title: HUVECs, ALP, 4

description: Library name: Sample8

data_processing:
Checked fastq reads quality using FastQC v0.11.8 (fastqc --noextract --format fastq -t 10 )
Trim adapter and remove low quality reads using fastp v0.20.0 with default setting
Trimmed sequence reads were mapped to transcriptome(hg38) and performed transcript quantification using kallisto (V0.46.2) quant with default settings.
Gene-level expression matrix based on transcript-level data were generated using Tximport v1.24.0
Read count data was imported to DEseq2 (v1.36.0) to perform differential expression analysis
Assembly: hg38
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample

extract_protocol:
Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure.  The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA).  The RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA) with RIN number >7.0, and confirmed by electrophoresis with denaturing agarose gel.
Poly (A) RNA is purified from 1μg total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) using two rounds of purification.   Then the poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.  e6150, USA) under 94℃ 5-7min.   Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat.   1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.  m0209, USA), RNase H (NEB, cat.  m0297, USA) and dUTP Solution (Thermo Fisher, cat.  R0133, USA）.   An A-base is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters.   Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA.   Single- or dual-index adapters are ligated to the fragments, and size selection was performed with AMPureXP beads.   After the heat-labile UDG enzyme (NEB, cat.  m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products are amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min;   8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec;   and then final extension at 72℃ for 5 min. The average insert size for the final cDNA library was 300±50 bp.   At last, we performed the 2×150bp paired-end sequencing (PE150) on an illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.


title: Male WT CV, PBS, bio rep 4

status: Public on Oct 27 2025

type: SRA

city: Saint Louis

first: Erica

middle: L

last: Scheller

state: MO

address: 660 S Euclid Ave

country: USA

institute: Washington University

description:
CV_682
Read_Counts.txt
TPM_values.txt

library_source: transcriptomic

data_processing:
The DNBSEQ platform was used to perform paired-end 100 bp sequence reads, generating an average of 4.84 to 6.87Gb per sample.
Sequencing data was filtered using SOAPnuke, a filtering algorithm that removes reads containing the adaptor, reads with N content >5%, and reads with base quality score <15 as the proportion of total bases in the reads that are greater than 20% as low-quality reads.
After filtering, HISAT and Bowtie2 were used to align clean reads to the Mus musculus reference genome version GCF_000001635.26_GRCm38.p6 with an average mapping ratio of 98% and 81-86%, respectively --> read counts.
Read counts were normalized per sample to generate TPM values
Assembly: GRCm39
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample
Supplementary files format and content: tab-delimited text file includes TPM values for each Sample

molecule: total RNA

organism: Mus musculus

source_name: Caudal vertebrae

growth_protocol: All mice were housed in a specific pathogen-free facility at a controlled temperature of 22–23 °C on a 12-hour light/dark cycle.

extract_protocol:
After endpoint dissection, tissues were homogenized and preserved in Trizol at -80 °C before RNA extraction. To purify RNA, samples were processed with PureLink™ RNA Mini Kit (Invitrogen, 12183025) according to the manufacturer’s instructions, and the RNA concentration and quality in the samples were checked with a spectrophotometer (Thermo Scientific, NanoDrop 2000).
After being enriched by oligo dT and fragmented, a cDNA library was generated by reverse-transcribing mRNA using random N6-primed RT.

treatment_protocol: Mice were treated with either ICV PBS or 100 ng/hr ICV leptin for 9 days. A group of mice were also subcutaneously supplemented with insulin for 9 days.

tag: tissue

value: Caudal vertebrae

tag: genotype

tag: treatment

value: ICV PBS


title: HUVECs, ALP, 3

city: Beijing

first: Wenting

last: Zhu

address: No.95 Yong'an Road, Xicheng District

country: China

institute: Beijing Friendship Hospital, Capital Medical University

description: Library name: Sample7

organism: Homo sapiens

source_name: Human Umbilical Vein Endothelial Cells

growth_protocol: Human umbilical vein endothelial cells (HUVECs) were cultured using endothelial cell medium containing Endothelial Cell Growth Supplement, 5% fetal bovine serum and 1% penicillin/streptomycin.Cells were cultured at 37℃ at 5% CO2.

tag: cell type

value: Human Umbilical Vein Endothelial Cells


title: HUVECs, ALP, 2

description: Library name: Sample6


title: Male WT CV, leptin + insulin, bio rep 4

description:
CV_452
Read_Counts.txt
TPM_values.txt

value: ICV leptin + SubQ insulin


title: Male WT CV, leptin + insulin, bio rep 3

description:
CV_374
Read_Counts.txt
TPM_values.txt


title: Mouse Gut EEC 36 GEX

city: New York

first: Robert

last: Lovelett

state: New York

address: 430 E 29th Street, 10th Floor

institute: Kallyope

description:
Library name: K_10X_mGUT036_GEX
10X Genomics

data_processing:
Cell Ranger 8.0.0 mkfastq, count, and multi utilties were used to demultiplex and quantify the reads using pipelines implemented in nf-core demultiplex v1.5.3 and nf-core/scrnaseq v3.0.0.
Assembly: Custom version of mm10 where 3' region was extended up to 5Kbp when it did not collide with another known gene on the same strand
Supplementary files format and content: h5ad files with gene expression count matrices, 10X h5 matrices with gene expression and antibody counts for the antibody tagged libraries (K_10X_mGUT035 and K_10X_mGUT036)
Library strategy: CITE-seq

molecule: polyA RNA

source_name: Gut - EEC enriched

extract_protocol:
EEC protocol: Samples included Neurod1-Cre and Neurog3-Cre mice that were crossed with Ai9 reporters, Chga-GFP, Tph1-CFP, Ffar3-mRFP, and wild type mice. Intestines (stomach, duodenum, jejunum, ileum, and colon) were digested to single cell suspensions following the protocol described in Roberts et al (Diabetes, 2019). They were then FACSed sorted to the cells that emit the fluorophore or wild type mice were sorted with antibodiesto Epcam+/Cldn- cells. For two libraries (K_10X_mGUT035 and K_10X_mGUT036), cell hashing was employed to multiplex multiple samples following the protocol at https://www.protocols.io/view/cite-seq-5qpvo4rxg4o1/v1  Nodose protocol: Wild type mice were used. Nodose ganglia were dissected and as much nerve as possible we trimmed off. 10-11 ganglia were pooled and dissociated with 500 ul of dissociation solution, (5 mg/ml Collegenase II,1 mg/ml Dispace II, in Hank's buffered salt solution) and stored on ice. After dissections were complete, ganglia in dissociation solution were placed in a 37 C water bath for 1 hour with gentle mixing and pipetting every 10 minutes. Triteration was performed with a 1 ml pipette. Undissociated tissue was visualized by microscope to check for the presence of neurons. Dissociation continued until all neurons were visibly dissociated. Then, 500 ul of nodose medium (10% heat inactivated fetal bovine serum in DMEM) was added to the dissociated tissue, mixed 5 times with a 1 ml pippette and spun at 420 RCF for 5 minutes at 4 C. Supernatatants were discarded and pellets were resuspended in nodose medium. Resuspended cells were layered on top of 1ml of BSA gradient in DPBS on ice. Tubes with BSA and cell suspections were spun at 200 RCF for 5 minutes at 4 C. Supernatants were discarded; pellets were resuspended in 500 ul DPBS with 0.4% BSA and filtered through a 70 um cell strainer. Cells were counted and diluted following the 10X Genomics protocol
Single cell suspensions were loaded on to a 10x Chromium Controller as quickly as possible. Approximately 20,000 cells were loaded per reaction. LIbraries were generated with V2 or V3 chemistry following 10x Genomics protocols.

value: Gut - EEC enriched

tag: marker sort

tag: library type

value: GEX


title: HUVECs, ALP, 1

description: Library name: Sample5


title: HUVECs, Ad-SRSF1, 1 (IP)

description:
Library name: Sample3, IP
library strategy: eCLIP-seq
SRSF1_eCLIP.reproducible_enriched_windows.tsv

data_processing:
Raw Reads were first clustered according to the UMI sequences, in which reads with the same UMI sequence were grouped into the same cluster.Reads in the same cluster were compared to each other by pairwise alignment, and then reads with sequence identity over 95% were extracted to a new sub-cluster. After all sub-clusters were generated, multiple sequence alignment was performed to get one consensus sequence for each sub-clusters. After these steps, the errors and biases introduced by PCR amplification and sequencing were eliminated. De-duplicated consensus sequences mapped to rRNAs were removed with SortMeRNA (version 4.3.3).
Raw sequencing data quality was checked with FastQC (version 0.11.9). Reads was then filtered by fastp (version 0.22.0), of which low-quality reads were discarded and the reads contaminated with adaptor sequences were trimmed.
Filtered reads were aligned to GRCh38_release110 reference genome using STAR (v2.7.4a) with the following configurations --alignEndsType EndToEnd --genomeLoad NoSharedMemory --outBAMcompression 10 --winAnchorMultimapNmax 100 --outFilterMultimapNmax 100 --outFilterMultimapScoreRange 1 --outSAMmultNmax 1 --outMultimapperOrder Random --outFilterScoreMin 10 --outFilterType BySJout --limitOutSJcollapsed 5000000 --outReadsUnmapped None --outSAMattributes All --outSAMmode Full --outSAMtype BAM SortedByCoordinate --outSAMunmapped Within --readFilesCommand zcat --outStd Log --runMode alignReads.
BAM files were filtered to retain uniquely mapped reads (NH:i:1) with mapping quality ≥10 using samtools. Command:  samtools view -h "$bam_file" |  awk '($0 ~ /^@/ || ($5 >= 10 && $0 ~ /NH:i:1/))' | samtools view -Sb -o "$filtered_bam". Coverage at 5' positions was calculated using bedtools genomecov with scaling factors, excluding non-canonical chromosomes. Command:  factor=$(samtools idxstats "$filtered_bam" | cut -f 3 | paste -sd+ | bc | xargs -I {{}} echo 'scale=6; 10^6 / {{}}' | bc);  bedtools genomecov -scale $factor -5 -bg -ibam "$filtered_bam" | sort -k1,1 -k2,2n | grep -v -E "EBV|KI|GL" | sed 's/^/chr/g' > "${SCALED_DIR}/${base_name}_unique_scaled.bg"
Differential peaks between CLIP and INPUT samples were identified using Skipper with default parameters.
Assembly: GRCh38
Supplementary files format and content: Tab-delimited text files include differential enriched peaks for each Sample
Supplementary files format and content: Bigwig files for each Sample

extract_protocol:
The samples were irradiated twice with 254 nm UV-C light on ice at 400mJ, and lysed on ice for 15 min with lysis buffer (20 mM Tris, pH 8.0, 1 mM EDTA, 400 mM NaCl, 0.1% SDS, 0.5% Triton X-100 (Merck, cat. no. X100), 1×Protease Inhibitor (Thermo Fisher, cat. no. 87785), 200 U/mL RNasin Plus (Promega, cat. no. N2611) and 20 U/mL DNase I (NEB, cat. no. M0303S)). The lysate was further treated with 40 U RNase I (Thermo Fisher, cat. no. EN0601) and rotary incubated at 37°C for 5 min. The reaction was quenched by adding RNasin® Plus. The mixture was centrifuged at 4°C for 15 minutes at 15,000 g, and 2% of the supernatant was was taken for Input sample. The remain of supernatant was incubated with Invitrogen 32-4500 antibody for immunoprecipitation, rotating slowly at 4°C for 4 h. 10 μL protein A/G magnetic beads (Thermo Fisher, cat. no. 88802) was added and incubated with immune complexes for 2 h followed by successive washes. The 3' phosphate groups on the RNA were removed using FastAP enzyme (Thermo Fisher, cat. no. EF0654) to facilitate subsequent ligation steps. A 3' App-DNA linker is ligated to the RNA molecules using T4 RNA ligase (NEB, cat. no. M0204). The RNA-protein complexes were then separated by Nupage 4-12% Bis-Tris protein gels (Thermo Fisher, cat. no. WG1401BX10) electrophoresis and transferred to a nitrocellulose (NC) membrane. The NC membrane was cut to extract the RNA bound to the protein of interest. The protein-bound RNA was extracted from the NC membrane by incubation with proteinase K (NEB, cat. no. P8107) at 37°C for 20 min and subsequent TRIzol (Thermo Fisher, cat. no. 15596018CN) treatment.
The purified RNA was subsequently concentrated, desalted, reverse transcribed. The resulted cDNA was recovered, ligated with a 3' UMI (Unique Molecular Identity) adapter, and amplified by PCR to generate sequencing libraries, which was sequenced on a Novaseq X Plus sequencer (Illumina) using PE150 mode.


