

description: Library name: Sample_3


title: Disome_Gly

description: Library name: Sample_66


title: WT.BLEO_1

description: Library name: 15157-PV-5


title: SeV2 CL-7 Dox-OFF day 4 rep1

description: Library name: con_d4_32


title: Disome_Glu

description: Library name: Sample_65


title: TN_CD_male_9d_133_heart

description: Library name: HDP-019071_heart


title: WT.PBS_3

description: Library name: 15157-PV-3

value: PBS


title: TN_WD_male_9d_132_heart

description: Library name: HDP-019081_heart


title: SeV2 CL-7 Dox-OFF day 3 rep2

description: Library name: con_d3_42


title: WT.PBS_4

description: Library name: 15157-PV-4


title: TN_CD_male_27d_131_heart

description: Library name: HDP-018980_heart


title: Disome_Gln

description: Library name: Sample_64


title: SeV2 CL-7 Dox-OFF day 2 rep2

description: Library name: con_d2_42


title: SeV2 CL-7 Dox-OFF day 3 rep1

description: Library name: con_d3_32


title: Disome_Cys

description: Library name: Sample_63


title: FMO3KO.BLEO_4

description: Library name: 15157-PV-17


title: Disome_Asp

description: Library name: Sample_62


title: RT_CD_female_17w_130_heart

description: Library name: HDP-018966_heart


title: SeV2 CL-7 Dox-OFF day 2 rep1

description: Library name: con_d2_32


title: RT_CD_female_17w_129_heart

description: Library name: HDP-018878_heart


title: WT.PBS_2

description: Library name: 15157-PV-2


title: FMO3KO.BLEO_3

description: Library name: 15157-PV-16


title: FMO3KO.BLEO_2

description: Library name: 15157-PV-15


title: TN_CD_male_9w_127_heart

description: Library name: HDP-018795_heart


title: SeV2 CL-7 Dox-OFF day 1 rep1

description: Library name: con_d1_32


title: Disome_Asn

description: Library name: Sample_61


title: TN_CD_male_27d_128_heart

description: Library name: HDP-018982_heart


title: Disome_Ala

description: Library name: Sample_60


title: Disome_Pro

description: Library name: Sample_59


title: SeV2 CL-7 Dox-OFF day 1 rep2

description: Library name: con_d1_42


title: FMO3KO.BLEO_1

description: Library name: 15157-PV-14


title: FMO3KO.PBS_4

description: Library name: 15157-PV-13


title: SeV2 CL-7 Dox-OFF day 0 rep2

description: Library name: d0_42


title: SeV2 CL-7 Dox-OFF day 0 rep1

description: Library name: d0_32


title: FMO3KO.PBS_3

description: Library name: 15157-PV-12


title: Disome_Arg

description: Library name: Sample_58


title: RT_CD_male_17w_125_heart

description: Library name: HDP-018879_heart


title: TN_CD_female_17w_126_heart

description: Library name: HDP-018735_heart


title: cArg_20-80

description: Library name: Sample_57


title: HAEC static flow

city: Boston

first: Aryan

last: Kaveh

state: MA

address: 60 Fenwood Road

department: Medicine

institute: Brigham and Women's Hospital

description: 10x Genomics generated processed data in barcode, feature and matrix files

data_processing:
Processed data files including the mapped barcode, feature and matrix files were generated using CellRanger software (10x Genomics). For zebrafish data processing, cellranger-6.0.1 was used. For HAEC data processing cellranger-4.0.0 was used.
Assembly: hg38 (HAEC) and danRer10 (zebrafish)
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Primary human aortic endothelial cells

extract_protocol:
Tg(flk:GFP) zebrafish embryos were digested using preheated 0.25% Trypsin-EDTA at 28°C for 20 minutes, mechanically aspirating every 5 minutes. Disassociated embryos were subsequently washed with PBS and 1% FBS. Cell pellets were obtained by centrifugation which were resuspended in PBS and 1% FBS in preparation for fluorescence-activated cell sorting of GFP+ endothelial cells and GFP- non-endothelial cells. HAECs were washed in PBS and 0.05% Trypsin-EDTA. Cell pellets were obtained by centrifugation which were resuspended in complete medium prior to scRNA-seq analysis.
The library was constructed according to the manufacturer’s instructions (single cell 3’ v3 protocol, 10x Genomics) for both zebrafish and HAEC datasets.

treatment_protocol: To inhibit cardiac contractility and generate embryonic zebrafish with reduced or static blood flow, tnnt2 morpholino was injected into the single-cell stage at a 1:100 or 1:20 dose respectively. HAECs were stimulated with unimodal shear stress (physiologic flow, 30 dynes/cm2; reduced flow, 2.5dynes/cm2 or static flow, 0 dynes/cm2) for 48 hours.

