title: STm LT2, MUC2 glycans, 3 h, Sequencing Run 2, Bio Rep 1

tag: treatment


title: 16-20 h embryo, ft-GFP[int], Replicate 2, NovaSeq 6000 (Input)

status: Public on Sep 25 2025

type: SRA

city: St. Louis

first: Helen

last: McNeill

state: Missouri

address: 660 S. Euclid Avenue

department: Developmental Biology

country: USA

institute: Washington University School of Medicine in St. Louis

description: Library name: 16-20 h embryo, ft-GFP[int], Replicate 2 (Input)

library_source: genomic

data_processing:
ChIP-seq reads were trimmed using Trimmomatic v0.33 with default settings.  ChIP-seq reads were algined to dm6 genome using Burrows-Wheeler Aligner v0.7.16a with default settings. Only uniquely mapping reads with at most two mismatches were retained.
To create ChIP-seq coverage plots, the locations of the mapped ChIP-seq reads were extended to 150 bp to represent sequnced fragments, renormalized (to reads per million, rpm) and reformatted in the bigWig file format using HOMER 4.11.1.
Peaks were called using MACS v2.1.2 with the significance cut-off q-value <= 0.01 and were annotated with ChIPseeker and motifs identified with HOMER v4.11.1.
Assembly: dm6
Supplementary files format and content: bigWig

molecule: genomic DNA

organism: Drosophila melanogaster

source_name: 16-20 h embryo

growth_protocol: Drosophila were reared at 25C on standard fly food.  Embryos were collected and aged at 25C on grape juice agar plates.

extract_protocol:
Embryos were collected for 4 hrs, aged for 16 hrs at 25C, and dechorionated in 50% bleach.  Eye-brain complexes were dissected from 3rd instar larvae in ice-cold PBS.  Tissues were crosslinked in 2 mM EGS for 45 min, followed by paraformaldehyde for 15 min, quenched, washed, flash frozen in liquid nitrogen and stored at -80C until use.  50 ml settled embryos or 100 eye-brain complexes were used per sample.  Crosslinked tissues were homogenized by douncing, followed by nuclear lysis and sonication of the genomic DNA using a Bioruptor or Qsonica Q700 sonicator.  Fragmented chromatin was incubated with anti-GFP or anti-FLAG antibodies and precipitated using MagnaChIP A+G beads or Dynabeads G.
ChIP-seq libraries were prepared from 10 ng DNA.  DNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends.  Size selection was performed with AMPure beads to limit the fragment size to 150-700 bp.  Ligated fragments were then amplified for 15 cycles using primers incorporating unique dual index tags.

tag: tissue

value: 16-20 h embryo

tag: genotype

value: Ft-GFP[int]

tag: chip antibody

value: a-GFP (ab290)


title: CRCHUG_112_tumor

city: Geneva

first: MELIVOIA

last: RAPTI

address: Rue du Général-Dufour

department: Medicine

country: Switzerland

institute: University of Geneva

library_source: transcriptomic

data_processing:
GeoMX transcriptomic data was used to identify genes associated with response to treatment according or independently from their spatial localization (tumor or stroma segments). We fitted a mixed linear model for gene expression looking for association to the response alone or together with localization. Patient IDs were added as random effects to account for patient and batch technical variability. We used the DREAM method from the R package VariancePartition18 to fit the mixed linear model, and to extract the statistical test result table. Reported p-values are adjusted for multiple testing with the Benjamini-Hochberg procedure. The averaged measurements of tumor and stroma segments were calculated to give a pseudo bulk estimate of the data. Expression of the samples that had information on either one or the other segment didn’t change. A log2 transformation was applied afterwards. Genes with low expression were filtered according to the Nanostring guidelines.
Library strategy: GeoMX transcriptomic

molecule: total RNA

organism: Homo sapiens

source_name: FFPE tissue

growth_protocol: not relevant to this study

extract_protocol:
The FFPE tissue sections were incubated with cocktails of unique DNA probes (18’677 targeted genes). The region of interest (ROI) was selected using up to 4 morphologic antibodies combined with fluorochromes. Compartments were chosen for high-resolution multiplex profiling, and oligonucleotide tags from the selected region were released upon exposure to UV light. Photocleaved oligos were then collected via microcapillary tube inspiration into in microplates and analyzed using Ncounter platform or sequencing. Digital counts were then imported back into the GeoMx DSP instrument for QC and data analyses using the GeoMx DSP analysis suite version (v2.4). All raw data have also been exported for downstream analysis within R statistical language.
DNA oligonucleotide probes were designed to bind mRNA targets. From 5′ to 3′, they are each comprised of a 35- to 50-nucleotide target complementary sequence, a ultraviolet (UV) photocleavable linker and a 66-nucleotide indexing oligonucleotide sequence containing a unique molecular identifier (UMI), RNA ID sequence and primer binding sites. AOIs were exposed to 385 nm light (UV), releasing the indexing oligonucleotides which were collected with a microcapillary and deposited in a 96-well plate for subsequent processing. The indexing oligonucleotides were dried down overnight and resuspended in 10 μl of DEPC-treated water. Sequencing libraries were generated by PCR from the photo-released indexing oligos and AOI-specific Illumina adapter sequences, and unique i5 and i7 sample indices were added.
Pooled libraries were paired-sequenced at 2 × 27 base pairs and with the single-index workflow on an Illumina HiSeq 3000/4000 instrument. FastQ files were converted into Digital Count Conversion (DCC) files according to manufacturer’s pipeline. DCC files are imported back into the GeoMx DSP instrument for QC and data analyses.

treatment_protocol: All patients included into this study received nCRT as a standard of care treatment. Formalin-fiyed and paraffin embedded blocks from these patiens were used to build a next generation TMA (ngTMA).

value: FFPE tissue

value: TMA1 | 067 | tumor

tag: slidename

tag: roilabel

tag: segmentlabel

value: tumor

tag: aoisurfacearea

tag: aoinucleicount

tag: roicoordinatex

tag: roicoordinatey

tag: type

value: Biopsy

tag: status

value: Alive

value: TRG 3

tag: code

tag: response

value: Mid responders

tag: gender

value: Male


title: ATAC-seq_mixed H7 cells,negative,Week 5

city: Macau

first: Ning-Yi

last: Shao

address: University of Macau

department: Faculty of Health Sciences

country: China

institute: University of Macau

data_processing:
ATAC-seq Guidelines from Harvard FAS informatics
Quality control, alignment, and format transformation were the same as those mentioned in bulk RNA-seq data
Peaks were called by Genrich v0.6.1, which can set ATAC-seq analysis mode to run all the post-alignment steps (removal of mitochondrial reads, removal of PCR duplicates, analysis of multimapping reads, and analysis of multiple replicates) through peak-calling within one command. The parameter sets like this: -j, -r, -e chrM ,-v,-E human blacklist,-q 0.01. Multiple replicates in the same biological condition were used together to call peaks for the replicates collectively.
Peaks were combined before being quantified by FeatureCounts.
The differential enriched region test was performed by DESeq2 v1.37.4 (pvalue<0.05 & logFC >1 or logFC <-1) and these regions were annotated by CHIPseeker v1.32.0 and enriched functionally by GeneAnswers v1.16.0 (pvalue<0.05) (Yu et al., 2015).
Assembly: hg38
Supplementary files format and content: tab-delimited text files include raw counts for each Sample
Supplementary files format and content: tab-delimited text files include DApeaks results

growth_protocol: To generate mixed organoids, S6K1 knockout H7 cells were labeled with eGFP through infection with eGFP-expressing AAV2 viral particles (Gene Copoeia, AA001) followed by fluorescence-activated cell sorting (FACS). Dorsal forebrain organoids were generated with 1:1 mixture of eGFP-labeled S6K1 knockout cells and non-labeled wild-type cells.

extract_protocol:
For the ATAC sequencing, the mixed organoids were dissociated into single cells with Papain dissociation kit. Then, the dissociated cells were divided into eGFP-positive and eGFP-negative portions using a BD FACS Aria, and 50,000 of sorted cells were lysed with 50 ul of nuclei isolation buffer (10mM Tris-HCl (pH7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% NP-40, 0.01% Digitonin, 1% BSA, 1 mM DTT) for 5 min on ice. The 500 ul of chilled Wash Buffer (10mM Tris-HCl (pH7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA, 1 mM DTT) were added to lysed cells, and cells were centrifuged at 500 x g for 5 min at 4°C. The supernatant was removed, and transposition was performed using Tn5 (illumina).
The fragmented DNA by Tn5 was purified using MinElute reaction Cleanup kit (Qiagen) and amplified with predesigned primers containing adaptors and barcodes. Next, the ATAC-seq libraries were purified using AMPure XP beads and final libraries were sequenced with a paired end read of 150 bp on the NovaSeq 6000 illumina platform.

treatment_protocol: S6K1 knockout H7 cells were generated by targeting exon 9 and 11 of RPS6KB1. The guide RNAs targeting exon 9 (E9-For: TTAAACATAATTGGTTTGGGGG, E9-Rev: GCAGCTTTTAACTGTGCACCTA) & exon 11 (E11-For: AAAAGAAATGCTGCTTCTCGTC, E11-Rev: AGGAAGAAAAGTTGAGGGGACT) of S6K1 were designed using online CRISPR design tool (https://chopchop.cbu.uib.no/) and cloned into the pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene, #62988). The 1ug of cloned plasmids were transfected into H7 ESCs using Lipofectamin 3000 reagent. After two days, GPF-expressing transfected cells were sorted with a BD FACS Aria. The sorted cells were seeded on 6 well plates and expanded clonally. To confirm genotype of each clone, genomic DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen), and PCR was conducted using PrimeSTAR GXl DNA Polymerase (TaKaRa).