title: Mouse Gut EEC 38

description:
Library name: K_10X_mGUT038
10X Genomics

library_source: transcriptomic single cell

data_processing:
Cell Ranger 8.0.0 mkfastq, count, and multi utilties were used to demultiplex and quantify the reads using pipelines implemented in nf-core demultiplex v1.5.3 and nf-core/scrnaseq v3.0.0.
Assembly: Custom version of mm10 where 3' region was extended up to 5Kbp when it did not collide with another known gene on the same strand
Supplementary files format and content: h5ad files with gene expression count matrices, 10X h5 matrices with gene expression and antibody counts for the antibody tagged libraries (K_10X_mGUT035 and K_10X_mGUT036)

source_name: Colon - EEC enriched

value: Colon - EEC enriched


title: HUVECs, DMSO, 4

description: Library name: Sample4


title: Mouse Gut EEC 35 GEX

description:
Library name: K_10X_mGUT035_GEX
10X Genomics

value: NeuroD1 or Tph-1-CFP


title: Male WT CV, PBS + insulin, bio rep 5

description:
CV_373
Read_Counts.txt
TPM_values.txt

value: ICV PBS + SubQ insulin


title: HUVECs, DMSO, 3

description: Library name: Sample3


title: HUVECs, DMSO, 1

description: Library name: Sample1


title: Male WT CV, PBS + insulin, bio rep 4

description:
CV_372
Read_Counts.txt
TPM_values.txt


title: Mouse Gut EEC 35 ADT

description:
Library name: K_10X_mGUT035_ADT
10X Genomics

library_source: other

molecule: protein

value: ADT

tag: adt

value: EpCAM antibody (GeneTex GTX80323)


title: Male WT CV, PBS + insulin, bio rep 3

description:
CV_370
Read_Counts.txt
TPM_values.txt


title: HUVECs, DMSO, 2

description: Library name: Sample2


title: Mouse Gut EEC 33

description:
Library name: K_10X_mGUT033
10X Genomics


title: Male WT CV, PBS + insulin, bio rep 2

description:
CV_337
Read_Counts.txt
TPM_values.txt


title: KO3F

city: Zurich

first: Burkhard

last: Becher

address: Winterthurerstrasse 190

country: Switzerland

institute: University of Zurich

data_processing:
Reads were trimmed with fastp v0.23.4. Non-default trimming options were --trim_poly_x
Gene expression values were computed with kallisto v0.46.1 with the options "-bias --bootstrap-samples 10 --seed 42  --rf-stranded"
Assembly: GRCm39/ with GENCODE Annotation Release_M31, downloaded 2023-01-30
Supplementary files format and content: tabular text files holding expression values

source_name: Salivary Gland

extract_protocol:
Qiagen RNeasy Plus Micro Kit Ref# 74034
Takara SMARTer Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian

value: Salivary Gland

value: Myoepithelial cell

tag: age

value: 1 month

tag: Sex

value: male


title: Mouse Gut EEC 34

description:
Library name: K_10X_mGUT034
10X Genomics

source_name: Stomach - EEC enriched

value: Stomach - EEC enriched


title: KO1F

value: female


title: KO4M


title: HUVECs, Ad-SRSF1, 2

data_processing:
Checked fastq reads quality using FastQC v0.11.8 (fastqc --noextract --format fastq -t 10 )
Trim adapter and remove low quality reads using fastp v0.20.0 with default setting
Trimmed sequence reads were mapped to transcriptome(hg38) and performed transcript quantification using kallisto (V0.46.2) quant with default settings.
Gene-level expression matrix based on transcript-level data were generated using Tximport v1.24.0
Read count data was imported to DEseq2 (v1.36.0) to perform diffrential expression analysis
Assembly: hg38
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample

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

value: SRSF1 overexpression


title: HUVECs, Ad-SRSF1, 3


title: Mouse Gut EEC 31

description:
Library name: K_10X_mGUT031
10X Genomics

source_name: Ileum - EEC enriched

value: Ileum - EEC enriched


title: Ctrl3M


title: HUVECs, Ad-SRSF1, 1

value: Human Umbilical Vein Endothelial Cells


title: KO2F


title: Mouse Gut EEC 29

description:
Library name: K_10X_mGUT029
10X Genomics


title: HUVECs, Ad-GFP, 3

value: control


title: Male WT CV, leptin + insulin, bio rep 1

description:
CV_331
Read_Counts.txt
TPM_values.txt

value: Caudal vertebrae

value: ICV leptin + SubQ insulin


title: Ctrl4M


title: Ctrl2F


title: Mouse Gut EEC 28

description:
Library name: K_10X_mGUT028
10X Genomics

value: GEX


title: HUVECs, Ad-GFP, 2


title: Mouse Gut EEC 32

description:
Library name: K_10X_mGUT032
10X Genomics

source_name: Duodenum - EEC enriched

value: Duodenum - EEC enriched


title: Male WT CV, leptin, bio rep 5

description:
CV_185
Read_Counts.txt
TPM_values.txt

value: ICV leptin


title: Mouse Gut EEC 27

description:
Library name: K_10X_mGUT027
10X Genomics

source_name: Small Intestine - EEC enriched

value: Small Intestine - EEC enriched


title: Male WT CV, PBS + insulin, bio rep 1

description:
CV_336
Read_Counts.txt
TPM_values.txt

value: ICV PBS + SubQ insulin


title: HUVECs, Ad-GFP, 1


title: Control 7, Male, Day 3

city: Aurora

first: Kristen

last: Wells

state: CO

address: 1775 Aurora Ct,

department: Barbara Davis Center

institute: University of Colorado Anschutz

description:
Library name: control_7
83_B_cell

data_processing:
Trimming (cutadapt V. 4.8)
Alignment STAR v. 2.7.11b
Gene expression qualitifcation featureCOunts V. 2.0.6
Analysis DESeq2 v. 1.44.0
Batch correction CombatSeq 3.52.1
Samples were viewed in IGV. Those that did not have the proper PTPN2 KO were removed before PCA and differential expression (15, 985, and 998)
Assembly: GRCm38
Supplementary files format and content: Raw read counts from featureCounts, tsv
Supplementary files format and content: Sample information, csv

source_name: Spleen

extract_protocol:
Rneasy Plus Mini Kit (74134 Qiagen)
TruSeq Staranded

value: Spleen

value: Male

value: B cell

value: Control

tag: batch

value: Day 3


title: Male WT CV, leptin, bio rep 3

description:
CV_183
Read_Counts.txt
TPM_values.txt


title: Male WT CV, leptin + insulin, bio rep 2

description:
CV_332
Read_Counts.txt
TPM_values.txt


title: Male WT CV, leptin, bio rep 4

description:
CV_184
Read_Counts.txt
TPM_values.txt


title: Ctrl1F

value: Salivary Gland

value: Myoepithelial cell

value: 1 month

value: female


title: Control 6, Male, Day 3

description:
Library name: control_6
44_B_cell


title: Mouse Gut EEC 26

description:
Library name: K_10X_mGUT026
10X Genomics

source_name: Dudodenum - EEC enriched

value: Dudodenum - EEC enriched


title: Targetseq_OTOF-reporter-ACA36+DKC1+tRNA-2

first: Wenqing

last: Liu

email: puyulwq@163.com, liuwq21@gamils.com

address: No. 5 Summer Palace Road, Haidian District, Beijing

institute: Peking University

description: detect modification level of PTC sites in wildtype HEK293T cells with RESTART v3 treatment

data_processing:
*library strategy: Target-seq
First, sequencing reads with the same unique molecular identifier (UMI) were grouped and UMI groups with less than 3 reads were discarded.
Then, PCR duplicates were removed by retaining the most consensus sequence in each UMI group.
Adapter sequences were further trimmed from deduplicated reads with cutadapt software (version 4.2) using the key parameters -e 0.1 -O 3 --quality-cutoff 25 -m 55. The UMIs were removed by umi_tools (version 1.0.0).
The cleaned reads were mapped to the targeted sequences using PRAISE-tool. Samtools was used to filter out the reads containing more than 2 mismatches.
Finally, the deletion rates of target sites were calculated.
Assembly: GRCh38, GRCm39
Supplementary files format and content: The processed data file format is xlsx. Its content is the pseudouridylation level of target sites.

source_name: Epithelial

growth_protocol: HEK293T cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
total RNA was extracted from cells using TRIzol reagent (Life Technologies)
PRAISE-labeled RNA was used for reverse transcription with Maxima H minus Reverse Transcriptase (Thermo Fisher Scientific, EP0753). Target amplicons were amplificated by two round of PCR.

treatment_protocol: For transfection, 2.5 x 10^5 of HEK293T cells were seeded on 24-well plates (Corning), and all transfections were conducted when cells reached approximately 80% confluency after 20-24h. The plasmids were extracted using EndoFree Mini Plasmid Kit II (TIANGEN BIOTECH, DP118), and were transfected into cells by Lipofectamine LTX reagent (Invitrogen) supplemented with PLUS reagent (Invitrogen) according to the manufacturer’s instructions.

value: Epithelial

tag: cell line

value: human embryonic kidney 293 cells

value: WT


title: SMARTseq_pIC_iMSCL_H5

first: James

last: Swann

state: NY

address: 650 W 168th Street

institute: Columbia University

data_processing:
10X Genomics: The demultiplexing, barcode processing, gene counting, and aggregation were performed using CellRanger software (version 7.0.1).
SMARTseq: Fastq quality was assessed with FastQC and MultiQC. After trimming adapters with TrimGalore sequences were pseudoaligned to the murine mm10 genome and counted using Salmon.
Assembly: mm10
Supplementary files format and content: 10X Genomics: Tab-separated values files and matrix files; oligo-hashtag IDs as comma separated file
Supplementary files format and content: SMARTseq: Counts table and cell identifiers for SMARTseq as comma separated files