value: Primary human aortic endothelial cells

value: wt


title: TN_CD_male_9w_124_heart

description: Library name: HDP-018821_heart


title: FMO3KO.PBS_2

description: Library name: 15157-PV-11


title: cQ-P_mix

description: Library name: Sample_56


title: HAEC reduced flow


title: cArg_AGC

description: Library name: Sample_55


title: RT_WD_male_17w_122_heart

description: Library name: HDP-018887_heart


title: cK-A

description: Library name: Sample_54


title: HAEC high flow


title: TN_CD_female_27d_123_heart

description: Library name: HDP-019017_heart


title: WT.PBS_1

description: Library name: 15157-PV-1


title: cArg_CGT

description: Library name: Sample_52


title: FMO3KO.PBS_1

description: Library name: 15157-PV-10


title: TN_CD_female_9d_121_heart

description: Library name: HDP-019054_heart


title: cA-K

description: Library name: Sample_53


title: TN_WD_female_9d_120_heart

description: Library name: HDP-019117_heart


title: KCP_LY_H3K27Ac_CR

city: New York

first: Wilfred

last: Wong

state: NY

address: 430 E 67th St

institute: Memorial Sloan Kettering Cancer Center

data_processing:
Cut&Run Tools was used to align reads and call peaks
Resulting pileup files were used to generate bigwigs from default macs2 settings in Cut&Run Tools.
Assembly: hg19
Supplementary files format and content: bigwig coverage tracks
Library strategy: CUT&RUN

source_name: Pancreatic (hPSC derived)

growth_protocol: POs were suspended in 30 ul of Matrigel dome and applied to the bottom of each well of non-tissue culture treated 48 well plate to solidify. The Matrigel dome was overlaid with 0.4 ml of DMEM-F12 base medium supplied with Glutamax, Penicillin/Streptomycin, B27TM supplement, 1mM N-acetyle-L-cystein, 10mM Nicotinamide, 0.5uM A83-01, 100ng/ml hNoggin, 50ng/ml hEGF, 10nM hGastrin I, 100ng/ml hFGF10, 500ng/ml Rsp1.

extract_protocol:
Organoids were extracted from the matrigel by cell recovery solution, trypsinized into single cell suspension. 500,000 cells were used per CUT&RUN assay.
CUT&RUN library was prepared with no more than 10 ng CUT&RUN DNA using NEBNext Ultra II DNA Library Prep Kit (NEB) according to manufacturer’s protocol.

treatment_protocol: In case of ERK inhbitor treatment,  LY3214996 was used at 1uM and POs were incubated for 4 days. DMSO was used as control.

value: Pancreatic (hPSC derived)

value: Pancreatic progenitor organoid

value: KCP

value: ERKi


title: RA_1d-rep2

first: Weipeng

last: Mo

address: 58 Zhongshaner Rd

department: The First Affiliated Hospital

institute: Sun Yat-sen University

description: Library name: RA_1d-rep2

data_processing:
HiChIP data processing using HiC-Pro
Assembly: mm10
Supplementary files format and content: hic format
Library strategy: HiChIP

source_name: cell line

growth_protocol: ESCs were grown in culture dishes coated with 0.1% gelatin (Sigma) in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 15% fetal bovine serum (FBS, AusGeneX), 1× nonessential amino acids (100×, Gibco), 1× L-glutamate (100×, Gibco), 1× penicillin-streptomycin (100×, Gibco), 50 μM β-mercaptoethanol (Sigma), 103 units /ml LIF (ESGRO), 1 μM PD0325901 (MedChemExpress) and 3 μM CHIR99021 (MedChemExpress). The medium was replaced daily when cells were not passaged. Cells are maintained at 37℃ in the 5% CO2 incubator.

extract_protocol:
ESCs were lysed with Trizol reagent (Life Technologies), and RNA was extracted based on the manufacturer’s instructions.
Crosslink & isolate nuclei: Fix cells with formaldehyde, quench, and prepare nuclei.  Digest chromatin: Cut DNA in nuclei with a restriction enzyme (commonly MboI/DpnII).  Fill-in + biotin label: Fill ends with biotinylated nucleotides.  Proximity ligation (in situ): Ligate DNA ends in nuclei to capture 3D contacts.  Shear DNA: Sonicate to ~200–500 bp fragments.  ChIP enrichment: Immunoprecipitate chromatin using an antibody to the target protein/mark (e.g., H3K27ac).  Reverse crosslinks & purify DNA: Proteinase digestion, clean up DNA.  Pull down biotin DNA: Enrich ligation junctions with streptavidin beads.  End repair / A-tailing / adapter ligation: Prepare fragments and ligate sequencing adapters.  PCR amplify & QC: Limited-cycle PCR, size selection if needed, quantify, then paired-end sequencing.