tag: cell line

tag: cell type

value: human dorsal forebrain organoids

tag: cell marker

value: GFP-negative

value: WT


title: CRCHUG_104_stroma

tag: tissue

value: TMA1 | 062 | stroma

value: stroma

value: Dead

value: TRG 2

value: Good responders


title: 16-20 h embryo, ft-GFP[int], Replicate 2, NovaSeq 6000 (ChIP)

description: Library name: 16-20 h embryo, ft-GFP[int], Replicate 2 (ChIP)

value: 16-20 h embryo

tag: genotype

value: Ft-GFP[int]

tag: chip antibody

value: a-GFP (ab290)


title: STm LT2, Medium alone, 3 h, Sequencing Run 2, Bio Rep 5

city: Cambridge

first: Katharina

last: Ribbeck

state: Massachusetts

address: 21 Ames St

department: Biological Engineering

institute: Massachusetts Institute of Technology

data_processing:
Reads were analyzed on the Galaxy platform (24.1.2) with the Burrows-Wheeler algorithm for alignment and HTseq-count for read counts. DESeq2 was used for differential expression analysis between the medium alone control and treated cells from the same experiment with the recommended settings. For all analyses of the sequencing data, we considered FDR-adjusted P values < 0.05 as significant.
For paired end reads, the fastq files from the two lanes were concatenated prior to mapping.
Assembly: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, GCF_000006945.2
Supplementary files format and content: excel workbooks with raw read counts

source_name: Bacterial culture

growth_protocol: S. Typhimurium LT2 overnight cultures were diluted 1:100 in 100 µL of SPI-1-inducing LB.3 medium with or without treatments and then cultured for 3 h at 37 °C under static conditions.

extract_protocol:
RNA was extracted with the MasterPure RNA Purification Kit (Biosearch Technologies), and DNA was eliminated using the Turbo DNA-Free Kit (Ambion). The Agilent 2100 Bioanalyzer (Agilent Technologies) was used to assess RNA integrity.
rRNA was depleted with the NEBNext rRNA Depletion Kit for bacteria (NEB). The Illumina HiSeq platform was used for the sequencing mucin-, glycan-treated, and monosaccharide pool-treated samples with a single-end protocol and read lengths of 40 nucleotides. The Illumina NovaSeq platform was used for sequencing HexNAc-treated samples with a paired-end protocol and read lengths of 50 nucleotides.

treatment_protocol: For treated bacterium, LB.3 contained either 0.1% MUC2, 0.1% MUC2 glycans, 0.2% GalNAc, 0.2% GlcNAc, 0.1% monosaccharide pool containing equal volumes of D-galactose, D-GalNAc, D-GlcNAc, L-fucose, and Neu5AC.

value: Bacterial culture

tag: treatment

value: Medium alone control


title: 16-20 h embryo, ft-GFP[int], Replicate 1, NovaSeq 6000 (Input)

description: Library name: 16-20 h embryo, ft-GFP[int], Replicate 1 (Input)


title: SGC-7901 shNC-1

city: Fujian

first: Yi

last: Zeng

address: Fu Ma Road 420,Fuzhou,Fujian

institute: Fujian Medical University Cancer Hospital, Fujian Cancer Hospital

data_processing:
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In 1 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. We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels.
Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.20.0). DESeq2 provide statistical routinesfor determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate . Genes with an adjusted P-value
Assembly: hg38
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

source_name: stomach

growth_protocol: HGC-27 and SGC-7901 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) (HyClone, South Logan, UT, USA), penicillin (100 U/ml), and streptomycin (100 U/ml) at 37℃ in a humidified 5% CO2 incubator.

extract_protocol:
Cells were washed twice with pre-chilled PBS, and then total RNAs were harvested using Trizol reagent.
RNA libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: HGC-27 and SGC-7901 cells were transfected with pSIH-H1-Puro-shSMYD3 or pSIH-H1-Puro-shNC respectively for 24 hours,then added puro screening for 7days

value: stomach

value: gastric cancer cells

value: transfected with pSIH-H1-Puro-shNC

tag: time

value: Day 7


title: 16-20 h embryo, ft-GFP[int], Replicate 1, NovaSeq 6000 (ChIP)

description: Library name: 16-20 h embryo, ft-GFP[int], Replicate 1 (ChIP)


title: SGC-7901 shNC-2


title: 16-20 h embryo, ft-GFP[C], Replicate 1, NovaSeq 6000 (ChIP)

description: Library name: 16-20 h embryo, ft-GFP[C], Replicate 1 (ChIP)

value: Ft-GFP[C]


title: STm LT2, Medium alone, 3 h, Sequencing Run 2, Bio Rep 4

organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2


title: STm LT2, MUC2, 3 h, Sequencing Run 1, Bio Rep 2


title: 16-20 h embryo, ft-GFP[C], Replicate 1, NovaSeq 6000 (Input)

description: Library name: 16-20 h embryo, ft-GFP[C], Replicate 1 (Input)


title: CRCHUG_096_2_tumor

value: FFPE tissue

value: TMA1 | 048 | tumor

tag: slidename

tag: roilabel

tag: segmentlabel

value: tumor

tag: aoisurfacearea

value: Biopsy

value: Alive

value: TRG 4

value: Non responders

value: Male


title: SGC-7901 shSMYD3-1

value: SMYD3 knockdown

value: transfected with pSIH-H1-Puro-shSMYD3


title: STm LT2, MUC2, 3 h, Sequencing Run 1, Bio Rep 3

value: Bacterial culture

value: WT


title: STm LT2, Medium alone, 3 h, Sequencing Run 2, Bio Rep 6

value: Medium alone control


title: HGC-27 shNC-2

value: stomach

value: gastric cancer cells


title: CRCHUG_096_1_tumor

tag: aoinucleicount

tag: roicoordinatex

tag: roicoordinatey

tag: type


title: CRCHUG_096_stroma

value: TMA1 | 048 | stroma

value: stroma

tag: status

tag: code

tag: response

tag: gender


title: STm LT2, Medium alone, 3 h, Sequencing Run 1, Bio Rep 3

tag: cell type


title: HGC-27 shNC-1

tag: tissue

tag: cell line

tag: genotype

value: transfected with pSIH-H1-Puro-shNC

value: Day 7


title: SGC-7901 shSMYD3-2

tag: treatment

tag: time


title: DBDSR4_1

city: Duarte

first: Yate-Ching

last: Yuan

state: CA

address: 1500 East Duarte

institute: City of Hope

description: Library name: Sample 4

data_processing:
Partek PGS v6.6 release 6.14.0402
Sequence reads were mapped  to GRCm38/mm10 using TopHat v 2.1.1 with validation of quality through pre-alignment and post-alignment quality assurance (QA)/quality control (QC) using Partek Flow
Aligned reads were further subjected to quantification using the Partek E/M algorithm and normalization to counts per million (CPM) with 0.001 added to each.
Normalized read were subjected for differential expression analysis using ANOVA compare the means of  SR4 treated versus vehicle control  after 5 weeks
Assembly: GRCm38/mm10
Supplementary files format and content: Bulk RNAseq raw counts

organism: Mus musculus

source_name: Liver

extract_protocol:
RNA extraction with the RNeasy Mini kit (Qiagen). 500 ng of total RNA were used for the construction of sequencing libraries.
Sequencing libraries were prepared with the TruSeq RNA Sample Prep Kit V2 (Illumina, San Diego , CA)  according to the manufacturer's protocol.

treatment_protocol: After one week of acclimatization, mice were treated with  vehicle control  and SR4 (10 mg/kg/body weight) via oral gavage  for 5 weeks.

value: Liver

value: SR4 treated

value: 5 weeks after SR4 treatment


title: DBDSR4_2

description: Library name: Sample 5


title: STm LT2, MUC2, 3 h, Sequencing Run 1, Bio Rep 1


title: CRCHUG_100_stroma

value: FFPE tissue

value: TMA1 | 053 | stroma

tag: slidename

tag: roilabel

value: Biopsy

value: Alive

value: TRG 4

value: Female


title: CRCHUG_104_tumor

value: TMA1 | 062 | tumor

tag: segmentlabel

value: tumor

tag: aoisurfacearea

value: Dead

value: TRG 2

value: Good responders

value: Male


title: CRCHUG_100_tumor

value: TMA1 | 053 | tumor

tag: aoinucleicount

tag: roicoordinatex

value: Non responders


title: HGC-27 shSMYD3-2

value: stomach

value: gastric cancer cells

value: SMYD3 knockdown

value: transfected with pSIH-H1-Puro-shSMYD3

value: Day 7


title: CRCHUG_094_tumor

value: FFPE tissue

value: TMA1 | 043 | tumor

tag: roicoordinatey

tag: type

value: Biopsy

value: TRG 3

value: Mid responders


title: DBD3

description: Library name: Sample 3

value: Liver

value: Control

value: 5 weeks after control (vehicle ) treatment


title: STm LT2, Medium alone, 3 h, Sequencing Run 1, Bio Rep 2

value: Bacterial culture

value: WT

value: Medium alone control


title: STm LT2, Medium alone, 3 h, Sequencing Run 1, Bio Rep 1


title: CRCHUG_086_stroma

value: TMA1 | 038 | stroma

value: stroma

tag: status

value: Alive

value: TRG 2-3

tag: code

value: Female


title: RNA-seq_KO3

data_processing:
paired-end reads were trimmed and filtered with default parameters via trim_galore v0.6.5 software and then aligned to the human genome GRCh38 (hg38) using Bowtie2 v2.4.5
Aligned reads were converted into the bam format and sorted by sambamba v0.8.2, followed by featureCounts for quantification.
The differentially expressed genes was detected by the DESeq2 v1.37.4 R package with the threshold adjust pvalue<0.001 and the functional enrichment test was conducted by the GeneAnswers v1.16.0 R package (pvalue<0.05)
Assembly: hg38
Supplementary files format and content: tab-delimited text files include raw counts for each Sample
Supplementary files format and content: tab-delimited text files include DEG results

source_name: H7 human embryonic stem cells, S6K1-depleted,Week 5

growth_protocol: H7 human embryonic stem cells were grown in feeder-free conditions on Matrigel (Corning)-coated plates with TeSR-E8 medium (Stem Cell Technologies).