source_name: Central marrow stroma

extract_protocol:
Combined stromal and hematopoietic cells isolation  Mice were euthanized in a rising concentration of carbon dioxide, followed by cervical dislocation. For each mouse, both femurs, tibiae, hemipelves, and humeri were dissected and thoroughly cleaned using KimWipes (Kimtech) and were used for combined isolation of stromal and hematopoietic cells. To isolate central marrow (CM) stromal cells, the proximal and distal epiphyses were removed from 1 femur. Intact marrow plugs were flushed with Hank’s balanced saline solution (HBSS) without calcium or magnesium into 5 ml polypropylene tubes by inserting a 3 ml syringe with 22G needle into the distal end of the femur. The plugs were digested with 1 ml of a solution of 3 mg/ml type I collagenase (Worthington) dissolved in HBSS for 10 min at 37 C with 110 rpm shaking. The tube was then vortexed briefly and the supernatant was removed into a new tube through a 100 m mesh, taking care not to disturb the marrow plug. A further 1 ml of 3 mg/ml type I collagenase solution was then added to the marrow plug and the digestion repeated. After the second incubation, the plug was dissociated by pipetting up and down with a P1000 pipette before passing the cell suspension through a 100 m mesh into the tube containing the previous digestion supernatant. To isolate endosteal (Endo) stromal cells and hematopoietic cells, the flushed femur was combined with the other bones, gently crushed up to 10 times using a mortar and pestle, and thoroughly washed with 10 ml HBSS until all the non-adherent BM cells were removed and collected in a separate 15 ml polypropylene tube. The bone chips were then digested in a 15 ml polypropylene tube with 3 ml of 3 mg/ml type I collagenase solution for 1 hour at 37 C with 110 rpm shaking. After digestion, the tube was vortexed briefly and the cell suspension was filtered through a 100 m mesh into a new tube. The bone chips were then washed with HBSS, and the washing was collected into the same tube as the digestate. For cell suspensions acquired for both stromal compartments, red blood cells were removed by adding 1 ml ACK lysis buffer (150 mM NH4Cl and 10 mM KHCO3) and incubated on ice for 3 minutes before washing with HBSS containing 4% fetal bovine serum (HI FBS, Gibco). During the optimization of the protocol outlined above, the digestion with 3 mg/mL type I collagenase was performed in parallel to and compared with 3 mg/mL type I collagenase + 4 mg/mL type II Dispase (Roche), 250 μg/mL Liberase DL (Roche) + 200 U/mL DNAse I (Sigma), and mechanical dissociation with P1000 pipette. The BM cells collected from crushed bones were resuspended in HBSS with 2% FBS and RBCs were removed by lysis with ACK buffer. The BM cell suspension was further purified on a density gradient (Histopaque 1119, Sigma-Aldrich), by layering 2 ml of the cell suspension under 2 ml of Histopaque solution. For profiling of CM plug and Endo marrow, only femurs were used and were flushed once with a 3 ml syringe and 21G needle to collect BM plugs, while the flushed bones were crushed to collect the remaining Endo-associated BM cells. No ACK RBC lysis was performed for these analysis. Both stromal and hematopoietic cells were finally counted using a Vicell automated cell counter (Beckman Coulter).  RNA sequencing - Droplet-based scRNA-seq for stroma and hematopoietic analyses For Endo and CM stromal cell preparations, 30,000–50,000 Ter119-/CD45- cells were sorted on purity mode into 1.5 ml tubes containing 300 μl of 50% FBS and 50% HBSS using a BD FACS Aria II instrument (gating shown in Supplementary Fig. 2a, 3b). For whole BM cells, 40,000 live (propidium iodide negative) cells from n=2 mice were sorted into similar tubes from PBS or pIC-injected mice. Cells were rested for 1 hour on ice then pelleted at 350 x g for 5 minutes at 4°C before the supernatant was removed down to a volume of 40 μl.  For sequencing of defined HSPCs, 8,000 cells of 10 different populations (HSC, stHSC, MPP2, FcγR- MPP3, FcγR+ MPP3, MPP4, GMP, MkP, CLP, CFU-E, see Supplementary Fig. 1 for sorting scheme) were sorted separately into 1.5 ml tubes. In this experiment, there were 2 biological replicates with BM from n=5 mice, and 2 technical replicates were generated per sample. Sorted cell populations were labelled with 0.05 μg of oligo-hashed antibodies specific for MHC class I and CD45 (BioLegend, TotalSeq B1-10) for 30 minutes on ice before washing three times with HBSS containing 2% FBS. All 10 populations were then pooled for subsequent partitioning. Cells were rested for 1 hour on ice then pelleted at 350 x g for 5 minutes at 4°C before the supernatant was removed down to a volume of 40 μl.  For sequencing of purified stromal populations, samples of central marrow or endosteal stromal cells pooled from n=7 mice were labelled with oligo-hashed antibodies at a concentration of 0.375 μg of antibody per 106 cells. These antibodies were mixed with the fluorophore-conjugated surface antibodies, and cells were then washed three times with HBSS and 4% FCS. Specific stromal cell populations were then sorted by FACS (gating scheme shown in Fig. 1e), and mixed according to the following scheme to produce two combined samples, each containing a total of 25-40,000 cells: Sample type Oligo hash antibody Sample 1 (cells) Sample 2 (cells) CM TotalSeq B1 MSC-L (6899) SEC (2228) CM TotalSeq B2 mMPr (2775) SEC (2097) CM TotalSeq B3 mMPr (2406) SEC (2529) CM TotalSeq B4 MPr (847) SEC (1041) CM TotalSeq B5 MPr (802) SEC (1353) CM TotalSeq B6 MSC-L (7642) AEC (438) CM TotalSeq B7 - AEC (496) Endo TotalSeq B8 MPr (10048) MSC-S (8212) Endo TotalSeq B9 AEC (5046) MSC-L (2319) Endo TotalSeq B10 SEC (3537) mMPr (6823)  Cells were rested for 1 hour on ice then pelleted at 350 x g for 5 minutes at 4°C before the supernatant was removed down to a volume of 40 μl. GEM generation and 3' RNA library preparation was performed according to 10X Genomics protocol CG000315 Rev E, targeting 5000 cell data recovery. RNA libraries were pooled 1:1:1:etc, sequenced on an Illumina NovaSeq 5000, and alignment was performed using Cellranger (v.7.0.1) to mouse genome mm10. For hashing samples, hashing libraries were pooled with RNA libraries at a ratio of 1:4. Library concentrations and fragment sizes were evaluated using Qubit dsDNA HS assay kit (ThermoFisher Scientific) and TapeStation D5000 DNA ScreenTape analysis (Agilent).  RNA sequencing - Plate-based SMART-seq for stromal cells and MSC-L For Smart-seq analyses of Endo (AEC, MSC-S, mMPr, MPr) and CM (SEC, MSC-L) stromal cells isolated from young WT mice, we utilized our own published data (Mitchell et al., 2023) that was processed using the same analytical pipeline. For Smart-seq of iMSC-L from pIC-injected young WT mice and aged 24-month-old WT mice, we utilized the SMART-Seq Single Cell PLUS kit (96 reactions, Takara Bio). Single iMSC-L were sorted into 96 well plates (see Supplementary Fig. 3c for gating strategy) containing 12.5 μl of CDS sorting solution provided in the kit. Preparation and amplification of cDNA and single cell libraries were completed according to the manufacturer’s instructions. Single cell libraries that passed quality control (n=43 from pIC-injected mice and n=30 from aged mice) were pooled at a 1:1:1etc ratio before sequencing on an Illumina NextSeq 500/550 instrument using a version 2 kit (Illumina). Fastq files were generated for each library in Illumina BaseSpace.
For 10X Genomics: Library was constructed according to the manufacturer's instructions (single cell 3', version 3 protocol, 10X Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscribed to cDNA, which contains an Illumina R1 primer sequence, unique molecular identifier (UMI) and the 10X barcode. The pooled barcoded cDNA was cleaned up with Silane Dynabeads, amplified by PCR, and the appropriate size fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction, paired-end constructs with P5 and P7 sequences and a sample index were added.  SMARTseq: Library construction was performed according to the manufacturer's instructions (SMART-Seq Single Cell PLUS kit (96 reactions, Takara Bio).

treatment_protocol: Where indicated: polyinosinic:polycytidylic acid (pIC) administered at 10 mg/kg IP in mice every 48 hours for 3 days before cells were isolated for sequencing.

value: Central marrow stroma

value: Inflammatory MSCL

value: Pooled male and female

value: 8-12 weeks

value: Poly(I:C) 10 mg/kg ip q48 hours for 3 days


title: PTPN2 KO 7, Male, Day 3

description:
Library name: ptpn2_ko_7
85_B_cell

value: Spleen

value: Male


title: Targetseq_OTOF-reporter-ACA19+DKC1+tRNA-2


title: Mouse Gut EEC 25

description:
Library name: K_10X_mGUT025
10X Genomics

value: Stomach - EEC enriched

value: GEX


title: Targetseq_OTOF-reporter-ACA36+DKC1-2

description: detect modification level of PTC sites in wildtype HEK293T cells with RESTART v2 treatment


title: Control 5, Female, Day 3

description:
Library name: control_5
77_B_cell

value: Female

value: B cell

value: Control

value: Day 3


title: Male WT CV, leptin, bio rep 1

description:
CV_180
Read_Counts.txt
TPM_values.txt

value: Caudal vertebrae

value: ICV leptin


title: SMARTseq_pIC_iMSCL_H4

description: SMART-Seq Single Cell PLUS kit (96 reactions, Takara Bio)


title: Control 4, Female, Day 3

description:
Library name: control_4
82_B_cell


title: SMARTseq_pIC_iMSCL_H3


title: Targetseq_OTOF-reporter-ACA19+DKC1-2

value: Epithelial

value: human embryonic kidney 293 cells


title: Male WT CV, leptin, bio rep 2

description:
CV_182
Read_Counts.txt
TPM_values.txt


title: Targetseq_OTOF-reporter-ACA36+DKC1+tRNA-1

value: WT


title: Male WT CV, PBS, bio rep 3

description:
CV_178
Read_Counts.txt
TPM_values.txt

value: ICV PBS


title: Male WT CV, PBS, bio rep 2

description:
CV_177
Read_Counts.txt
TPM_values.txt


title: PTPN2 KO 6, Female, Day 3

description:
Library name: ptpn2_ko_6
30_B_cell


title: Targetseq_OTOF-reporter-ACA19+DKC1+tRNA-1


title: SMARTseq_pIC_iMSCL_H1

value: Central marrow stroma

value: Inflammatory MSCL

value: Pooled male and female


title: Male WT CV, PBS, bio rep 1

description:
CV_176
Read_Counts.txt
TPM_values.txt


title: SMARTseq_pIC_iMSCL_G7

value: 8-12 weeks

value: Poly(I:C) 10 mg/kg ip q48 hours for 3 days


title: PTPN2 KO 5, Female, Day 3

description:
Library name: ptpn2_ko_5
33_B_cell

value: Spleen


title: Male KO CV, leptin, bio rep 1

description:
CV_165
Read_Counts.txt
TPM_values.txt


title: Targetseq_OTOF-reporter-ACA36+DKC1-1


title: Targetseq_OTOF-reporter-ACA19+DKC1-1


title: Male KO CV, PBS, bio rep 2

description:
CV_163
Read_Counts.txt
TPM_values.txt


title: SMARTseq_pIC_iMSCL_G6


title: PTPN2 KO 2, Male, Day 1

description:
Library name: ptpn2_ko_2
24_B_cell

value: Male

value: Day 1


title: Targetseq_OTOF-reporter-ACA19+tRNA-2

description: detect modification level of PTC sites in wildtype HEK293T cells with RESTART v3-mini treatment


title: Targetseq_OTOF-reporter-ACA36+tRNA-2


title: PTPN2 KO 3, Male, Day 1

description:
Library name: ptpn2_ko_3
9_B_cell

value: B cell


title: SMARTseq_pIC_iMSCL_G5


title: Female KO LV, PBS, bio rep 4

description:
LV_692
Read_Counts.txt
TPM_values.txt

source_name: Lumbar Vertebrae

value: Lumbar Vertebrae


title: Control 3, Female, Day 2

description:
Library name: control_3
998_B_cell

value: Female

value: Control

value: Day 2


title: Male KO CV, PBS, bio rep 1

description:
CV_162
Read_Counts.txt
TPM_values.txt

value: Caudal vertebrae


title: SMARTseq_pIC_iMSCL_F6


title: PTPN2 KO 4, Male, Day 3

description:
Library name: ptpn2_ko_4
68_B_cell

value: Day 3


title: Targetseq_OTOF-reporter-ACA36-2

description: detect modification level of PTC site in wildtype HEK293T cells with RESTART v1 treatment

value: Epithelial

value: human embryonic kidney 293 cells

value: WT


title: Targetseq_OTOF-reporter-ACA19-2


title: SMARTseq_pIC_iMSCL_G3

value: Central marrow stroma

value: Inflammatory MSCL


title: Control 2, Female, Day 2

description:
Library name: control_2
985_B_cell


title: SMARTseq_pIC_iMSCL_F7

value: Pooled male and female

value: 8-12 weeks


title: Female WT LV, PBS, bio rep 4

description:
LV_688
Read_Counts.txt
TPM_values.txt

value: ICV PBS


title: SMARTseq_pIC_iMSCL_F4

value: Poly(I:C) 10 mg/kg ip q48 hours for 3 days


title: Control 1, Male, Day 1

description:
Library name: control_1
29_B_cell

value: Spleen

value: Male

value: Day 1


title: Female WT LV, PBS, bio rep 5

description:
LV_689
Read_Counts.txt
TPM_values.txt

value: Lumbar Vertebrae


title: Targetseq_OTOF-reporter-gctrl-2

description: detect modification level of PTC sites in wildtype HEK293T cells


title: Female WT LV, leptin, bio rep 5

description:
LV_687
Read_Counts.txt
TPM_values.txt

value: ICV leptin


title: PTPN2 KO 1, Female, Day 2

description:
Library name: ptpn2_ko_1
15_B_cell

value: Female

value: B cell

value: Day 2


title: SMARTseq_pIC_iMSCL_F3


title: Targetseq_OTOF-reporter-ACA36+tRNA-1

value: Epithelial


title: SMARTseq_pIC_iMSCL_F2


title: sgCdk10_3, replicate 3, RNAseq

city: wuhan

first: Gaoshan

last: Xu

state: -- Please Select --

address: wuhan

institute: wuhan university

description: Library name: sgCdk10_3

data_processing:
Transcriptome sequencing and analysis were performed by OE Biotech Co., Ltd. The RNA libraries were sequenced on an Illumina NovaSeq 6000 platform, generating 150 bp paired-end reads.
Assembly: These clean reads were then aligned to the reference M. musculus mm10 genome with HISAT2 (2.2.1).
Supplementary files format and content: The read counts for each gene were obtained using FeatureCounts (2.0.3) and differential gene expression analysis was performed using DESeq2 (1.40.2).