treatment_protocol: ESCs were induced to differentiate by removing LIF/2i or adding 2 μM RA (Sigma) for 12h and 24 h, respectively.

value: cell line

value: ESC

value: wildtype


title: ATAC_KCP_LY_2

data_processing:
Align to hg19 reference genome using bowtie2-2.4.1
Peak calling to generate signal tracks on on individual replicates using macs2 2.2.7.1 with options -B -p 1e-2 -f BAMPE
Assembly: hg19
Supplementary files format and content: Coverage track in bigWig format

extract_protocol:
Organoids were extracted from the matrigel by cell recovery solution, trypsinized into single cell suspension. 50,000 cells were lysed in ATAC lysis buffer and tagmented based on omni-ATAC-seq protocol.
ATAC libraries were prepared for sequencing using standard Illumina Nextera DNA Library Preparation protocols.


title: RT_WD_female_17w_118_heart

description: Library name: HDP-018954_heart

value: heart


title: KCP_DMSO_H3K27Ac_CR


title: cArg_CGG

description: Library name: Sample_51


title: RA_1d-rep1

description: Library name: RA_1d-rep1


title: ATAC_KCP_LY_1


title: Cyp26a1-KO-RA_24h-scRNA

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software.
Assembly: mm10
Supplementary files format and content: Cell Ranger Feature Barcode Matrices (HDF5 Format).

source_name: ESC

extract_protocol:
1. Cell Counting & Quality Control: The prepared cell suspension was counted using TC20 Automated Cell Counter (Bio-Rad, USA) to obtain the information of cell size, concentration and viability, and to determine the sample loading volume. 2. GEM Generation and cDNA Amplification: Within the Chromium instrument, barcoded Single Cell 3' Gel Beads are mixed with cells or nuclei, enzymes, and partitioning oil to form tens of thousands of single cell emulsion droplets called "GEMs" (Gel Bead-in-emulsion). Immediately following GEM generation, the Gel Bead is dissolved and any co-partitioned cell is lysed. Oligonucleotides containing (i) an Illumina Read 1 (read 1 sequencing primer), (ii) a 16 nt 10x Barcode, (iii) a 12 nt unique molecular identifier (UMI), and (iv) 30 nt poly(dT) sequence are released and mixed with the cell lysate and a Master Mix containing reverse transcription (RT) reagents. Incubation of the GEMs produces 10x Barcoded, full length cDNA from poly-adenylated mRNA. Then, the first-strand cDNA of 10X Barcoded was purified by silane magnetic beads, and sufficient mass was obtained by PCR amplification. The PCR products were clean up by SPRI beads, and double-stranded cDNA was obtained. Then quantify and qualify the dsDNA by Qubit3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), so as to confirm the final PCR cycle number for library construction.
Library Construction: The 1/4 of the dsDNA was then treated with Fragmentation Mix to fragment cDNA, End Prep Enzyme Mix to repair both ends and add a dA-tailing in one reaction, followed by double sided size selection to recover ~300 bp of the cDNA. Each sample was then added adaptors to both ends by a T-A ligation. The purified Ligation was amplified by PCR using P5 and P7 primers, with both primers carrying sequences which can anneal with flow cell to perform Illumina bridge PCR. And P7 primers are a mixture of four primers, each carrying a eight-base index allowing for multiplexing. The PCR products were cleaned up using beads, validated using an Qsep100 (Bioptic, Taiwan, China), and quantified by Qubit3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA).


title: RA_0d-rep2

description: Library name: RA_0d-rep2


title: Ribo_cPro_eIF5A_KD

description: Library name: Sample_48


title: WT-RA_24h-scRNA


title: cPhe_SAMD9_+Dox_rep2

description: Library name: Sample_47


title: RA_0d-rep1

description: Library name: RA_0d-rep1


title: TN_WD_female_17w_119_heart

description: Library name: HDP-018715_heart


title: TN_CD_female_9d_117_heart

description: Library name: HDP-019055_heart


title: ATAC_KCP_DMSO_2


title: Ribo_cPro_scramble

description: Library name: Sample_49


title: Hoxb4-KO-RA_24h-scRNA


title: ATAC_KCP_DMSO_1


title: RT_WD_female_17w_114_heart

description: Library name: HDP-018951_heart


title: TN_CD_female_17w_116_heart

description: Library name: HDP-018754_heart


title: cAsp_GAT-GAC

description: Library name: Sample_50


title: cPhe_SAMD9_-Dox_rep2

description: Library name: Sample_46


title: cAsp_GAC

description: Library name: Sample_45


title: ESC-NoRA24h-scRNA