extract_protocol:
For the bulk RNA sequencing, total RNA was extracted from undifferentiated wild-type and S6K1-depleted embryonic stem cells using miRNeasy Kit (Qiagen).
A library construction for bulk RNA sequencing was conducted with TruSeq Stranded mRNA Sample Preparation Kit (Illumina) according to the manufacturer’s instruction.

treatment_protocol:
S6K1 knockout H7 cells were generated by targeting exon 9 and 11 of RPS6KB1. The guide RNAs targeting exon 9 (E9-For: TTAAACATAATTGGTTTGGGGG, E9-Rev: GCAGCTTTTAACTGTGCACCTA) & exon 11 (E11-For: AAAAGAAATGCTGCTTCTCGTC, E11-Rev: AGGAAGAAAAGTTGAGGGGACT) of S6K1 were designed using online CRISPR design tool (https://chopchop.cbu.uib.no/) and cloned into the pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene, #62988). The 1ug of cloned plasmids were transfected into H7 ESCs using Lipofectamin 3000 reagent. After two days, GPF-expressing transfected cells were sorted with a BD FACS Aria. The sorted cells were seeded on 6 well plates and expanded clonally. To confirm genotype of each clone, genomic DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen), and PCR was conducted using PrimeSTAR GXl DNA Polymerase (TaKaRa).
1uM dexamethasone (Sigma), and 0.5mM isobutylmethylxanthine (Sigma) in the presence of 10% FBS until day 2. Cells were then fed with DMEM supplemented with 10% FBS and 10ug/ml insulin for 2 days, after which they were fed every other day with DMEM containing 10% FBS

value: human embryonic stem cells (ESCs)

value: S6K1 knockdown

value: undifferentiation

value: Week 5


title: DBD2

description: Library name: Sample 2


title: HGC-27 shSMYD3-1

value: stomach

value: gastric cancer cells

value: SMYD3 knockdown

value: transfected with pSIH-H1-Puro-shSMYD3

value: Day 7


title: CRCHUG_086_tumor

value: FFPE tissue

value: TMA1 | 038 | tumor

value: tumor

value: Biopsy

tag: response

value: Mid responders

tag: gender


title: tumor tissue of Patient 6 [T6]

city: Guangzhou

first: xunjun

last: li

state: China

address: Guangzhou 510515, Guangdong Province, Ch

institute: Nanfang Hospital

data_processing:
CLC Genomics Workbench v 11.0.1
Sequence reads were trimmed for adaptor sequence/low-quality sequence using CLC genomic benchwork (parameter- Quality limit: 0.05)
Trimmed sequence reads were mapped to GrCM38/mm10 using CLC genomic benchwork (parameters- mismath cost: 2 insertion cost: 3 deletion cost: 3 length fraction: 0.8 similarity fraction: 0.8)
Read count extraction and normalization were performed using CLC genomic benchwork
Supplementary files format and content: CRch38.p13
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

extract_protocol:
Total RNA was extracted using the TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Then the libraries were constructed using VAHTS Universal V6 RNA-seq Library Prep Kit according to the manufacturer’s instructions. The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China).

treatment_protocol: The tissues were rinsed with normal saline, loaded in tissue cryopreservation tubes and stored in liquid nitrogen.

tag: tissue

value: female

value: non perineural invasion

tag: tumor stage

tag: age(years)


title: tumor tissue of Patient 39 [T39]

value: male

tag: genotype

value: perineural invasion


title: RNA-seq_KO2

tag: cell line

tag: cell type

value: human embryonic stem cells (ESCs)

value: S6K1 knockdown

tag: treatment

tag: time


title: Lgr5_f_Y_rep3 (WGBS)

city: Kiel

first: Seyed Mohammd Mahdi

last: Rasa

state: Schleswig-Holstein

address: Michaelisstrasse 5

department: Institut for Immunology

country: Germany

institute: Universitätsklinikum Schleswig-Holstein

data_processing:
Fastq files quality check was performed with FastQC v0.11.5
Sequencing reads were adapter clipped with fastx_clipper by using the following parameters: -a GATCGGAAGAGCACACGTCTGAACTCCAGTCAC -Q33 -l 20 -n -v -M 10 .
The fastq files were mapped to the mm9 genome using BSMAP v2.89
Duplicates were removed using a custom script.
The CpG methylation was calculated using methratio.py script in the BSMAP package with -z -g -x CG parameters.
Genome_build: mm9
Supplementary_files_format_and_content: The processed data is the output of the methraio.py indicating the methylation level in each CpG.

source_name: Lgr5 positive cells from small intestine(intestinal stem cells)

growth_protocol: Young (2 months old) and old (18-26 months old) male and female wild-type C57BL6/J mice were group housed and maintained in a specific pathogen-free animal facility in Fritz Lipmann Institute with 12 h of light/dark cycle and fed with a standard mouse chow.

extract_protocol:
For the Lgr5-eGFP ISCs isolation, the villi-free intestinal pieces (2 cm) were incubated in 5mM EDTA/PBS on ice for 45 min. The tissue was then shaken vigorously for 1 min, the crypt solution was filtered through a 100 µm cell strainer and centrifuged at 300xg for 5 min at 4°C. The crypts were dissociated with the mixture of 18 ml TrypLE Express, 2 ml of 10X DNase I Buffer (100 mM Tris HCl pH 7.5, 25 mM MgCl2, 5 mM CaCl2) and 1 ml DNase I (10 mg/ml) for 30 min at 37°C with brief vortexing every 10 min. The single-cell suspension was then passed through a 20 µm cell strainer and centrifuged at 450xg for 5 minutes at 4°C. The pellet was resuspended in 3 ml FSM (2% FBS, 2.5 mM EDTA) containing 10 µM Y27632 and DAPI (1:1000). The single cell suspension was applied to FACS LSRII (BD Biosciences) and the Lgr5-eGFPHi ISCs were sorted for downstream analysis.
For whole genome bisulfite sequencing (WGBS) library preparation, 10 ng of genomic DNA was used as input using Pico Methyl-Seq™ Library Prep Kit (Zymo Research). The bisulfite conversion was performed using the Methylation-Lightning Kit (Zymo Research) following manufacturer’s instructions.

tag: strain


title: CRCHUG_085_stroma

value: TMA1 | 033 | stroma

tag: slidename

tag: roilabel

tag: segmentlabel

value: stroma

tag: aoisurfacearea

tag: aoinucleicount

value: Alive

value: TRG 3

value: Female


title: tumor tissue of Patient 24 [T24]

value: stomach


title: RNA-seq_WT2

source_name: H7 human embryonic stem cells, wild-type,Week 5

value: WT

value: undifferentiation

value: Week 5


title: Lgr5_f_Y_rep2 (WGBS)


title: RNA-seq_WT3


title: CRCHUG_076_stroma

value: FFPE tissue

value: TMA1 | 028 | stroma

tag: roicoordinatex

tag: roicoordinatey

tag: type

value: Biopsy

tag: status

tag: code

value: Male


title: tumor tissue of Patient 19 [T19]

tag: tissue

tag: gender

value: male

value: non perineural invasion

tag: tumor stage

tag: age(years)


title: CRCHUG_076_tumor

value: TMA1 | 028 | tumor

value: tumor

tag: response

value: Mid responders


title: CRCHUG_075_stroma

value: TMA1 | 023 | stroma

value: stroma


title: ID40v0, SCI, Acute

city: Manhasset

first: Ona

last: Bloom

state: New York

address: 350 Community Dr

department: Molecular Medicine

institute: Feinstein Institute

description: Library name: ID40v0

data_processing:
Partek Genomics Flow software
trimmed reads aligned to hg38 genome assembly using STAR
filtered for expression >50
normalized using the Trimmed Mean of M-values (TMM) method using the edgeR package
log2 transformed
Assembly: hg38
Supplementary files format and content: Normalized Morrison 2025 processed data.txt

source_name: Whole blood

extract_protocol:
Total RNA was isolated using the PAXgene total RNA kit and the Qiagen QIAcube, acccording to manufacturer's protocol
RNA seq libraries were prepared using Illumina TruSeq Stranded Total RNA according to manufacturer's instructions.