source_name: cell line

extract_protocol:
Library preparation was carried out using the VAHTS Universal V6 RNA-seq Library Prep Kit, in accordance with the provided protocol
Library preparation was carried out using the VAHTS Universal V6 RNA-seq Library Prep Kit, in accordance with the provided protocol

value: cell line

value: Cancer cell


title: Female WT LV, leptin, bio rep 4

description:
LV_681
Read_Counts.txt
TPM_values.txt


title: Female WT LV, leptin, bio rep 3

description:
LV_680
Read_Counts.txt
TPM_values.txt


title: sgCdk10_2, replicate 2, RNAseq

description: Library name: sgCdk10_2


title: SMARTseq_pIC_iMSCL_F1

value: Central marrow stroma


title: Targetseq_OTOF-reporter-ACA36-1

value: human embryonic kidney 293 cells

value: WT


title: Female KO LV, PBS, bio rep 3

description:
LV_5484
Read_Counts.txt
TPM_values.txt


title: sgCdk10_1, replicate 1, RNAseq

description: Library name: sgCdk10_1


title: Targetseq_OTOF-reporter-ACA19-1


title: Female KO LV, leptin, bio rep 4

description:
LV_5482
Read_Counts.txt
TPM_values.txt


title: sgControl_3, replicate 3, RNAseq

description: Library name: sgControl_3


title: SMARTseq_pIC_iMSCL_E6

value: Inflammatory MSCL

value: Pooled male and female

value: 8-12 weeks


title: Female WT LV, PBS, bio rep 3

description:
LV_190
Read_Counts.txt
TPM_values.txt

value: ICV PBS


title: Targetseq_OTOF-FL-gctrl-2


title: SMARTseq_pIC_iMSCL_E7

value: Poly(I:C) 10 mg/kg ip q48 hours for 3 days


title: Targetseq_OTOF-reporter-gctrl-1


title: sgControl_1, replicate 1, RNAseq

description: Library name: sgControl_1


title: Female WT LV, PBS, bio rep 2

description:
LV_189
Read_Counts.txt
TPM_values.txt


title: sgControl_2, replicate 2, RNAseq

description: Library name: sgControl_2


title: SMARTseq_pIC_iMSCL_E4


title: SMARTseq_pIC_iMSCL_E3


title: Female WT LV, PBS, bio rep 1

description:
LV_188
Read_Counts.txt
TPM_values.txt


title: Cell_3, replicate 3

description: Library name: cell_3

library_source: genomic

data_processing:
The libraries were then sequenced on an Illumina NovaSeq 6000
Assembly: mmu10(Ensembl release 100)
Supplementary files format and content: Sequencing data were analyzed using MAGeCK-VISPR (v.0.5.3) to identify sgRNAs that were significantly enriched or depleted in each conditional comparison
Library strategy: CRISPR Screen

molecule: genomic DNA

source_name: Mouse subcutaneous tumor

extract_protocol:
Mice were euthanized on day 11 after implantation, cells and tumor genomic DNA was extracted from the whole tumor tissue.The DNA concentration was measured using a Nanodrop.
To ensure the accuracy of the sequencing results, the sgRNA library readout was performed using a two-step PCR strategy.

value: Mouse subcutaneous tumor


title: SMARTseq_pIC_iMSCL_E2


title: Targetseq_OTOF-FL-ACA19+DKC1-2


title: Female WT LV, leptin, bio rep 2

description:
LV_187
Read_Counts.txt
TPM_values.txt

value: Lumbar Vertebrae


title: Cell_2, replicate 2

description: Library name: cell_2


title: Targetseq_OTOF-reporter-ACA19+tRNA-1


title: SMARTseq_pIC_iMSCL_D5


title: Female WT LV, leptin, bio rep 1

description:
LV_186
Read_Counts.txt
TPM_values.txt

value: ICV leptin


title: SMARTseq_pIC_iMSCL_D2


title: Cell_1, replicate 1

description: Library name: cell_1


title: Female KO LV, leptin, bio rep 3

description:
LV_169
Read_Counts.txt
TPM_values.txt


title: SMARTseq_pIC_iMSCL_D1


title: Targetseq_OTOF-FL-gctrl-1


title: Nude_2, replicate 2

description: Library name: Nude_2


title: Targetseq_OTOF-FL-ACA19-2


title: Female KO LV, leptin, bio rep 1

description:
LV_167
Read_Counts.txt
TPM_values.txt


title: Nude_1, replicate 1

description: Library name: Nude_1


title: Targetseq_OTOF-FL-ACA19+DKC1-1


title: Targetseq_OTOF-FL-ACA19+DKC1+tRNA-1


title: SMARTseq_pIC_iMSCL_C6


title: SMARTseq_pIC_iMSCL_C5


title: Female KO LV, PBS, bio rep 2

description:
LV_166
Read_Counts.txt
TPM_values.txt


title: Targetseq_OTOF-FL-ACA19+tRNA-1


title: WT_3, replicate 3

description: Library name: WT_3


title: Female KO LV, PBS, bio rep 1

description:
LV_161
Read_Counts.txt
TPM_values.txt


title: WT_2, replicate 2

description: Library name: WT_2


title: Targetseq_OTOF-FL-ACA19+DKC1+tRNA-2


title: Targetseq_OTOF-FL-ACA19+tRNA-2


title: Targetseq_OTOF-FL-ACA19-1


title: SMARTseq_pIC_iMSCL_C4


title: Female KO CV, PBS, bio rep 4

description:
CV_692
Read_Counts.txt
TPM_values.txt

value: Caudal vertebrae


title: WT_1, replicate 1

description: Library name: WT_1


title: Normal colonic epithelial stem cell, scRNAseq

city: Kyoto

first: Makoto

middle: Mark

last: Taketo

state: Kyoto

address: Yoshida-Honmachi, Sakyo

department: Kyoto University Hospital

country: Japan

institute: Kyoto University

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v6.1.2 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger).Droplets with number of detected genes < 2500 and/or with mitochondrial genomes > 15% were discarded as low quality and/or dying cell droplets. UMI counts were then normalized so that each cell had a total of 10,000 UMIs across all genes and these normalized counts were logarithm-transformed with a pseudocount of 1 using the LogNormalize function in the Seurat 4.0 package (Hao et al., Cell 2022).
Assembly: hg38
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Large intestine of 70N

extract_protocol:
The NCE-SC were washed in PBS with 0.5 mM EDTA, resuspended in trypsin buffer (PBS supplemented with 0.25% w/v trypsin. Invitrogen, cat. No. 15090046) and incubated at 37 °C for 10 min. The NCE-SC were washed in DMEM/F-12 (nacalai, 08460-95) supplemented with 5% v/v FBS, filtered through a strainer (pluristrainer, SKU 43-50001-13) and re-suspended in PBS supplemented with 0.04% w/v BSA.
scRNA-seq analysis was performed by Rhelixa (Tokyo, Japan). A barcoded sequencing library was generated using the Chromium Single Cell 3’ v3.1 reagent Kit Dual. The NCE-SC were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.

value: Large intestine of 70N

value: NCE-SC

value: Spheroid line

value: 72 years old


title: SMARTseq_pIC_iMSCL_C3


title: Nude_3, replicate 3

description: Library name: Nude_3

value: Cancer cell


title: sgCdk10_2, replicate 2, scRNAseq

description:
Library name: sgCdk10_2
DNBSEQ-T7

data_processing:
The scRNA-seq library was prepared using the SeekOne® Single Cell Whole Transcriptome Kit according to the manufacturer's instructions. The libraries were then sequenced on an Illumina NovaSeq 6000 with a PE150 read length.
Assembly: mmu10(Ensembl release 100)
Supplementary files format and content: MTX fle and TSV fle contain counts for each barcodes and genes

source_name: Subcutaneous tumor

growth_protocol: 5 × 10⁵ CT26 (sgControl or sgCdk10) tumor cells were injected subcutaneously into 6-week-old female BALB/c WT mice,Fourteen days after injection, tumors were harvested from the subcutaneous tissue of the mice and dissociated into a single-cell suspension for scRNA-seq analysis.

extract_protocol:
Each sample was placed in a sterile Petri dish on ice. Tumor tissues were transferred into a separate Petri dish and cut into smaller pieces measuring 2–4 mm. The samples were then washed with phosphate-buffered saline (PBS) and further minced into pieces approximately 1 mm³ on ice. Enzymatic digestion was performed using collagenase II (Worthington) at 37°C for 60 minutes with continuous agitation.The resulting cell suspension was passed through a pre-wet 70 µm MACS SmartStrainer filter, and the filtrate was collected into a 50 ml centrifuge tube. The filter was rinsed with 10 ml of RPMI 1640 or DMEM to ensure maximum recovery, and the rinsate was added to the centrifuge tube containing the filtrate. The samples were centrifuged at 300 rcf for 7 minutes at room temperature. After centrifugation, the supernatant was carefully discarded, and erythrocyte lysis was performed immediately.Cell viability was assessed using Trypan blue staining. Finally, an appropriate volume of pre-cooled wash buffer was added, and the cell suspension was gently mixed until the final cell concentration reached 700–1200 cells/µl.
Fragmentation, end repair, and A-tailing were performed, followed by purification of the product using SPRIselect beads. Adaptor ligation was then carried out, with subsequent purification using SPRIselect beads. Index PCR was performed, followed by another round of SPRIselect purification. The library concentration was measured using the Qubit庐 3.0 Fluorometer (Life Technologies, CA, USA). The fragment size distribution of the library products was assessed using the Agilent 2100 Bioanalyzer with the High Sensitivity DNA Assay Kit (Agilent Technologies, CA, USA).

value: Subcutaneous tumor


title: Female KO LV, leptin, bio rep 2

description:
LV_168
Read_Counts.txt
TPM_values.txt


title: Female WT CV, PBS, bio rep 4

description:
CV_688
Read_Counts.txt
TPM_values.txt

source_name: Caudal vertebrae


title: SMARTseq_pIC_iMSCL_C7

value: Central marrow stroma


title: sgCdk10_1, replicate 1, scRNAseq

description:
Library name: sgCdk10_1
DNBSEQ-T7


title: SMARTseq_pIC_iMSCL_C1


title: Female WT CV, leptin, bio rep 5

description:
CV_687
Read_Counts.txt
TPM_values.txt


title: hzl-1 aak(0) PD S1

city: Montreal

first: Sabih

last: Rashid

state: Quebec

address: 3611 Rue Sainte Famille, Unit 11

department: Biology

country: Canada

institute: McGill University

description:
Library name: Sample 7
aak_hzl-1_PD_1

data_processing:
Reference genome assembly and DESeq2. Sequences were trimmed for sequencing adapters and low quality 3' bases using Trimmomatic version 0.35 and aligned to the reference C. elegans genome version WBcel235 (gene annotation from Ensembl version 104) using STAR version 2.7.1a. Gene expressions were obtained both as readcount directly from STAR as well as computed using RSEM in order to obtain gene and transcript level expression, either in TPM or FPKM values, for these stranded RNA libraries. DESeq2 version 1.30.1 was then used to normalize gene readcounts. Sample clustering based on normalized log readcounts produces the following hierarchy of samples
Assembly: WBcel235 (gene annotation from Ensembl version 104)
Supplementary files format and content: "daf2 vs aak vs hzl1 FKPM.tsv" contains read counts, pairwise comparisons in 2 separate files for fold change calculations.

organism: Caenorhabditis elegans

source_name: Whole animal

growth_protocol: Bleach-synchronized worms were put onto large NGM plates and grown at 25 °C. Dauer animals were collected after 96 hours (i.e. Day 2 in dauer) and washed with M9, post-dauer animals collected after 192 hours. Animals were immediately subjected to Trizol RNA extraction after collection.