value: Whole blood

tag: group

value: SCI

tag: treatment

value: Acute

tag: age

tag: Sex

tag: ais

value: D

tag: nli


title: tumor tissue of Patient 12 [T12]

value: stomach

value: female

tag: genotype

value: perineural invasion


title: RNA-seq_WT1

tag: cell line

tag: cell type

value: human embryonic stem cells (ESCs)

value: WT

value: undifferentiation

tag: time

value: Week 5


title: Lgr5_f_Y_rep1 (WGBS)

tag: strain


title: ID39v0, SCI, Acute

description: Library name: ID39v0

value: Male

value: C


title: SHR Joint Chondrocytes (3 months old) rep3

city: Hung Hom, Kowloon

first: Lanlan

last: Zhang

address: 11 Yuk Choi Road

country: Hong Kong

institute: The Hong Kong Polytechnic University

description: Library name: SHR_Joint_3m_C

data_processing:
Reads of low quality, with adaptor sequences, or high levels of N base were filtered with SOAPnuke (v1.5.6).
Bowtie2 (v2.4.4) was employed to align the clean reads to the reference genes.
Gene expression was quantified using RSEM (v1.2.28) .
Normalization of gene expression was performed with DESeq2 (v1.42.1).
Assembly: GCF_000001895.5_Rnor_6.0
Supplementary files format and content: countdata.csv: Raw read counts are provided for each sample.
Supplementary files format and content: exprs_Chon.csv: Normalized expression for each sample
Supplementary files format and content: exprs_5d.csv: Normalized expression for all samples harvested from 5-day-old rats
Supplementary files format and content: exprs_3m.csv: Normalized expression for all samples harvested from 3-month-old rats
Supplementary files format and content: exprs_WKY.csv: Normalized expression for all samples harvested from Wistar Kyoto rats
Supplementary files format and content: exprs_SHR.csv: Normalized expression for all samples harvested from spontaneously hypertensive rats

organism: Rattus norvegicus

source_name: articular cartilage

growth_protocol: Primary rat chondrocytes were cultured in Dulbecco's modified Eagle medium/F-12 (DMEM/F12, Gibco, 1:1, v:v) supplemented with 5% fetal bovine serum (FBS) (v/v) and 1% Penicillin/Streptomycin (P/S) (v/v)

extract_protocol:
Total RNA was extracted with E.Z.N.A.® Total RNA Kit (Omega Biotek)
Library preparation and sequencing were outsourced to BGI Genomics. Samples were sequenced using DNBSEQ platform with a paired-end read length of 100 bp (PE100)

value: articular cartilage

value: chondrocytes

value: spontaneously hypertensive

value: harvested at 3-month-old

tag: batch


title: tumor tissue of Patient 10 [T10]

tag: tissue

value: male

value: non perineural invasion


title: DBD1

description: Library name: Sample 1

value: Liver

value: Control

value: 5 weeks after control (vehicle ) treatment


title: CRCHUG_075_tumor

value: FFPE tissue

value: TMA1 | 023 | tumor

tag: slidename

tag: roilabel

tag: segmentlabel

value: tumor

tag: aoisurfacearea

tag: aoinucleicount

tag: roicoordinatex

tag: roicoordinatey

tag: type

value: Biopsy

tag: status

value: Alive

value: TRG 3

tag: code

value: Mid responders

tag: gender


title: RNA-seq_KO1

tag: cell line

value: human embryonic stem cells (ESCs)

tag: genotype

value: S6K1 knockdown

tag: treatment

value: undifferentiation

value: Week 5


title: CRCHUG_067_stroma

value: TMA1 | 021 | stroma

value: stroma

value: TRG 1

tag: response

value: Good responders

value: Female


title: ID38v0, SCI, Acute

description: Library name: ID38v0

value: Whole blood

tag: group

value: SCI

value: Acute

tag: age

tag: Sex

tag: ais

value: A

tag: nli


title: CRCHUG_085_tumor

tag: tissue

value: FFPE tissue

value: TMA1 | 033 | tumor

tag: slidename

tag: roilabel

tag: segmentlabel

value: tumor

tag: aoisurfacearea

value: Biopsy


title: ID37v0, SCI, Acute

description: Library name: ID37v0

value: C


title: adjacent normal tissues of Patient 6 [N6]

value: stomach

value: female

value: non perineural invasion

tag: tumor stage

tag: age(years)


title: SHR Joint Chondrocytes (3 months old) rep1

description: Library name: SHR_Joint_3m_A

value: articular cartilage

tag: cell type

value: chondrocytes

value: spontaneously hypertensive

value: harvested at 3-month-old

tag: batch


title: SHR Joint Chondrocytes (3 months old) rep2

description: Library name: SHR_Joint_3m_B

tag: genotype


title: tumor tissue of Patient 22 [T22]

tag: gender

value: male


title: SHR Joint Chondrocytes (5 days old) rep3

description: Library name: SHR_Joint_5d_C

tag: treatment

value: harvested at 5-day-old


title: adjacent normal tissues of Patient 24 [N24]


title: CRCHUG_059_stroma

value: TMA1 | 016 | stroma

value: stroma

tag: aoinucleicount

tag: roicoordinatex

tag: roicoordinatey

tag: type

tag: status

value: Alive

value: TRG 2

tag: code

tag: response

value: Good responders

value: Male


title: ID35v0, SCI, Acute

description: Library name: ID35v0

tag: tissue

value: Whole blood

tag: group

value: SCI

value: Acute

tag: age

tag: Sex

tag: ais

value: A

tag: nli


title: CRCHUG_059_tumor

value: FFPE tissue

value: TMA1 | 016 | tumor

tag: slidename

tag: roilabel

tag: segmentlabel

value: tumor

tag: aoisurfacearea

value: Biopsy


title: ID34v0, SCI, Acute

description: Library name: ID34v0

tag: treatment


title: SHR Joint Chondrocytes (5 days old) rep1

description: Library name: SHR_Joint_5d_A

value: articular cartilage

tag: cell type

value: chondrocytes

tag: genotype

value: spontaneously hypertensive

tag: batch


title: CRCHUG_057_stroma

value: TMA1 | 011 | stroma

tag: aoinucleicount

tag: roicoordinatex

tag: roicoordinatey

tag: type

tag: status

value: TRG 3

value: Mid responders

tag: gender

value: Female


title: ID36v0, SCI, Acute

description: Library name: ID36v0

tag: tissue

tag: group

tag: age

tag: Sex


title: ID33v0, SCI, Acute

description: Library name: ID33v0

tag: ais

tag: nli


title: SHR Joint Chondrocytes (5 days old) rep2

description: Library name: SHR_Joint_5d_B

value: harvested at 5-day-old


title: adjacent normal tissues of Patient 22 [N22]

value: stomach

value: male

value: non perineural invasion

tag: tumor stage

tag: age(years)


title: WKY Joint Chondrocytes (3 months old) rep3

description: Library name: WKY_Joint_3m_C

tag: cell type

value: Wistar Kyoto

tag: treatment

value: harvested at 3-month-old


title: adjacent normal tissues of Patient 19 [N19]

tag: genotype


title: CRCHUG_057_tumor

value: FFPE tissue

value: TMA1 | 011 | tumor

tag: slidename

tag: roilabel

tag: segmentlabel

tag: aoisurfacearea

tag: aoinucleicount

value: Alive

tag: code

tag: response


title: adjacent normal tissues of Patient 39 [N39]

tag: tissue

tag: gender

value: perineural invasion


title: ID32v0, SCI, Acute

description: Library name: ID32v0

value: Whole blood

tag: group

value: SCI

value: Acute

tag: age

tag: Sex

value: Male

value: A


title: WKY Joint Chondrocytes (3 months old) rep2

description: Library name: WKY_Joint_3m_B

value: articular cartilage

value: chondrocytes

tag: batch


title: CRCHUG_041_stroma

value: TMA1 | 001 | stroma

value: stroma

tag: roicoordinatex

tag: roicoordinatey

tag: type

value: Biopsy

tag: status

value: TRG 3-4

value: Non responders


title: CRCHUG_041_tumor

value: TMA1 | 001 | tumor

tag: slidename

value: tumor


title: WKY Joint Chondrocytes (3 months old) rep1

description: Library name: WKY_Joint_3m_A

tag: cell type

tag: genotype

value: Wistar Kyoto

tag: treatment

value: harvested at 3-month-old


title: adjacent normal tissues of Patient 40 [N40]

value: stomach

value: female

value: perineural invasion

tag: tumor stage

tag: age(years)


title: CRCHUG_067_tumor

value: FFPE tissue

value: TMA1 | 021 | tumor

tag: roilabel

tag: segmentlabel

tag: aoisurfacearea

tag: aoinucleicount

value: Alive

value: TRG 1

tag: code

tag: response

value: Good responders

value: Female


title: ID30v0, SCI, Acute

description: Library name: ID30v0

tag: tissue

value: Whole blood

tag: group

value: SCI

value: Acute

tag: age

tag: Sex

tag: ais

value: D

tag: nli


title: adjacent normal tissues of Patient 4 [N4]

tag: gender


title: CRCHUG_065_stroma

value: TMA1 | 028 | stroma

tag: slidename

value: stroma

tag: roicoordinatex

tag: roicoordinatey

tag: type

value: Biopsy

tag: status

value: Dead

value: Male


title: WKY Joint Chondrocytes (5 days old) rep3

description: Library name: WKY_Joint_5d_C

value: articular cartilage

tag: cell type

value: chondrocytes

tag: genotype

value: Wistar Kyoto

tag: treatment

value: harvested at 5-day-old

tag: batch


title: ID31v0, SCI, Acute

description: Library name: ID31v0

value: A


title: ID29v0, SCI, Acute

description: Library name: ID29v0


title: CRCHUG_065_tumor

value: FFPE tissue

value: TMA1 | 028 | tumor

tag: roilabel

tag: segmentlabel

value: tumor

tag: aoisurfacearea

tag: aoinucleicount

tag: code

tag: response


title: WKY Joint Chondrocytes (5 days old) rep2

description: Library name: WKY_Joint_5d_B

tag: tissue


title: adjacent normal tissues of Patient 12 [N12]

value: stomach

tag: gender

value: female

value: perineural invasion

tag: tumor stage

tag: age(years)


title: CRCHUG_066_stroma

value: TMA1 | 027 | stroma

tag: slidename

tag: roicoordinatex

tag: roicoordinatey

value: Alive

value: Good responders

value: Female


title: adjacent normal tissues of Patient 30 [N30]

value: male


title: adjacent normal tissues of Patient 28 [N28]


title: ID28v0, SCI, Acute

description: Library name: ID28v0

value: Whole blood

tag: group

value: SCI

value: Acute

tag: age

tag: Sex

value: Male

tag: ais

tag: nli


title: adjacent normal tissues of Patient 26 [N26]

tag: genotype

value: non perineural invasion


title: smRNAseq_Flower_b11_pRIM22_RIM22B3mut-3xFLAG_line1

city: La Jolla

first: Julie

middle: A

last: Law

address: 10010 North Torrey Pines Rd

department: Plant Molecular and Cellular Biology

institute: Salk Institute for Biological Studies

description: Library name: sample178

library_source: other

data_processing:
For each sample, fastq files were trimmed using cutadapt (v1.18) with the -a AGATCGGAAGAGC option to remove the adapter sequence and the -m 15 option to discard reads shorter than 15 nt. Clean reads were then mapped to the TAIR10 reference genome using ShortStack (v3.8.5) allowing one mismatch (--mismatches 1). The --mmap f option was also included to assign multiple-mapped reads to a specific locus using the fractional-seeded algorithm. The resulting bam files were then filtered to keep perfectly mapped reads or reads with a single mismatch at the 3’ end using bamtools (v2.5.1) together with an in-house script (JSON_findPerfectMatches_and_TerminalMisMatches_v3) to meet the characteristics of Pol-IV transcription. To facilitate downstream analyses, the filtered bam files were used to generate Tag Directories using the makeTagDirectory function of HOMER (v4.10) with the following options: -format sam -mis 1 -keepAll. To quantify the expression of smRNAs with specific sizes, the tag directories were split by size (20nt-25nt) using a custom perl script (splitTagDirectoryByLength.dev2.pl) and all subsequent analyses utilized the Tag Directories corresponding to 24nt siRNAs.
Assembly: TAIR10
Supplementary files format and content: bedGraph file, including header and chr#, start position, end position and normalized value (rp10M).

molecule: other

organism: Arabidopsis thaliana

source_name: flower bud (stage12 and younger)

growth_protocol: For transgenic lines that required drug selection, seeds were sterilized in sterilization buffer (70% ethanol, 0.05% SDS) for 10 minutes, followed by plating on half strength Linsmaier and Skoog medium (1/2 LS; Caisson Labs, Cat# LSP03) supplemented with 0.6% agar (Phyto Agar, Cat# P1003). The plates were placed in a 4˚C cold room for 4 days before growing in a growth chamber with 16h-light and 8h-dark cycles for 7 days. The seedlings were transplanted and grown under Salk greenhouse conditions. For the HEM EMS lines, the T-DNA insertion lines, and their corresponding control lines, seeds were stratified in 0.1% agar (Phyto Agar, Cat# P1003) at 4˚C for 4 days before sowing directly onto soil and grown under Salk greenhouse conditions. Approximately five weeks after planting, flower buds (stage 12 and younger), ovules (stage 12 unfertilized ovules), and anthers (stage 9) were collected and frozen in liquid nitrogen. Flower development stages were determined according to Smyth et al. Ovule collection was performed using a vacuum pump-based collection device that was engineered at the Salk Institute and was described previously.

extract_protocol:
Total RNA extraction and smRNA enrichment was performed following a previously reported protocol. Briefly, 10 μg of total RNA extracted from flower buds (stage 12 and younger), ovules (stage 12 unfertilized ovules), and anthers (stage 9) were used for each smRNA library prep.
The smRNA-seq libraries were constructed using the NEBNext Small RNA Library Prep Kit (NEB, Cat# E7330L). The smRNA libraries were further purified using an 8% acrylamide gel and size selected (130-160 bp) relative to the O'RangeRuler 10 bp DNA Ladder (Thermo Scientific, Cat# SM1313). The size selected smRNA libraries were pooled for sequencing with single-end 50 bp kit (SE50) on Illumina Nextseq 2000 or Novaseq X plus platforms.

value: flower bud (stage12 and younger)

tag: ecotype

value: Columbia

tag: batch

value: experiment batch11


title: ID26v0, SCI, Acute

description: Library name: ID26v0

tag: treatment

value: A


title: adjacent normal tissues of Patient 27 [N27]

value: stomach


title: WKY Joint Chondrocytes (5 days old) rep1

description: Library name: WKY_Joint_5d_A

value: articular cartilage

tag: cell type

value: chondrocytes

value: Wistar Kyoto

value: harvested at 5-day-old


title: CRCHUG_066_tumor

tag: tissue

value: FFPE tissue

value: TMA1 | 027 | tumor

tag: roilabel

tag: segmentlabel

value: tumor

tag: aoisurfacearea

tag: aoinucleicount

tag: type

value: Biopsy

tag: status

value: Alive

tag: code

tag: response

value: Good responders


title: ID25v0, SCI, Acute

description: Library name: ID25v0

value: Whole blood

tag: group

value: SCI

value: Acute

tag: age

tag: Sex

value: Female

value: D


title: smRNAseq_Flower_b11_clsy1,2

description: Library name: sample177

value: flower bud (stage12 and younger)

value: Columbia

tag: genotype

value: experiment batch11


title: adjacent normal tissues of Patient 7 [N7]

value: stomach

tag: gender

value: female

value: perineural invasion

tag: tumor stage

tag: age(years)


title: adjacent normal tissues of Patient 25 [N25]

value: male


title: ID24v0, SCI, Acute

description: Library name: ID24v0

tag: treatment

value: Male

tag: ais

value: B

tag: nli


title: ID23v0, SCI, Acute

description: Library name: ID23v0

tag: tissue


title: smRNAseq_Flower_b11_pol-iv

description: Library name: sample176

tag: ecotype

tag: batch


title: adjacent normal tissues of Patient 23 [N23]


title: smRNAseq_Flower_b11_clsy3,4

description: Library name: sample175


title: adjacent normal tissues of Patient 21 [N21]


title: adjacent normal tissues of Patient 2 [N2]


title: smRNAseq_Flower_b11_WT_rep3

description: Library name: sample173


title: smRNAseq_Flower_b11_pRIM22_RIM22B3mut-3xFLAG_line2

description: Library name: sample179


title: ID11v0, SCI, Acute

description: Library name: ID11v0

value: A


title: CRCHUG_037_tumor

value: FFPE tissue

value: TMA1 | 015 | tumor

tag: slidename

tag: roilabel

tag: segmentlabel

value: tumor

tag: aoisurfacearea

tag: aoinucleicount

tag: roicoordinatex

tag: roicoordinatey

tag: type

value: Biopsy

value: Dead

value: TRG 3

value: Mid responders


title: ID27v0, SCI, Acute

description: Library name: ID27v0

value: Whole blood

tag: group

value: SCI

value: Acute

tag: age

tag: Sex

value: Female

value: D


title: ID12v0, SCI, Acute

description: Library name: ID12v0

tag: treatment


title: CRCHUG_017_stroma

value: TMA1 | 010 | stroma

value: stroma

tag: status

tag: code

tag: response

tag: gender


title: smRNAseq_Flower_b11_clsy3

description: Library name: sample174

tag: tissue

value: flower bud (stage12 and younger)

tag: ecotype

value: Columbia

tag: genotype

tag: batch

value: experiment batch11


title: CRCHUG_061_stroma

value: FFPE tissue

value: TMA1 | 025 | stroma

tag: slidename

tag: roilabel

tag: segmentlabel

tag: aoisurfacearea

value: Male


title: adjacent normal tissues of Patient 17 [N17]

value: stomach

value: male

value: perineural invasion

tag: tumor stage

tag: age(years)


title: CRCHUG_030_tumor

value: TMA1 | 013 | tumor

value: tumor

tag: aoinucleicount

tag: roicoordinatex

tag: roicoordinatey

tag: type

value: Biopsy

value: Dead

value: Mid responders


title: smRNAseq_Flower_b11_WT_rep2

description: Library name: sample172


title: CRCHUG_030_stroma

value: TMA1 | 013 | stroma

tag: status

tag: code

value: Female


title: CTL_iPSC_d90_VPA_n_2

city: L'Hospitalet de Llobregat

first: Sandra

last: Acosta

address: Feixa Llarga

department: Pathology and Experimental Therapeutics

country: Spain

institute: University of Barcelona

data_processing:
Transcript alignment was performed on STAR software using the human reference GRCh38 and Annotation Release 109 from Ensembl. Counts were obtained using HTSeq software, and MultiQC software was employed for quality assessment.
Assembly: GRCh38
Supplementary files format and content: ImPheNet_counts1.csv: Comma separated values file with raw count matrices of CTL and DS samples at days 21, 35 and 90, and VPA treatment. Replicates 1 and 2.
Supplementary files format and content: ImPheNet_counts2.csv: Comma separated values file with raw count matrices of CTL and DS samples at days 35 and 90. Replicate 3.

molecule: polyA RNA

source_name: Brain organoid

extract_protocol:
Brain organoids were harvested and snap-frozen in liquid nitrogen and stored at -80ºC upon their processing. RNA extraction was performed as followed: mechanic homogeneization in 700 µl Qiazol (Qiagen), phase separation in 140 µl Chloroform (C2432, Merck) followed by 15 minutes centrigution at 12000rpm; precipitation in Isopropanol (19516, Merck) and rehydration in 70%, final dilution in 100 µl of ultraPure water. Next, a column purification with the RNeasy Miki Kit (Qiagen) was performed  following the supplier protocol. QC was validated in a Bioanalyzer.
RNAseq libraries were prepared using polyA capture and QC validated in Bioanalyzer. Libraries were pooled together, and sequenced on a NexSeq 500, using a 2x75 cycles High Output run, in addition to a 2x75 cycles Mid Output run.