extract_protocol:
Standard Trizol RNA extraction was carried out, approximately 200 ul per 20 ul packed worms. Freeze-thawing with liquid nitrogen was carried out to separate RNA, followed by heavy centrifugation. Two rounds of chloform extraction were carried out (50 ul per 200 ul Trizol). Samples were washed with isopropanol followed by 70% EtoH, and air-dryed before dissolving in ultrapure water. Sample concentration was measured using Nanodrop, and run on agarose gels to verify integrity of RNA. RNA was stored at -80 °C for up to 3 days before being sent for sequencing. Minimum of 100 ng/ul RNA was sent for sequencing.
mRNA-Seq with PolyA selection, 150 cycles Paired-End (maximum 2x80nt), 400M fragments, using KAPA mRNA Hyper Prep Hyperprep library per manufacturer instructions

value: Whole animal

value: Hermaphrodite

value: daf-2; aak(0); hzl-1

value: Trizol

tag: time

value: 192 hrs


title: SMARTseq_pIC_iMSCL_B7

value: Inflammatory MSCL

value: Pooled male and female

value: 8-12 weeks

value: Poly(I:C) 10 mg/kg ip q48 hours for 3 days


title: sgControl_2, replicate 2, scRNAseq

description:
Library name: sgControl_2
DNBSEQ-T7


title: sgControl_1, replicate 1, scRNAseq

description:
Library name: sgControl_1
DNBSEQ-T7


title: SMARTseq_pIC_iMSCL_B6


title: hzl-1 aak(0) Dauer S1

description:
Library name: Sample 5
aak_hzl-1_Dauer_1

value: 96 hrs


title: Female WT CV, leptin, bio rep 3

description:
CV_680
Read_Counts.txt
TPM_values.txt

value: Caudal vertebrae

value: ICV leptin


title: SMARTseq_pIC_iMSCL_B5


title: hzl-1 daf-2 PD S2

description:
Library name: Sample 4
daf-2_hzl-1_PD_2

value: daf-2; hzl-1


title: Female KO CV, PBS, bio rep 3

description:
CV_5484
Read_Counts.txt
TPM_values.txt

value: ICV PBS


title: SMARTseq_pIC_iMSCL_B4


title: Female WT CV, PBS, bio rep 5

description:
CV_689
Read_Counts.txt
TPM_values.txt


title: Control_Library_397223

data_processing:
Gene expression matrix was computed using CellRanger v7.0.0 with the option '--include-introns=true'
Assembly: GRCm39 with the annotation Annotation Release 106 from ENSEMBL, downloaded 2022-07-05
Supplementary files format and content: Filtered count matrix from cellranger count is stored in three files: barcodes.tsv.gz, genes.tsv,gz and matrix.mtx.gz

molecule: nuclear RNA

extract_protocol:
Nuclei extraction from total SG tissue according to study design.
Library construction was performed according to the 10x Chromium Single Cell 3' Reagent Kit v2.

value: Salivary Gland

value: 20 days

value: Unselected


title: Female KO CV, leptin, bio rep 4

description:
CV_5482
Read_Counts.txt
TPM_values.txt


title: H-CDs3

city: jinan

first: zhongyao

last: li

address: qingdao road 6699

institute: shandong first medical university

data_processing:
The sequencing data was filtered with SOAPnuke (v1.5.2)   by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 5) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterwards clean reads were obtained and stored in FASTQ format.
Assembly: GRCm38/mm10
Supplementary files format and content: Text Document  include Raw read counts for each Sample

source_name: Ascites

extract_protocol:
Total RNA was extracted using the TRIzol reagent. RNA quality and quantity were determined using a Nano Drop and Bioanalyzer 2100 system
mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

value: Ascites

value: macrophage

value: WT

value: treated with LPS (1 ug/ml) and H-CDs (400 ug/ml)


title: SMARTseq_pIC_iMSCL_C2

value: Central marrow stroma


title: hzl-1 daf-2 Dauer S2

description:
Library name: Sample 2
daf-2_hzl-1_Dauer_2

value: Whole animal

value: Hermaphrodite

value: Trizol


title: SMARTseq_pIC_iMSCL_B3

value: Inflammatory MSCL

value: Pooled male and female

value: 8-12 weeks

value: Poly(I:C) 10 mg/kg ip q48 hours for 3 days


title: hzl-1 aak(0) PD S2

description:
Library name: Sample 8
aak_hzl-1_PD_2

value: daf-2; aak(0); hzl-1

value: 192 hrs


title: Female WT CV, PBS, bio rep 3

description:
CV_190
Read_Counts.txt
TPM_values.txt

value: Caudal vertebrae

value: ICV PBS


title: H-CDs2


title: Female WT CV, leptin, bio rep 4

description:
CV_681
Read_Counts.txt
TPM_values.txt

value: ICV leptin


title: hzl-1 daf-2 Dauer S1

description:
Library name: Sample 1
daf-2_hzl-1_Dauer_1

value: daf-2; hzl-1

value: 96 hrs


title: hzl-1 aak(0) Dauer S2

description:
Library name: Sample 6
aak_hzl-1_Dauer_2


title: SMARTseq_pIC_iMSCL_B1


title: Female WT CV, PBS, bio rep 1

description:
CV_188
Read_Counts.txt
TPM_values.txt


title: Female WT CV, leptin, bio rep 2

description:
CV_187
Read_Counts.txt
TPM_values.txt


title: hzl-1 daf-2 PD S1

description:
Library name: Sample 3
daf-2_hzl-1_PD_1


title: SMARTseq_pIC_iMSCL_A6


title: 43-SS-CO

city: Boston

first: Ionita

middle: Calin

last: Ghiran

state: MA

address: 3 Blackfan Circle

department: Medicine

institute: Beth Israel Deaconess Medical Center

data_processing:
All FASTQ files were processed uniformly through the extra-cellular RNA processing toolkit (exceRpt). Information about exceRpt is available at https://github.gersteinlab.org/exceRpt/.
Assembly: hg19
Supplementary files format and content: Each processed data file is a core results archive generated by exceRpt. The core results archive contains read counts for alignments to the different transcriptome libraries used by exceRpt (miRNA / piRNA / tRNA / GENCODE annotations / circRNA), taxonomy trees for exogenous rRNA and genomic reads aligned by exceRpt, and a variety of other files generated by exceRpt.

source_name: Plasma

extract_protocol:
RNA Isolation method was RNA was isolated from serum using QiaZol/Chloroform and cleaned using Qiagen miRNeasy Mini Kit. RNA Isolation kit was miRNeasy (Qiagen).
Library construction kit was NEBNext small RNA library prep (NEB). Samples were not multiplexed. Type of Starting Material was small RNA.

tag: condition

value: Sickle Cell Disease

tag: anatomical location

value: Entire cardiovascular system

tag: rna source

value: total cell-free biofluid RNA


title: LPS3

value: Ascites

value: macrophage

value: WT

value: treated with LPS (1 ug/ml)


title: Female WT CV, leptin, bio rep 1

description:
CV_186
Read_Counts.txt
TPM_values.txt

data_processing:
The DNBSEQ platform was used to perform paired-end 100 bp sequence reads, generating an average of 4.84 to 6.87Gb per sample.
Sequencing data was filtered using SOAPnuke, a filtering algorithm that removes reads containing the adaptor, reads with N content >5%, and reads with base quality score <15 as the proportion of total bases in the reads that are greater than 20% as low-quality reads.
After filtering, HISAT and Bowtie2 were used to align clean reads to the Mus musculus reference genome version GCF_000001635.26_GRCm38.p6 with an average mapping ratio of 98% and 81-86%, respectively --> read counts.
Read counts were normalized per sample to generate TPM values
Assembly: GRCm39
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample
Supplementary files format and content: tab-delimited text file includes TPM values for each Sample


title: 9-SS-Co


title: LPS1


title: Female KO CV, leptin, bio rep 3

description:
CV_169
Read_Counts.txt
TPM_values.txt


title: 8-SS-Co


title: SMARTseq_pIC_iMSCL_A5

value: Central marrow stroma

value: Inflammatory MSCL

value: Pooled male and female


title: SMARTseq_pIC_iMSCL_A4

value: 8-12 weeks

value: Poly(I:C) 10 mg/kg ip q48 hours for 3 days


title: 61-SS


title: Female KO CV, leptin, bio rep 2

description:
CV_168
Read_Counts.txt
TPM_values.txt

value: Caudal vertebrae


title: SMARTseq_pIC_iMSCL_A3


title: LPS2


title: control3

value: untreated


title: control2


title: KO_Library_397224

source_name: Salivary Gland

value: Salivary Gland

value: 20 days

value: Unselected


title: SMARTseq_pIC_iMSCL_B2


title: H-CDs1

value: treated with LPS (1 ug/ml) and H-CDs (400 ug/ml)


title: 45-SS-CO

value: Sickle Cell Disease

value: Entire cardiovascular system


title: Female WT CV, PBS, bio rep 2

description:
CV_189
Read_Counts.txt
TPM_values.txt

value: ICV PBS


title: control1

value: Ascites

value: macrophage


title: 33-SS

value: total cell-free biofluid RNA


title: Female KO CV, leptin, bio rep 1

description:
CV_167
Read_Counts.txt
TPM_values.txt

value: ICV leptin


title: SMARTseq_pIC_iMSCL_A2

value: Central marrow stroma

value: Inflammatory MSCL


title: 11-SS


title: SMARTseq_pIC_iMSCL_A7

value: Pooled male and female

value: 8-12 weeks


title: SMARTseq_pIC_iMSCL_A1

value: Poly(I:C) 10 mg/kg ip q48 hours for 3 days


title: 87-SS


title: Female KO CV, PBS, bio rep 1

description:
CV_161
Read_Counts.txt
TPM_values.txt

value: Caudal vertebrae


title: SMARTseq_Old_iMSCL_H12

value: male

value: 24 months


title: Female WT iWAT, PBS, bio rep 5

description:
iWAT_689
Read_Counts.txt
TPM_values.txt

source_name: Inguinal white adipose tissue

value: Inguinal white adipose tissue


title: 15-AA


title: HSCPHash4_scRNAseq

description: 10X Genomics

source_name: Bone marrow stem and progenitor cells

value: Bone marrow stem and progenitor cells

value: Sorted HSPC populations

value: female


title: HSCPHash2_scRNAseq

description: 10X Genomics


title: 38-AA

value: Sickle Cell Disease

value: Entire cardiovascular system


title: Female WT iWAT, PBS, bio rep 4

description:
iWAT_688
Read_Counts.txt
TPM_values.txt

value: ICV PBS


title: SMARTseq_Old_iMSCL_H11


title: HSCPHash1_scRNAseq

description: 10X Genomics


title: SMARTseq_Old_iMSCL_H9


title: 37-AA

value: total cell-free biofluid RNA


title: HSCPHash3_scRNAseq

description: 10X Genomics


title: SMARTseq_Old_iMSCL_H8


title: Female WT iWAT, leptin, bio rep 4

description:
iWAT_681
Read_Counts.txt
TPM_values.txt

value: ICV leptin


title: 1-AA


title: Female KO CV, PBS, bio rep 2

description:
CV_166
Read_Counts.txt
TPM_values.txt


title: SMARTseq_Old_iMSCL_G11

value: Central marrow stroma


title: StromaHash1_scRNAseq

description: 10X Genomics

source_name: Central marrow and endosteal stroma, sorted populations

value: CD45-, Ter119- cells


title: pIC_WBM_scRNAseq

description: 10X Genomics

source_name: Whole bone marrow

value: Whole bone marrow

value: Live bone marrow cells

value: Pooled male and female


title: 98-AA-Co

tag: rna isolation kit

value: miRNeasy (Qiagen)


title: SMARTseq_Old_iMSCL_G10

value: Inflammatory MSCL

value: male


title: Female WT iWAT, leptin, bio rep 3

description:
iWAT_680
Read_Counts.txt
TPM_values.txt

value: Inguinal white adipose tissue


title: 27-AA-CO


title: pIC_Endo2_scRNAseq

description: 10X Genomics

source_name: Endosteal stroma

value: Endosteal stroma

value: 8-12 weeks

value: Poly(I:C) 10 mg/kg ip q48 hours for 3 days


title: SMARTseq_Old_iMSCL_F11

value: 24 months


title: Female WT iWAT, PBS, bio rep 2

description:
iWAT_189
Read_Counts.txt
TPM_values.txt


title: 30-AA-CO

value: Sickle Cell Disease

value: Entire cardiovascular system


title: SMARTseq_Old_iMSCL_F10


title: pIC_Endo1_scRNAseq

description: 10X Genomics


title: 97-AA-Co

value: total cell-free biofluid RNA


title: Female WT iWAT, PBS, bio rep 1

description:
iWAT_188
Read_Counts.txt
TPM_values.txt

value: ICV PBS


title: Female WT iWAT, leptin, bio rep 5

description:
iWAT_687
Read_Counts.txt
TPM_values.txt

value: ICV leptin


title: SMARTseq_Old_iMSCL_F9

value: Central marrow stroma


title: TP53 knock-down sample,  oscillatory hypoxia condition, replicate 3

city: Farmington

first: Xihua

last: Qiu

state: CT

address: 263 Farmington Ave

department: Biomedical Engineering

institute: University of Connecticut

description: Library name: YL19_3

data_processing:
Alignment with GRCh38
Gene counting with featureCounts
Transcripts per Million calculations
Assembly: GRCh38
Supplementary files format and content: TPM values in Excel

source_name: Breast

extract_protocol:
mRNA Extraction
Standard Illumina mRNA library prep

value: Breast

value: Epithelial

value: TP53 knock-down

value: oscillatory hypoxia


title: StromaHash2_scRNAseq

description: 10X Genomics

value: Central marrow and endosteal stroma, sorted populations

value: CD45-, Ter119- cells


title: SMARTseq_Old_iMSCL_F8

source_name: Central marrow stroma

value: Inflammatory MSCL

value: male


title: Female WT iWAT, leptin, bio rep 2

description:
iWAT_187
Read_Counts.txt
TPM_values.txt

extract_protocol:
After endpoint dissection, tissues were homogenized and preserved in Trizol at -80 °C before RNA extraction. To purify RNA, samples were processed with PureLink™ RNA Mini Kit (Invitrogen, 12183025) according to the manufacturer’s instructions, and the RNA concentration and quality in the samples were checked with a spectrophotometer (Thermo Scientific, NanoDrop 2000).
After being enriched by oligo dT and fragmented, a cDNA library was generated by reverse-transcribing mRNA using random N6-primed RT.

value: Inguinal white adipose tissue


title: pIC_CM1_scRNAseq

description: 10X Genomics

value: Pooled male and female

value: 8-12 weeks

value: Poly(I:C) 10 mg/kg ip q48 hours for 3 days


title: TP53 knock-down sample,  oscillatory hypoxia condition, replicate 2

description: Library name: YL19_2


title: Set3_Endo_scRNAseq

description: 10X Genomics

value: Endosteal stroma


title: SMARTseq_Old_iMSCL_E12

value: 24 months


title: TP53 knock-down sample,  oscillatory hypoxia condition, replicate 1

description: Library name: YL19_1


title: TP53 knock-down sample,  lactate condition, replicate 3

description: Library name: YL18_3

value: lactate


title: Set3_CM_scRNAseq

description: 10X Genomics


title: SK-OV-3_mCherry-EGFP

city: Tianjin

first: Jianying

last: Lv

state: Tianjin

address: weijin Road No.94

department: School of medicine

institute: Nankai University

description:
Library name: SK-OV-3_control
V300080615_L*_HOMkidRAAABAAA-548

data_processing:
Sequencing of the samples is performed on the Illumina platform. Since the raw sequencing data may contain low-quality sequences, adapter sequences, and other contaminants, it is necessary to filter these impurities to obtain high-quality CleanData, ensuring the reliability of the data analysis results
control-VS-sample: Based on the sgRNA count levels of each sample, we can detect the differences in sgRNA (DEG) between control and sample. In this project, the test method in the Mageck software is used for differential analysis.
Assembly: hg38
Supplementary files format and content: control-VS-sample.*.txt
Library strategy: CRISPR Screen

extract_protocol:
The sorted cells (mCherry+ EGFP-) and the parental cells (unsorted cells) were subjected to genomic DNA extraction, PCR-based amplification of sgRNA-encoding sequences and the high-throughput DNA sequencing
Agarose gel electrophoresis is used to analyze the degree of DNA degradation and check for RNA or protein contamination. The DNA concentration is then precisely quantified using Qubit. DNA samples with a concentration above 0.5 µg are used for library construction. After end repair and adding an A-tail to the DNA template, adapters are ligated to both ends of the fragments without PCR amplification in the middle, preparing a PCR-free library

value: human ovarian cancer cell line

tag: sample type

value: control


title: Female WT iWAT, PBS, bio rep 3

description:
iWAT_190
Read_Counts.txt
TPM_values.txt

tag: tissue

value: ICV PBS


title: SMARTseq_Old_iMSCL_E11

value: Central marrow stroma

tag: genotype


title: SK-OV-3_mCherry

description:
Library name: SK-OV-3_sample
V300080615_L*_HOMkidRAAACAAA-565

value: sample


title: Set2_Endo_scRNAseq

description: 10X Genomics

tag: treatment


title: TP53 knock-down sample,  lactate condition, replicate 2

description: Library name: YL18_2

value: Breast

tag: cell type

value: Epithelial

value: TP53 knock-down


title: SMARTseq_Old_iMSCL_E10

value: Inflammatory MSCL


title: SK-OV-3_shRRN3 (INPUT)

description: Library name: SK-OV-3_shRRN3, INPUT

data_processing:
After sequencing data was delivered, we did data filtering first, which could remove low quality reads, N reads and adaptor sequences, and get clean data. Then, we mapped clean data to reference genome when we made sure the data quantity of clean data was qualified. Also, we needed to make a quality test about the alignment. Then we used the alignment result to calling peak. Later, we used the peak information for standard bioinformatics analysis and personalized bioinformatics analysis.
Assembly: hg38
Supplementary files format and content: wig  bed

extract_protocol:
Using the ChIP-IT Express Kit according to the manufacturer's instruction (Active motif)
(1) DNA-end repair, 3'-dA overhang and ligation of methylated sequencing adaptor.(2) PCR amplification and size selection (usually 100-300bp, including adaptor sequence).(3) Qualified library for sequencing.


title: SMARTseq_Old_iMSCL_E9

value: male


title: TP53 knock-down sample,  lactate condition, replicate 1

description: Library name: YL18_1


title: SK-OV-3_shRRN3, Pol II(ChIP)

description: Library name: SK-OV-3_shRRN3, ChIP

tag: chip antibody

value: RNA polymerase II (Active motif #91151)


title: pIC_CM2_scRNAseq

description: 10X Genomics

value: CD45-, Ter119- cells

value: Pooled male and female


title: Set1_Endo_scRNAseq

description: 10X Genomics

value: 8-12 weeks


title: Female WT iWAT, leptin, bio rep 1

description:
iWAT_186
Read_Counts.txt
TPM_values.txt

value: Inguinal white adipose tissue

value: ICV leptin


title: TP53 knock-down sample,  hypoxia condition, replicate 3

description: Library name: YL17_3

value: hypoxia


title: SMARTseq_Old_iMSCL_D12

value: 24 months


title: Set1_CM_scRNAseq

description: 10X Genomics


title: SMARTseq_Old_iMSCL_D11


title: TP53 knock-down sample,  hypoxia condition, replicate 2

description: Library name: YL17_2


title: Otof_c.1315_CtoT_RESTARTv3_Targetseq-2

description: detect modification level of PTC sites in Otof c. 1315 C > T (p.R439X) mice with RESTART v3 treatment

organism: Mus

source_name: cochlear

value: cochlear


title: SMARTseq_Old_iMSCL_D10


title: TP53 knock-down sample,  hypoxia condition, replicate 1

description: Library name: YL17_1


title: SMARTseq_Old_iMSCL_D9


title: TP53 knock-down sample,  normoxia condition, replicate 3

description: Library name: YL16_3

value: normoxia


title: SK-OV-3_shLacZ, Pol II( ChIP)

description: Library name: SK-OV-3_shLacZ, ChIP

value: human ovarian cancer cell line

value: shLacZ


title: Heart, MR2694

city: Guangzhou

first: Xiang

last: Li

state: Guangdong

address: 11 fengxin road, guangzhou science city

institute: Guangdong Laboratory Animals Monitoring Institute

data_processing:
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts.In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5.
Assembly: mm10
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample

source_name: Heart

extract_protocol:
Snap frozen heart tissues were ground to powder under liquid nitrogen. Pulverized samples were used for total RNA extraction using TRIzol reagent (Magen, China), and RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, USA).
Sequencing libraries were generated using a ABclonal mRNA-seq Lib Prep Kit for ABclonal (ABclonal, China) following manufacturer's recommendations and index codes were added to attribute sequences to each sample.

value: Heart

tag: strain

value: MR


title: Heart, MR2691


title: Set2_CM_scRNAseq

description: 10X Genomics

value: Central marrow stroma


title: SMARTseq_Old_iMSCL_C12

value: Inflammatory MSCL


title: TP53 knock-down sample,  normoxia condition, replicate 2

description: Library name: YL16_2

value: Breast

value: Epithelial

value: TP53 knock-down


title: SMARTseq_Old_iMSCL_D8

value: male


title: Otof_c.1315_CtoT_Targetseq-2

description: detect modification level of PTC sites in Otof c. 1315 C > T (p.R439X) mice


title: Heart, MR2682


title: Otof_c.1315_CtoT_RESTARTv3_Targetseq-1


title: Otof_c.1315_CtoT_Targetseq-1


title: SK-OV-3_shLacZ (INPUT)

description: Library name: SK-OV-3_shLacZ, INPUT


title: NOTCH1 knock-down sample,  oscillatory hypoxia condition, replicate 3

description: Library name: YL15_3

value: NOTCH1 knock-down

value: oscillatory hypoxia


title: TP53 knock-down sample,  normoxia condition, replicate 1

description: Library name: YL16_1


title: RNA-seq-RESTARTv3_2

description: detect global RNA expression level in cochlear tissue of Otof c. 1315 C > T (p.R439X) mice with RESRTART treatment

data_processing:
Only read 2 was used for subsequent analysis. Raw sequencing reads were subjected to cutadapt software (version 4.2) for quality control and adapter trimming. The key parameters were: -e 0.1 -O 3 --quality-cutoff 25 -m 55.
Seqkit were used to remove the duplicated reads. The 8bp UMI in the deduplication reads were revomed by umi_tools (version 1.0.0). During the library construction, six bases were respectively added to the 5 'end and the 3' end thus umi_tools3 was used to remove them. The key parameters were: umi_tools extract --bc-pattern=NNNNNNNNNNNNNN --3prime --bc-pattern=NNNNNN.
Then the cleaned reads were mapped to the mouse genome (GRCm39, NCBI Refseq) using STAR (version 2.7.10b) with default parameters. Mapped reads were filtered by samtools (version 1.14). The key parameters were: samtools view -f 3 -F 256.
The gene counts were quantified by HTSeq (version 1.14),  and genes with low biological repeatability (log2 fold change(replication 1/replication 2)>1) were discarded. The gene expression level was quantified by RPKM.
Assembly: GRCm39
Supplementary files format and content: The processed data file format is xls. Its content is the genome-wide expression levels of the mouse cochlear tissues.

extract_protocol:
total RNA was extracted from cells using TRIzol reagent (Life Technologies)
RNA libraries were prepared using SMARTer Stranded Total RNA-Seq kits (v3) - Pico Input mammalian (Takara) Library Prep Kit following manufacturer's protocols.

value: with RESRTART treatment


title: Heart, C2766

value: Sham


title: RNA-seq-RESTARTv3_1


title: SMARTseq_Old_iMSCL_C10

value: 24 months


title: Heart, C2765


title: SMARTseq_Old_iMSCL_C9


title: NOTCH1 knock-down sample,  oscillatory hypoxia condition, replicate 1

description: Library name: YL15_1


title: Heart, MR2696


title: RNA-seq-Otof_c.1315_CtoT_2

description: detect global RNA expression level in cochlear tissue of Otof c. 1315 C > T (p.R439X) mice without RESRTART treatment

value: cochlear

value: without RESRTART treatment


title: SMARTseq_Old_iMSCL_B12


title: NOTCH1 knock-down sample,  lactate condition, replicate 3

description: Library name: YL14_3

value: lactate


title: Heart, C2764


title: SMARTseq_Old_iMSCL_B8


title: BMDC with whole body Aldh1a2 KO

city: Plainsboro

first: Yong

last: Wei

state: NJ

address: LTL227 Wathington Road

department: Molecular Biology

institute: Princeton University

description:
BMDC_KOvsWT_FPKM+1.txt
2203__508

data_processing:
The library pools were denatured and sequenced on Illumina NovaSeq 6000 using the S Prime flowcells as pair-end 65 nt reads according to the manufacturer’s protocol.  Raw sequencing reads were filtered by Illumina NovaSeq Control Software, only the Pass-Filter (PF) reads were de-multiplexed allowing 1 mismatch and used for further analysis.
Demultiplexed raw sequence file (FASTQ) were analyzed using Partek® Flow® software package. Raw reads were aligned using STAR 2.7.8a, followed by quantification to annotation model GeneCode release M27 (GRCm39). After noise filtering, counts were normalized as FPKM+1.
Assembly: GeneCode release M27 (GRCm39)
Supplementary files format and content: tab-delimited text files with FPKM+1 value

source_name: Bone marrow

growth_protocol: Bone marrow derived dendritic cells (BMDCs) were generated by culturing mouse bone marrow cells isolated from both femur and tibias in RPMI1640+10% fresh thaw FBS. The culture is carried in the presence of 10ng/ml GM-CSF and 10ng/ml IL-4 for 7 days, with supplementation of 1 volume of fresh media at day3.