value: Brain organoid

value: iPSC control (CW70019)

tag: treatment


title: CRCHUG_017_tumor

tag: tissue

value: TMA1 | 010 | tumor

value: TRG 3

tag: response

tag: gender


title: CTL_iPSC_d90_VPA_n_1

tag: genotype


title: adjacent normal tissues of Patient 15 [N15]


title: smRNAseq_Flower_b11_WT_rep1

description: Library name: sample171

value: flower bud (stage12 and younger)

tag: ecotype

value: Columbia

tag: batch

value: experiment batch11


title: CRCHUG_061_tumor

value: FFPE tissue

value: TMA1 | 025 | tumor

tag: slidename

tag: roilabel

tag: segmentlabel

value: Male


title: CRCHUG_037_stroma

value: TMA1 | 015 | stroma

value: stroma

tag: aoisurfacearea


title: smRNAseq_Ovules_b10_pUBQ10_3xFLAG-RIM12_col-0_line2_rep2

description: Library name: sample170

source_name: Unfertilized ovules (stage12)

value: Unfertilized ovules (stage12)

value: experiment batch10


title: adjacent normal tissues of Patient 13 [N13]

value: stomach

value: male

value: perineural invasion

tag: tumor stage

tag: age(years)


title: CRCHUG_019_stroma

value: TMA1 | 009 | stroma

tag: aoinucleicount

tag: roicoordinatex

tag: roicoordinatey

value: Biopsy

value: Dead

value: Mid responders

value: Female


title: CTL_iPSC_d90_n_2

value: Brain organoid

value: iPSC control (CW70019)


title: ID22v0, SCI, Acute

description: Library name: ID22v0

value: Whole blood

tag: group

value: SCI

value: Acute

tag: age

tag: Sex

tag: ais

value: D

tag: nli


title: adjacent normal tissues of Patient 11 [N11]

tag: tissue

tag: gender

tag: genotype


title: CRCHUG_019_tumor

value: FFPE tissue

value: TMA1 | 009 | tumor

tag: slidename

value: tumor

tag: type

tag: status

value: TRG 3

tag: code

tag: response


title: smRNAseq_Ovules_b10_pUBQ10_3xFLAG-RIM12_col-0_line2_rep1

description: Library name: sample169

value: Unfertilized ovules (stage12)

tag: ecotype

value: Columbia

tag: batch

value: experiment batch10


title: CTL_iPSC_d90_n_1

tag: treatment


title: smRNAseq_Ovules_b10_pUBQ10_3xFLAG-RIM12_col-0_line1_rep2

description: Library name: sample168


title: adjacent normal tissues of Patient 1 [N1]

value: stomach

value: male

value: non perineural invasion

tag: tumor stage

tag: age(years)


title: CTL_iPSC_d35_n_2


title: CRCHUG_028_stroma

value: TMA1 | 007 | stroma

tag: roilabel

tag: segmentlabel

value: stroma

tag: aoisurfacearea

tag: aoinucleicount

tag: roicoordinatex

tag: roicoordinatey

value: Biopsy

value: Dead

value: Mid responders

value: Female


title: adjacent normal tissues of Patient 14 [N14]

tag: tissue

tag: gender

tag: genotype

value: perineural invasion


title: CRCHUG_002_stroma

value: FFPE tissue

value: TMA1 | 002 | stroma

tag: slidename

tag: type

tag: status

value: Male


title: tumor tissue of Patient 4 [T4]

value: female


title: smRNAseq_Ovules_b10_3-396WT_rep2_rep2

description: Library name: sample166

value: Unfertilized ovules (stage12)

tag: ecotype


title: tumor tissue of Patient 7 [T7]


title: CTL_iPSC_d35_n_1

value: Brain organoid

value: iPSC control (CW70019)


title: smRNAseq_Ovules_b10_3-396_rep2

description: Library name: sample164

value: Columbia

tag: batch

value: experiment batch10


title: smRNAseq_Ovules_b10_pUBQ10_3xFLAG-RIM12_col-0_line1_rep1

description: Library name: sample167


title: DS_iPSC_d90_VPA_n_2

value: iPSC Dravet Syndrome (PFIZi014)

tag: treatment


title: DS_iPSC_d90_n_2


title: tumor tissue of Patient 40 [T40]

value: stomach

tag: tumor stage


title: CRCHUG_002_tumor

value: TMA1 | 002 | tumor

value: tumor

value: TRG 3

tag: code

tag: response


title: tumor tissue of Patient 30 [T30]

value: male

tag: age(years)


title: smRNAseq_Ovules_b10_3-396WT_rep1_rep1

description: Library name: sample165


title: DS_iPSC_d90_VPA_n_1


title: CRCHUG_064_stroma

value: TMA1 | 029 | stroma

tag: roilabel

tag: segmentlabel

value: stroma

tag: aoisurfacearea

value: Biopsy

value: Alive

value: TRG 2

value: Good responders

value: Female


title: smRNAseq_Ovules_b10_3-396_rep1

description: Library name: sample163

value: Unfertilized ovules (stage12)


title: CRCHUG_060_stroma

value: FFPE tissue

value: TMA1 | 024 | stroma

tag: aoinucleicount

tag: roicoordinatex

tag: roicoordinatey

value: Dead

value: TRG 4-5

value: Non responders

value: Male


title: DS_iPSC_d90_n_1

tag: tissue

value: Brain organoid

tag: genotype

value: iPSC Dravet Syndrome (PFIZi014)


title: DS_iPSC_d35_n_2


title: tumor tissue of Patient 29 [T29]

value: stomach

tag: gender

value: female

value: perineural invasion


title: smRNAseq_Ovules_b10_4-336WT_rep2

description: Library name: sample162

value: Columbia

value: experiment batch10


title: CRCHUG_028_tumor

value: TMA1 | 007 | tumor

tag: slidename

value: tumor

tag: type

value: Biopsy

tag: status

value: TRG 3

value: Mid responders


title: smRNAseq_Ovules_b10_4-336WT_rep1

description: Library name: sample161

value: Unfertilized ovules (stage12)

tag: ecotype

tag: batch


title: tumor tissue of Patient 28 [T28]

tag: tumor stage


title: CRCHUG_060_tumor

value: FFPE tissue

value: TMA1 | 024 | tumor

value: Dead

tag: code

tag: response


title: smRNAseq_Ovules_b10_4-336_rep2

description: Library name: sample160


title: tumor tissue of Patient 27 [T27]

value: male

tag: age(years)


title: CRCHUG_055_stroma

value: TMA1 | 022 | stroma

tag: roilabel

tag: segmentlabel

value: stroma

value: Alive

value: TRG 4

value: Non responders

value: Male


title: CTL_iPSC_d21_n_2

value: Brain organoid

value: iPSC control (CW70019)

tag: treatment


title: CRCHUG_055_tumor

value: TMA1 | 022 | tumor

value: tumor

tag: aoisurfacearea

tag: aoinucleicount

tag: roicoordinatex


title: smRNAseq_Ovules_b10_4-336_rep1

description: Library name: sample159

tag: tissue

value: Columbia

tag: genotype

value: experiment batch10


title: tumor tissue of Patient 26 [T26]

value: stomach

tag: gender

value: female

value: non perineural invasion


title: CRCHUG_044_stroma

value: FFPE tissue

value: TMA1 | 021 | stroma

tag: slidename

tag: roicoordinatey

tag: type

value: Biopsy


title: CRCHUG_064_tumor

value: TMA1 | 029 | tumor

tag: status

value: TRG 2

value: Good responders

value: Female


title: tumor tissue of Patient 3 [T3]

value: perineural invasion

tag: tumor stage


title: CRCHUG_044_tumor

value: TMA1 | 021 | tumor

value: Alive

value: TRG 4

tag: code

value: Non responders

value: Male


title: smRNAseq_Ovules_b10_pol-iv

description: Library name: sample158

value: Unfertilized ovules (stage12)

tag: ecotype

value: Columbia

tag: batch

value: experiment batch10


title: smRNAseq_Ovules_b10_clsy3

description: Library name: sample156


title: smRNAseq_Ovules_b10_clsy3,4

description: Library name: sample157


title: tumor tissue of Patient 21 [T21]

value: stomach

value: male

tag: age(years)


title: tumor tissue of Patient 25 [T25]


title: CTL_iPSC_d35_n_3

value: Brain organoid

value: iPSC control (CW70019)


title: CTL_iPSC_d90_n_3


title: smRNAseq_Ovules_b10_WT_rep3

description: Library name: sample155


title: DS_iPSC_d35_n_3

value: iPSC Dravet Syndrome (PFIZi014)


title: tumor tissue of Patient 23 [T23]


title: CRCHUG_045_tumor

value: FFPE tissue

value: TMA1 | 020 | tumor

tag: roilabel

tag: segmentlabel

value: tumor

value: Biopsy

value: TRG 4-5

tag: response


title: tumor tissue of Patient 20 [T20]

value: perineural invasion


title: DS_iPSC_d35_n_1

tag: treatment


title: DS_iPSC_d90_n_3


title: smRNAseq_Ovules_b10_WT_rep2

description: Library name: sample154


title: CRCHUG_047_stroma

value: TMA1 | 019 | stroma

value: stroma

tag: aoisurfacearea

tag: aoinucleicount

tag: roicoordinatex

value: Alive

value: TRG 1

value: Good responders


title: tumor tissue of Patient 2 [T2]

tag: tissue

tag: gender

tag: genotype


title: CTL_iPSC_d21_n_1


title: smRNAseq_Ovules_b10_WT_rep1

description: Library name: sample153

value: Unfertilized ovules (stage12)

value: Columbia

value: experiment batch10


title: NHA_Ribo-depleted_RNA-seq

city: Kensington

first: Irina

last: Voineagu

state: NSW

address: High St

department: Biotechnology and Biomolecular Sciences

country: Australia

institute: The University of New South Wales

data_processing:
For the PolyA+ RNA-seq library, reads were mapped to hg38 using STAR v2.5.2b (Dobin et al. 2013) with the following parameters: --outSJfilterOverhangMin 15 15 15 15 --alignSJoverhangMin 15 --alignSJDBoverhangMin 15 --outFilterMultimapNmax 1 --outFilterScoreMin 1 --outFilterMatchNmin 1 --outFilterMismatchNmax 2  --chimSegmentMin 15 --chimScoreMin 15 --chimScoreSeparation 10 --chimJunctionOverhangMin 15. GENCODE v32 (Frankish et al. 2019) was used for gene coordinates.
For ribo-depleted RNA-seq and TT-seq, FASTQs were processed using the nf-core RNA-seq pipeline (https://nf-co.re/rnaseq/3.13.2) (Ewels et al. 2020). Reads were mapped to hg38 using the STAR aligner (2.7.10a) (Dobin et al. 2013), where genomic origin was identified (STable 11). Candidate peak expression was determined using FeatureCounts (Dobin et al. 2013) implemented in the Rsubread package (Liao et al. 2019). The following parameters were set: useMetaFeatures = FALSE, allowMultiOverlap = TRUE, isPairedEnd = TRUE, strandSpecific = 2, minMQS = 10, checkFragLength = FALSE, countChimericFragments = FALSE. Candidate size was set to a standard length of by adjusting start and end coordinates to be ±500bp centred around the midpoint.
Assembly: hg38
Supplementary files format and content: PolyA.csv - comma separated values with ENSEMBL Gene ID, gene symbol, counts and CPM for the mRNA-seq sample
Supplementary files format and content: TTseq_RiboDep.csv - comma separated values with enhancer ID from our CRISPRi screen, chromosome, enhancer start and end locations (+-500bp from enhancer midpoint), and postive strand, negative strand and unstranded counts for TT-seq and ribo-depleted RNA-seq

source_name: Normal Human Astrocytes (Lonza, #CC-2565)

growth_protocol: NHA were grown in DMEM media supplemented with 10% foetal bovine serum and 1% streptomycin/penicillin at 37°C, 5% CO2

extract_protocol:
RNA was extracted using the Qiagen miRNeasy kit, according to manufacturer's instructions
For TT-seq samples, cells were treated with 4sU for 5 mins before RNA extraction as above
For TT-seq samples, RNA was sonicated to ~1.5kb (Schwalb et al. 2016), then 4sU-labeled RNA was biotinylated (Duffy et al 2016) before isolation using streptavidin conjugated magnetic beads (Rädle et al. 2013)
For mRNA-seq samples, libraries were prepared using the Illumina TruSeq Stranded mRNA Prep Kit, according to manufacturer's instructions
For ribo-depleted RNA-seq samples, libraries were prepared using the Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus, according to manufacturer's instructions

treatment_protocol: NHAs were transduced with lenti-dCas9-KRAB and selected using 10 µg/mL blasticidine S hydrochloride

tag: cell line


title: tumor tissue of Patient 17 [T17]

value: stomach

value: male


title: tumor tissue of Patient 15 [T15]


title: smRNAseq_Anthers_b9_pUBQ10_3xFLAG-RIM12_col-0_line1_rep2

description: Library name: sample152

source_name: anthers (stage9)

value: anthers (stage9)

value: experiment batch9


title: CRCHUG_033_stroma

value: FFPE tissue

value: TMA1 | 014 | stroma

tag: slidename

tag: roicoordinatey

tag: type

tag: status

value: Dead

value: TRG 2

value: Female


title: CRCHUG_033_tumor

value: TMA1 | 014 | tumor

value: tumor

value: Biopsy

tag: code


title: tumor tissue of Patient 14 [T14]

value: perineural invasion

tag: tumor stage

tag: age(years)


title: H7 human embryonic stem cells,Replicate 2,S6K1-depleted,Week 14

description:
10x Genomics
sce.all_int_0.35.rds

library_source: transcriptomic single cell

data_processing:
Cells produced by the Chromium Next GEM Single Cell 3ʹ v3.1 kit from 10X Genomics were aligned to the human GRCh38 transcriptome using the 10x Genomics Cell Ranger 3.1.0, and all procedures were guided by 10x Genomics protocols with default parameters. The analysis workflow including quality control, normalization, feature selection, integration, scaling, dimensional reduction, unsupervised clustering, and differential expression, was performed by Seurat v 4.0.6 on the RStudio, and the pseudotime trajectory analysis was conducted via the Monocle3 R package.
Samples were filtered by the certain thresholds (500 < nFeature_RNA < 7,000 and 500 < nCount_RNA, 5%<ribosomal reads, and mitochondrial reads <20%). Normalization and 3000 variable features identification for each dataset respectively via the “vst” method, followed by different integration for different analyses.
As for specific time point analysis, samples from the same time point were integrated. Uniform Manifold Approximation and Projection (UMAP) dimensional reduction was then computed using the selected significant Principal components (PCs) (20 for each time point) after scaling (percentage of mitochondrial and ribosomal were treated as variables to regress out). Clustering of the cells was performed via FindNeighbors and FindClusters to choose the better resolution (0.2 for the 5th week and 0.3 for the 14th week, respectively). A differentially expressed genes test was conducted by the FindAllMarkers function with the default parameters, except that the parameter “only.pos” is true.
In terms of pseudotime trajectory analysis, samples across biological conditions and time points were integrated. Scaling, dimensional reduction, and differential gene expression were the same as those mentioned above, except that the resolution of unsupervised clustering is 0.3. The cells clustered by UMAP were separated via biological conditions, then were used to infer the cell differentiation trajectories by as.cell_data_set, cluster_cells with these parameters (reduction_method= “UMAP”, k=20) and learn_graph command imported from Monocle3 R package.
Assembly: hg38
Supplementary files format and content: rds files of different time points

source_name: dorsal forebrain organoids

growth_protocol: To generate dorsal forebrain organoids, H7 cells were dissociated with Accutase (Gibco) and reaggregated in ultra-low-cell adhesion 96 well V-bottom plates (10,000 cells per well) with cortical differentiation medium (CDM) I (Glasgow-MEM containing 20% KSR, 0.1 mM MEM-NEAA, 1mM sodium pyruvate, 0.1 mM β-mercaptoethanol, and penicillin/streptomycin) supplemented with 20 μM Y-27632 (day 0-6), 3 μM IWR-1 (day 0-18) and 5 μM SB431542 (day 0-18). Media was changed every 3 days until day 18. From day 18 to day 34, cell aggregates were moved to 60 mm ultra-low-attachment culture dishes on the orbital shaker (70 rpm) in CDM II (DMEM/F12 containing 2 mM Glutamax, 1% N2, 1% CD lipid concentrate, 0.25 μg/ml fungizone, and penicillin/streptomycin). From day 35 to day 69, organoids were incubated with CDM III (CDM II with 10% FBS, 5 μg/ml Heparin, and 1% Matrigel). From day 70, organoids were incubated CDM IV (CDM III with B27 and 2% Matrigel). CDM II, CDM III, and CDM IV were changed every 2 days.

extract_protocol:
For the single cell RNA sequencing, organoids were dissociated with Papain dissociation kit (Worthington Biochemical Corporation, #LK003150). (PN-120263;120212).
The single cell droplets were barcoded using Chromium Next GEM Single Cell 3’ Kit v3.1 (10x Genomics, cat# 1000121) by subjecting the cells to Chromium Single Cell Controller. Then, cDNA with barcodes was synthesized through reverse-transcription in the droplet and amplified using Chromium Next GEM Single Cell 3ʹ v3.1 kit .

tag: cell type

value: human embryonic stem cells (ESCs)

value: dorsal forebrain organoids

tag: age

value: Week 14


title: NHA_TT-seq

value: Normal Human Astrocytes (Lonza, #CC-2565)

tag: treatment


title: CRCHUG_045_stroma

value: TMA1 | 020 | stroma

tag: roilabel

tag: segmentlabel

value: stroma

tag: aoisurfacearea

value: Alive

value: TRG 4-5

tag: response

value: Non responders


title: smRNAseq_Anthers_b9_pol-iv

description: Library name: sample149

tag: tissue

value: anthers (stage9)

tag: ecotype

value: Columbia

tag: batch

value: experiment batch9


title: tumor tissue of Patient 13 [T13]

value: stomach

tag: gender

value: male

tag: genotype


title: H7 human embryonic stem cells,Replicate 1,S6K1-depleted,Week 14

description:
10x Genomics
sce.all_int_0.35.rds


title: CRCHUG_016_tumor

value: FFPE tissue

value: TMA1 | 011 | tumor

tag: slidename

value: tumor

tag: aoinucleicount

tag: roicoordinatex

tag: roicoordinatey

tag: type

value: Dead

value: TRG 2

value: Good responders

value: Female


title: CRCHUG_016_stroma

value: TMA1 | 011 | stroma

value: Biopsy

tag: status

tag: code


title: tumor tissue of Patient 11 [T11]

value: perineural invasion

tag: tumor stage

tag: age(years)


title: tumor tissue of Patient 1 [T1]

value: non perineural invasion


title: smRNAseq_Anthers_b9_pUBQ10_3xFLAG-RIM12_col-0_line1_rep1

description: Library name: sample151


title: smRNAseq_Anthers_b9_clsy3,4

description: Library name: sample148


title: H7 human embryonic stem cells,Replicate 2, wild-type,Week 14

description:
10x Genomics
sce.all_int_0.35.rds

tag: cell type

value: human embryonic stem cells (ESCs)

value: dorsal forebrain organoids

value: WT

tag: age

value: Week 14


title: CRCHUG_015_stroma

value: TMA1 | 006 | stroma

tag: roilabel

tag: segmentlabel

value: stroma

tag: aoisurfacearea

tag: response

value: Male


title: smRNAseq_Anthers_b9_clsy1,2

description: Library name: sample150

tag: tissue

value: anthers (stage9)

tag: ecotype

value: Columbia

tag: batch

value: experiment batch9


title: CRCHUG_047_tumor

value: FFPE tissue

value: TMA1 | 019 | tumor

tag: slidename

value: tumor

tag: aoinucleicount

value: Alive

value: TRG 1

tag: gender


title: H7 human embryonic stem cells,Replicate 2,S6K1-depleted,Week 5

description:
10x Genomics
sce.all_int_0.2.rds

tag: genotype

value: Week 5


title: Ss72h

city: Shanghai

first: Wenfang

last: Wang

address: 100 Guilin Road

institute: Shanghai Normal University

data_processing:
Illumina NovaSeq 6000 User Guide
Sequence reads were trimmed using fastp software (parameter-Quality limit:0.01)
Bowtie2 software for alignment
Assembly: GCF_014490055.1
Supplementary files format and content: tab-delimited xls file includes raw counts for each sample
Supplementary files format and content: tab-delimited xls file includes RPKM values for each sample

organism: Saccharopolyspora spinosa

source_name: Bacteria

growth_protocol: S. spinosa was grown on 2CMC agar medium at 30℃.