extract_protocol:
Total RNA were extracted using RNAeasy mini-prep kit from Qiagen
The integrity of total RNA samples were assessed on Bioanalyzer 2100 using RNA 6000 Pico chip (Agilent Technologies, CA).  Messenger RNA was enriched from these samples using QIAseq FastSelect - rRNA HMR Kit (Qiagen, CA), fragmented and converted to cDNA and Illumina sequencing library using the PrepX RNA-seq library preparation protocol on the Apollo 324TM NGS Library Prep System (Takara Bio, CA).

treatment_protocol: The regular FBS was changed to charcoal-stripped FBS to induce high maturation of BMDC; 10nM retino acid (RA) supplement (with 0.1%DMSO as vehicle control) was used to induce low maturation of BMDC.

value: Bone marrow

value: Bone marrow derived dendritic cells

value: Aldh1a2ko/ko by UBC-CreERT2 with tamoxifen tx


title: RNA-seq-Otof_c.1315_CtoT_1


title: NOTCH1 knock-down sample,  lactate condition, replicate 2

description: Library name: YL14_2


title: PRAISE-RESTARTv3_2

description: transcriptome-wide quantitative Ψ detection in  cochlear tissue of Otof c. 1315 C > T (p.R439X) mice with RESRTART treatment

data_processing:
This data needed to combine with the data of RNA-seq for analysis.Briefly, the samples with PRAISE treatment were defined as PRAISE(+) groups, and the RNA-seq samples are defined as PRAISE(-) groups.
Only read 2 was used for subsequent analysis. Cutadapt (version 4.2) first subjected raw to adaptor trimming and filtering of low-quality reads. Duplicated reads were removed by Seqkit (version 0.13.2). The key parameters were: seqkit rmdup -s.
umi_tools (version 1.0.0) was used to remove the 8bp UMI in the deduplication reads. Six bases after the UMI added during library construction at the 5’ end of inserted sequences and the six bases at the 3’ end of inserted sequences were also removed, using the key parameters: umi_tools extract --extract-method=string --bc-pattern=NNNNNNNNNNNNNN and umi_tools extract --extract-method=string --3prime --bc-pattern=NNNNNN.
Then the cleaned reads were downsampled to 117Mb and mapped to the reference transcriptome(GRCm39, NCBI Refseq) using PRAISE-tool(https://github.com/Zhe-jiang/PRAISE), and the pseudouridine signals as deletion rate in the transcriptome could be calculated.
We identified off-target Ψ sites using the following approach. (1) The deletion rate difference in RESTART v3(-) and RESTART v3(+) groups must be > 5%.(2) The deletion ratio in the PRAISE(-) samples must be < 5%. (3) For off-target Ψ sites, we applied the statistical test to each of them (contingency table test between PRAISE(+) sample and PRAISE (-) sample), and the P value between the two groups must be <0.005. (4) Compared with the RESTART v3(-) samples, the increasing level of deletion ratio in the RESTART v3(+) group must be > 10%.
Assembly: GRCm39
Supplementary files format and content: The processed data file format is xls. Its content is the information on off-target sites of RESTART v3 in mouse cochlear tissues.

value: mouse cochlear tissue cells


title: SMARTseq_Old_iMSCL_A12

data_processing:
10X Genomics: The demultiplexing, barcode processing, gene counting, and aggregation were performed using CellRanger software (version 7.0.1).
SMARTseq: Fastq quality was assessed with FastQC and MultiQC. After trimming adapters with TrimGalore sequences were pseudoaligned to the murine mm10 genome and counted using Salmon.
Assembly: mm10
Supplementary files format and content: 10X Genomics: Tab-separated values files and matrix files; oligo-hashtag IDs as comma separated file
Supplementary files format and content: SMARTseq: Counts table and cell identifiers for SMARTseq as comma separated files

value: Central marrow stroma

value: Inflammatory MSCL


title: BMDC with wild type Aldh1a2 (floxed) #510

description:
BMDC_KOvsWT_FPKM+1.txt
2203__510

value: Aldh1a2 wt (fl/fl), no Cre


title: Heart, C2767

value: Heart


title: SMARTseq_Old_iMSCL_C11

value: male


title: NOTCH1 knock-down sample,  lactate condition, replicate 1

description: Library name: YL14_1

value: Breast

value: Epithelial

value: NOTCH1 knock-down


title: PRAISE-RESTARTv3_1


title: BMDC with DC specific Aldh1a2 KO

description:
BMDC_KOvsWT_FPKM+1.txt
2203__432

value: Aldh1a2ko/ko by Cd11c-Cre


title: SMARTseq_Old_iMSCL_A11

value: 24 months


title: NOTCH1 knock-down sample,  hypoxia condition, replicate 3

description: Library name: YL13_3

value: hypoxia


title: PRAISE-Otof_c.1315_CtoT_2

description: transcriptome-wide quantitative Ψ detection in  cochlear tissue of Otof c. 1315 C > T (p.R439X) mice without RESRTART treatment

value: cochlear


title: NOTCH1 knock-down sample,  oscillatory hypoxia condition, replicate 2

description: Library name: YL15_2

value: oscillatory hypoxia


title: SMARTseq_Old_iMSCL_A10


title: PRAISE-Otof_c.1315_CtoT_1


title: NOTCH1 knock-down sample,  hypoxia condition, replicate 2

description: Library name: YL13_2


title: High maturation subpopulation of BMDC, CD11c+MHCIIhiCD86hi, bio rep 3

description:
BMDC_CSvsCS+RA_FPKM+1.txt
2044__CS-3

value: Bone marrow

value: wild type

tag: treatment 1

value: charcoal-stripped (CS) FBS

tag: treatment 2


title: NOTCH1 knock-down sample,  hypoxia condition, replicate 1

description: Library name: YL13_1


title: SKBR3 human breast cancer cell line, EPZ6438, rep2 (Input)

first: William

last: Muller

address: 1160 Av des Pins O

department: Biochemistry

description: Library name: P-E_rep2_Input

data_processing:
ChIP-seq reads were first trimmed for adapter sequences and low quality score bases using Trimmomatic (Bolger et al., 2014). The resulting reads were mapped to their respective reference genomes (mouse mm10 or human GRCh38) using BWA-MEM (Li and Durbin, 2009) in paired-end mode at default parameters. Only reads that had a unique alignment (mapping quality > 20) were retained and PCR duplicates were marked using Picard tools (http://broadinstitute.github.io/picard/). Reads mapping to ENCODE blacklist regions (Amemiya et al., 2019) were further removed.
For spike-in normalization, reads were aligned separately to the D. melanogaster BDGP6 genome. For each sample, count the number of reads that align to the spike-in DNA and identify the sample with the lowest number of spike-in reads. The scale factor for each sample is calculated by dividing the minimum spike-in read count by the number of spike-in reads in that sample.
Assembly: mouse mm10 or human GRCh38
Supplementary files format and content: bigWig

source_name: human cell line

growth_protocol: DMEM (Wisent) for SK-BR-3 supplemented with 10% FBS, 1% penicillin/streptomycin (Wisent), 1% Amphotericin B (Wisent) and 0.1% Gentamycin (Wisent) and incubated at 37°C in 5% CO2.

extract_protocol:
Tissue samples were first minced and resuspended in ice-cold PBS with protease inhibitor cocktail before further being disaggregated with a dounce homogenizer, then filtered through a 70mm filter to collect a single cell suspension. Collected cell pellets were resuspended in PBS containing 1% of formaldehyde for 10 min and the cross-linking reaction was quenched with glycine.
Libraries were generated with NEBNext Ultra II DNA Library Prep kits (NEB, E7103L)

treatment_protocol: cells were treated with DMSO and 2μM EPZ6438 for 7 days, medium and drug were replenished every 2-3 days

value: human cell line

value: Human breast cancer

value: EPZ6438 treatment for 7 days


title: High maturation subpopulation of BMDC, CD11c+MHCIIhiCD86hi, bio rep 2

description:
BMDC_CSvsCS+RA_FPKM+1.txt
2044__CS-2

value: Bone marrow derived dendritic cells


title: SMARTseq_Old_iMSCL_A9

extract_protocol:
Combined stromal and hematopoietic cells isolation  Mice were euthanized in a rising concentration of carbon dioxide, followed by cervical dislocation. For each mouse, both femurs, tibiae, hemipelves, and humeri were dissected and thoroughly cleaned using KimWipes (Kimtech) and were used for combined isolation of stromal and hematopoietic cells. To isolate central marrow (CM) stromal cells, the proximal and distal epiphyses were removed from 1 femur. Intact marrow plugs were flushed with Hank’s balanced saline solution (HBSS) without calcium or magnesium into 5 ml polypropylene tubes by inserting a 3 ml syringe with 22G needle into the distal end of the femur. The plugs were digested with 1 ml of a solution of 3 mg/ml type I collagenase (Worthington) dissolved in HBSS for 10 min at 37 C with 110 rpm shaking. The tube was then vortexed briefly and the supernatant was removed into a new tube through a 100 m mesh, taking care not to disturb the marrow plug. A further 1 ml of 3 mg/ml type I collagenase solution was then added to the marrow plug and the digestion repeated. After the second incubation, the plug was dissociated by pipetting up and down with a P1000 pipette before passing the cell suspension through a 100 m mesh into the tube containing the previous digestion supernatant. To isolate endosteal (Endo) stromal cells and hematopoietic cells, the flushed femur was combined with the other bones, gently crushed up to 10 times using a mortar and pestle, and thoroughly washed with 10 ml HBSS until all the non-adherent BM cells were removed and collected in a separate 15 ml polypropylene tube. The bone chips were then digested in a 15 ml polypropylene tube with 3 ml of 3 mg/ml type I collagenase solution for 1 hour at 37 C with 110 rpm shaking. After digestion, the tube was vortexed briefly and the cell suspension was filtered through a 100 m mesh into a new tube. The bone chips were then washed with HBSS, and the washing was collected into the same tube as the digestate. For cell suspensions acquired for both stromal compartments, red blood cells were removed by adding 1 ml ACK lysis buffer (150 mM NH4Cl and 10 mM KHCO3) and incubated on ice for 3 minutes before washing with HBSS containing 4% fetal bovine serum (HI FBS, Gibco). During the optimization of the protocol outlined above, the digestion with 3 mg/mL type I collagenase was performed in parallel to and compared with 3 mg/mL type I collagenase + 4 mg/mL type II Dispase (Roche), 250 μg/mL Liberase DL (Roche) + 200 U/mL DNAse I (Sigma), and mechanical dissociation with P1000 pipette. The BM cells collected from crushed bones were resuspended in HBSS with 2% FBS and RBCs were removed by lysis with ACK buffer. The BM cell suspension was further purified on a density gradient (Histopaque 1119, Sigma-Aldrich), by layering 2 ml of the cell suspension under 2 ml of Histopaque solution. For profiling of CM plug and Endo marrow, only femurs were used and were flushed once with a 3 ml syringe and 21G needle to collect BM plugs, while the flushed bones were crushed to collect the remaining Endo-associated BM cells. No ACK RBC lysis was performed for these analysis. Both stromal and hematopoietic cells were finally counted using a Vicell automated cell counter (Beckman Coulter).  RNA sequencing - Droplet-based scRNA-seq for stroma and hematopoietic analyses For Endo and CM stromal cell preparations, 30,000–50,000 Ter119-/CD45- cells were sorted on purity mode into 1.5 ml tubes containing 300 μl of 50% FBS and 50% HBSS using a BD FACS Aria II instrument (gating shown in Supplementary Fig. 2a, 3b). For whole BM cells, 40,000 live (propidium iodide negative) cells from n=2 mice were sorted into similar tubes from PBS or pIC-injected mice. Cells were rested for 1 hour on ice then pelleted at 350 x g for 5 minutes at 4°C before the supernatant was removed down to a volume of 40 μl.  For sequencing of defined HSPCs, 8,000 cells of 10 different populations (HSC, stHSC, MPP2, FcγR- MPP3, FcγR+ MPP3, MPP4, GMP, MkP, CLP, CFU-E, see Supplementary Fig. 1 for sorting scheme) were sorted separately into 1.5 ml tubes. In this experiment, there were 2 biological replicates with BM from n=5 mice, and 2 technical replicates were generated per sample. Sorted cell populations were labelled with 0.05 μg of oligo-hashed antibodies specific for MHC class I and CD45 (BioLegend, TotalSeq B1-10) for 30 minutes on ice before washing three times with HBSS containing 2% FBS. All 10 populations were then pooled for subsequent partitioning. Cells were rested for 1 hour on ice then pelleted at 350 x g for 5 minutes at 4°C before the supernatant was removed down to a volume of 40 μl.  For sequencing of purified stromal populations, samples of central marrow or endosteal stromal cells pooled from n=7 mice were labelled with oligo-hashed antibodies at a concentration of 0.375 μg of antibody per 106 cells. These antibodies were mixed with the fluorophore-conjugated surface antibodies, and cells were then washed three times with HBSS and 4% FCS. Specific stromal cell populations were then sorted by FACS (gating scheme shown in Fig. 1e), and mixed according to the following scheme to produce two combined samples, each containing a total of 25-40,000 cells: Sample type Oligo hash antibody Sample 1 (cells) Sample 2 (cells) CM TotalSeq B1 MSC-L (6899) SEC (2228) CM TotalSeq B2 mMPr (2775) SEC (2097) CM TotalSeq B3 mMPr (2406) SEC (2529) CM TotalSeq B4 MPr (847) SEC (1041) CM TotalSeq B5 MPr (802) SEC (1353) CM TotalSeq B6 MSC-L (7642) AEC (438) CM TotalSeq B7 - AEC (496) Endo TotalSeq B8 MPr (10048) MSC-S (8212) Endo TotalSeq B9 AEC (5046) MSC-L (2319) Endo TotalSeq B10 SEC (3537) mMPr (6823)  Cells were rested for 1 hour on ice then pelleted at 350 x g for 5 minutes at 4°C before the supernatant was removed down to a volume of 40 μl. GEM generation and 3' RNA library preparation was performed according to 10X Genomics protocol CG000315 Rev E, targeting 5000 cell data recovery. RNA libraries were pooled 1:1:1:etc, sequenced on an Illumina NovaSeq 5000, and alignment was performed using Cellranger (v.7.0.1) to mouse genome mm10. For hashing samples, hashing libraries were pooled with RNA libraries at a ratio of 1:4. Library concentrations and fragment sizes were evaluated using Qubit dsDNA HS assay kit (ThermoFisher Scientific) and TapeStation D5000 DNA ScreenTape analysis (Agilent).  RNA sequencing - Plate-based SMART-seq for stromal cells and MSC-L For Smart-seq analyses of Endo (AEC, MSC-S, mMPr, MPr) and CM (SEC, MSC-L) stromal cells isolated from young WT mice, we utilized our own published data (Mitchell et al., 2023) that was processed using the same analytical pipeline. For Smart-seq of iMSC-L from pIC-injected young WT mice and aged 24-month-old WT mice, we utilized the SMART-Seq Single Cell PLUS kit (96 reactions, Takara Bio). Single iMSC-L were sorted into 96 well plates (see Supplementary Fig. 3c for gating strategy) containing 12.5 μl of CDS sorting solution provided in the kit. Preparation and amplification of cDNA and single cell libraries were completed according to the manufacturer’s instructions. Single cell libraries that passed quality control (n=43 from pIC-injected mice and n=30 from aged mice) were pooled at a 1:1:1etc ratio before sequencing on an Illumina NextSeq 500/550 instrument using a version 2 kit (Illumina). Fastq files were generated for each library in Illumina BaseSpace.
For 10X Genomics: Library was constructed according to the manufacturer's instructions (single cell 3', version 3 protocol, 10X Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscribed to cDNA, which contains an Illumina R1 primer sequence, unique molecular identifier (UMI) and the 10X barcode. The pooled barcoded cDNA was cleaned up with Silane Dynabeads, amplified by PCR, and the appropriate size fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction, paired-end constructs with P5 and P7 sequences and a sample index were added.  SMARTseq: Library construction was performed according to the manufacturer's instructions (SMART-Seq Single Cell PLUS kit (96 reactions, Takara Bio).

value: Central marrow stroma

value: Inflammatory MSCL

tag: Sex

tag: age


title: Juvenile_XL_ovary, biol_rep_3

city: Baltimore

first: Asya

last: Davidian

state: MARYLAND

address: 3520 San Martin Dr

department: Embryology

institute: Carnegie Institution for Science

description:
Library name: AD3
10x Genomics

data_processing:
Cell Ranger software (v8.0.1) from 10X Genomics was used for demultiplexing, barcode processing, gene counting, and generation of expression matrices. Reads were aligned to the Xenopus laevis genome assembly Xenla10.1.
Assembly: Xenla10.1
Supplementary files format and content: Processed files include a sparse gene expression matrix (matrix.mtx), cell barcodes (barcodes.tsv), and feature annotations (features.tsv), in standard 10X Genomics format. All files are tab-delimited.

organism: Xenopus laevis

source_name: ovary

growth_protocol: Individual ovaries were isolated from wild-type juvenile Xenopus laevis at developmental stages NF62–66.

extract_protocol:
Ovaries from juvenile Xenopus laevis (NF62–66) were chopped and enzymatically dissociated in a collagenase mix (L-15 with 3 mg/ml Collagenase I [Gibco 17018029], 2 mg/ml Collagenase IV [Gibco 17104019], 3 mg/ml Collagenase II [Sigma C6885], and 1 mM CaCl₂) for 40 min at room temperature with rotation. Cells were washed in PBS, treated with TrypLE Express (Gibco) for 10 min, washed again, re-suspended in cold PBS, and filtered through 70 μm and 40 μm strainers. The final cell suspension was prepared in PBS with 1% BSA for 10X Genomics loading.
Libraries were prepared using the 10X Genomics Chromium GEM-X Single Cell 3′ Reagent Kit v4, following the manufacturer’s protocol. Approximately 10,000 cells per sample were loaded onto the Chromium platform to generate Gel Beads-in-Emulsion (GEMs), followed by reverse transcription, cDNA amplification, and library preparation according to the 10X workflow.

treatment_protocol: No treatment

value: ovary

value: germline cells

value: WT


title: NOTCH1 knock-down sample,  normoxia condition, replicate 3

description: Library name: YL12_3

value: Breast

tag: cell line

value: Epithelial

value: NOTCH1 knock-down

value: normoxia


title: High maturation subpopulation of BMDC, CD11c+MHCIIhiCD86hi, bio rep 1

description:
BMDC_CSvsCS+RA_FPKM+1.txt
2044__CS-1


title: Juvenile_XL_ovary, biol_ rep_2

description:
Library name: AD2
10x Genomics


title: Low maturation subpopulation of BMDC, CD11c+MHCIIdimCD86dim, bio rep 3

description:
BMDC_CSvsCS+RA_FPKM+1.txt
2044__CS+RA-3

value: 10nM retino acid (RA) in 0.1% DMSO


title: Juvenile_XL_ovary, biol_rep_1

description:
Library name: AD1
10x Genomics


title: SKBR3 human breast cancer cell line,DMSO, rep2 (Input)

description: Library name: P-D_rep2_Input

value: DMSO treatment for 7 days


title: NOTCH1 knock-down sample,  normoxia condition, replicate 1

description: Library name: YL12_1


title: Low maturation subpopulation of BMDC, CD11c+MHCIIdimCD86dim, bio rep 2

description:
BMDC_CSvsCS+RA_FPKM+1.txt
2044__CS+RA-2

value: Bone marrow

value: wild type


title: SKBR3 human breast cancer cell line,DMSO, rep1 (Input)

description: Library name: P-D_rep1_Input

value: human cell line


title: SK-OV-3(V5-RRN3)_INPUT

description: Library name: SK-OV-3_INPUT

data_processing:
After obtaining the raw sequencing data, the first step is to filter the raw data to obtain high-quality sequencing data (clean data). The clean data is then aligned to the reference genome of the species of interest. Genome-wide peak calling is performed on the alignment results to study the binding preferences of proteins on RNA, and motif analysis is conducted on the binding sites
Assembly: GRCh38
Supplementary files format and content: peak files(txt)

extract_protocol:
V5-RRN3-expressing cells were grown in its complete medium supplemented with 100 μM 4-thiouridine (4-SU) for 14 h before crosslinking. After washing the cells with ice-cold phosphate-buffered saline (PBS), cells were cross-linked by exposure to 365-nm UV light for receiving a total energy of 0.2 J/cm2. The cells were then suspended in 3 volumes of NP40 lysis buffer [50 mM HEPES-KOH, pH 7.5, 150 mM KCl, 2 mM EDTA, 1 mM NaF, 0.5% (v/v) Nonidet P-40 (NP-40), 0.5 mM DTT, protease inhibitor cocktail (Roche, Swiss)] and incubate on ice for 10 min. The cell lysates were then treated with 10 μg/mL RNase A and 4 U/mL DNase I (Thermo-Fisher Scientific). Ten percent of cleared cell lysates were used as the input and the rest were subjected to immunoprecipitation with anti-V5 antibody (Thermo-Fisher Scientific), followed by the elution with V5 peptide (MedChemExpress, Shanghai, China). The eluted samples were digested with proteinase K before the RNA fragments were extracted by 25:24:2（v/v/v）phenol/chloroform/isoamyl alcohol (pH<5.2). The purified RNA samples were then subjected to the library construction and deep sequencing
1. Use RNA as a template and reverse transcribe it into cDNA using six-base random primers.2. Ligate adapters containing UID (Unique Molecular Identifier).3. Add primers, dNTPs, and DNA polymerase to perform PCR amplification, generating a library that meets the sequencing instrument's requirements.4. Use magnetic beads to recover fragments of the desired size and perform quality control on the constructed library.5. After the library passes quality control, pool different libraries according to effective concentration and the required sequencing data output, then load them onto the sequencing instrument for sequencing

value: human ovarian cancer cell line


title: NOTCH1 knock-down sample,  normoxia condition, replicate 2

description: Library name: YL12_2


title: Low maturation subpopulation of BMDC, CD11c+MHCIIdimCD86dim, bio rep 1

description:
BMDC_CSvsCS+RA_FPKM+1.txt
2044__CS+RA-1

value: charcoal-stripped (CS) FBS


title: BMDC with wild type Aldh1a2 (floxed) #430

description:
BMDC_KOvsWT_FPKM+1.txt
2203__430

value: Bone marrow derived dendritic cells

value: Aldh1a2 wt (fl/fl), no Cre


title: Wild-type sample, oscillatory hypoxia condition, replicate 2

description: Library name: YL11_2

value: Wild-type

value: oscillatory hypoxia


title: SKBR3 human breast cancer cell line, EPZ6438, rep1 (H3K72me3 ChIP)

description: Library name: P-E_rep1_IP

value: Human breast cancer

value: EPZ6438 treatment for 7 days


title: SK-OV-3(V5-RRN3)_IP (PAR-CLIP)

description: Library name: SK-OV-3_IP

molecule: other

tag: antibody

value: V5 (Invitrogen, Cat No. R960-25)


title: Wild-type sample, oscillatory hypoxia condition, replicate 3

description: Library name: YL11_3

value: Breast

value: Epithelial


title: SKBR3 human breast cancer cell line, EPZ6438, rep2 (H3K27me3 ChIP)

description: Library name: P-E_rep2_IP


title: Wild-type sample, oscillatory hypoxia condition, replicate 1

description: Library name: YL11_1


title: CTR_48h_3

city: Tallinn

first: Indrek

last: Koppel

address: Akadeemia tee 15-241

department: Department of Chemistry and Biotechnology

country: Estonia

institute: Tallinn University of Technology

data_processing:
fastq files were generated by the usage bcl2fastq (basecalling)
Reads were trimmed using cutadapt
Reads were aligned to the mouse genome build GRCm38 using STAR
Counting reads on reference genome GRCm38, Ensembl realese 82, was done with HTSeq-count
Differential analysis was carried out using DESeq2
Assembly: GRCm38
Supplementary files format and content: processed1_raw_counts.xlsx
Supplementary files format and content: processed2_normalized_counts.xlsx

growth_protocol: NIH-3T33T3 cells (mouse embryonic fibroblasts) were grown at 37°C and 5% CO2 in DMEM (Gibco), supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin (Biological Industries).

extract_protocol:
RNA was extracted using RNeasy Mini kit (Qiagen) according to manufacturer’s instructions. RNA integrity was verified using TapeStation analysis (Agilent) and high intergrity samples (RIN > 9) were processed.
Inhouse polyA based RNA seq

treatment_protocol: AS1411 (GGTGGTGGTGGTTGTGGTGGTGGTGG) and control DNA (CCTCCTCCTCCTTCTCCTCCTCCTCC) oligonucleotides (Integrated DNA Technologies) were dissolved in culture medium as 10x stock solutions and used at 10 µM final concentrations.

tag: time point

value: fibroblast

value: WT

value: Control aptamer