extract_protocol:
RNA samples were extracted using RNeasy Mini Kit (Qiagen, Germany) according to the procedure manual
The RNA library preparation and sequencing were performed at the Shanghai Sinotech Genomics Technology Co., Ltd. according Illumina NovaSeq 6000 user guide

treatment_protocol: stationary culture

value: Bacteria

tag: cell line

value: mycelium

tag: treatment

value: No treated


title: smRNAseq_Anthers_b9_clsy3

description: Library name: sample147


title: CRCHUG_015_tumor

value: TMA1 | 006 | tumor

tag: roicoordinatex

tag: roicoordinatey

tag: type

value: Biopsy

tag: status

value: Dead

value: TRG 2

tag: code

value: Good responders


title: H7 human embryonic stem cells,Replicate 1,S6K1-depleted,Week 5

description:
10x Genomics
sce.all_int_0.2.rds

tag: cell type

value: human embryonic stem cells (ESCs)

value: dorsal forebrain organoids

tag: age


title: smRNAseq_Anthers_b9_WT_rep3

description: Library name: sample146

value: anthers (stage9)

value: Columbia

value: experiment batch9


title: CRCHUG_008_stroma

value: FFPE tissue

value: TMA1 | 003 | stroma

tag: roilabel

tag: segmentlabel

value: stroma

tag: aoisurfacearea

value: TRG 4

tag: response

value: Non responders

value: Male


title: Ss36h

tag: tissue

value: Bacteria

value: mycelium

value: WT

value: No treated


title: H7 human embryonic stem cells,Replicate 2, wild-type,Week 5

description:
10x Genomics
sce.all_int_0.2.rds

value: Week 5


title: smRNAseq_Anthers_b9_WT_rep2

description: Library name: sample145

tag: ecotype

tag: batch


title: CRCHUG_008_tumor

value: TMA1 | 003 | tumor

tag: slidename

value: tumor

tag: aoinucleicount

value: Biopsy

value: Dead

tag: gender


title: H7 human embryonic stem cells,Replicate 1, wild-type,Week 14

description:
10x Genomics
sce.all_int_0.35.rds

value: human embryonic stem cells (ESCs)

value: dorsal forebrain organoids

tag: genotype

value: Week 14


title: H7 human embryonic stem cells,Replicate 1, wild-type,Week 5

description:
10x Genomics
sce.all_int_0.2.rds


title: smRNAseq_Anthers_b9_WT_rep1

description: Library name: sample144

value: anthers (stage9)

value: Columbia

value: experiment batch9


title: CFP male flea 2

status: Public on Sep 24 2025

city: Seville

first: Angela

middle: M.

last: García Sánchez

address: Profesor García González, 2

institute: Universidad de Sevilla

description: Library name: Sample CFP.m.2

data_processing:
Paired-end reads were merged using FLASH V1.2.7
Quality filtering on the raw tags was performed according to QIIME V1.9
Chimeric merged reads were detected and removed using UCHIME
Multiple sequence alignment was conducted using MUSCLE V 3.8.31
Assembly: NCBI genome assembly ASM342690v1
Supplementary files format and content: tab-delimited text file includes raw counts for each sample

organism: Ctenocephalides felis

extract_protocol:
Total DNA was extracted from fleas using flea abdomen by the DNeasy Blood  and Tissue Kit (Qiagen, Valencia, CA, U.S.A.) in accordance with the manufacturer’s instructions
Sequencing libraries were generated using TruSeq DNA PCR- Free Sample Preparation Kit (Illumina, USA) following manufacturer's recommendations

tag: host

value: Dog

tag: cell line


title: CFP male flea 1

description: Library name: Sample CFP.m.1

value: Male


title: CFP female flea 14

description: Library name: Sample CFP.f.14

value: Female


title: CFP female flea 15

description: Library name: Sample CFP.f.15


title: CFP female flea 13

description: Library name: Sample CFP.f.13


title: CFP female flea 11

description: Library name: Sample CFP.f.11


title: CFP female flea 16

description: Library name: Sample CFP.f.16


title: CFP female flea 12

description: Library name: Sample CFP.f.12


title: CFP female flea 10

description: Library name: Sample CFP.f.10


title: CFP female flea 7

description: Library name: Sample CFP.f.7


title: CFP female flea 8

description: Library name: Sample CFP.f.8


title: CFP female flea 9

description: Library name: Sample CFP.f.9


title: CFP female flea 6

description: Library name: Sample CFP.f.6


title: CFP female flea 3

description: Library name: Sample CFP.f.3


title: CFP female flea 2

description: Library name: Sample CFP.f.2


title: CFP female flea 5

description: Library name: Sample CFP.f.5


title: CFP female flea 4

description: Library name: Sample CFP.f.4


title: Ss48h

value: Bacteria

value: mycelium

tag: treatment


title: CFG male flea 4

description: Library name: Sample CFG.m.4

value: Cat


title: Ss24h

value: WT

value: No treated


title: CFG male flea 3

description: Library name: Sample CFG.m.3


title: CFG male flea 1

description: Library name: Sample CFG.m.1


title: CFG female flea 12

description: Library name: Sample CFG.f.12


title: CFG female flea 11

description: Library name: Sample CFG.f.11


title: CFG female flea 10

description: Library name: Sample CFG.f.10


title: CFG female flea 8

description: Library name: Sample CFG.f.8


title: CFG female flea 7

description: Library name: Sample CFG.f.7


title: CFG female flea 6

description: Library name: Sample CFG.f.6


title: CFG female flea 9

description: Library name: Sample CFG.f.9


title: CFG female flea 4

description: Library name: Sample CFG.f.4


title: CFG female flea 3

description: Library name: Sample CFG.f.3


title: CFG female flea 5

description: Library name: Sample CFG.f.5


title: CFG female flea 1

description: Library name: Sample CFG.f.1


title: CFG female flea 2

description: Library name: Sample CFG.f.2


title: CCP male flea 1

description: Library name: Sample CCP.m.1

organism: Ctenocephalides canis


title: CCP male flea 2

description: Library name: Sample CCP.m.2


title: AEP male flea 1

description: Library name: Sample AEP.m.1

organism: Archaeopsylla erinacei


title: AEP female flea 3

description: Library name: Sample AEP.f.3


title: AEP female flea 1

description: Library name: Sample AEP.f.1


title: CFP female flea 1

description: Library name: Sample CFP.f.1


title: AEE male flea 3

description: Library name: Sample AEE.m.3

value: Hedgehog


title: AEE male flea 2

description: Library name: Sample AEE.m.2


title: CFG male flea 2

description: Library name: Sample CFG.m.2


title: AEE female flea 2

description: Library name: Sample AEE.f.2


title: AEE female flea 1

description: Library name: Sample AEE.f.1


title: Cut&Tag-seq H3K4me1 in D6

city: hefei

first: wenxian

last: wang

address: No. 81, Meishan Road, Shushan District, Hefei City, Anhui Province

institute: Anhui Medical University

description: Library name: Sample18

data_processing:
Fastp was conducted for trimming raw fastq files to remove adaptors and low-quality bases.
The trimmed fastq files were then mapped to the mm10 reference genome utilizing Bowtie2.
Then uniquely mapped reads were obtained using SAMtools.
Picard was used to remove duplicates and For visualization, deepTools was utilized with the “bamCoverage” function to generate normalized RPM bigWig files. Then MACS2 was utilized for peak calling.
Assembly: mm10
Supplementary files format and content: Bigwig files for Cut&Run-seq and Cut&Tag-seq.
Library strategy: CUT&Tag

source_name: Differentiated motor neuron

growth_protocol: C57BL/6 mouse embryonic stem cells (mESCs) were cultured on a monolayer of Mitomycin-C–treated mouse embryonic fibroblasts in Knockout DMEM medium supplemented with 10% fetal bovine serum, 5% Knockout serum replacement , 100 U/ml penicillin-streptomycin solution, 2 mM L-glutamax, 1X MEM non-essential amino acid solution, 0.1 mM β-mercaptoethanol and 1000 U/ml leukemia inhibitory factor.

extract_protocol:
Cells were harvested at indicated stages for Cut&Run assay using Hyperactive pG-MNase Cut&Run Assay Kit for Illumina (Vazyme HD102), for Cut&Tag assay using Hyperactive Universal Cut&Tag Assay Kit for Illumina Pro (Vazyme TD904) according to manufacture's protocols.
Regarding Cut&Run-seq,  VAHTS multiplex Oligos Set 5 (Vazyme N322) was used for library construction. As for Cut&Tag-seq, Trueprep Index Kit V2 for Illumina (Vazyme TD202) was employed for library construction. For RNA-seq, 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. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Prime and lastly PCR products were purified.

treatment_protocol: mESCs were dissociated with 0.25% trypsin into single cell suspension at 2.5x104 cells/ml in ADFNK medium (Advanced DMEM/F12:Neurobasal (1:1) medium, 10% Knockout Serum Replacement, 100 U/ml penicillin-streptomycin solution, 2 mM L-glutamax, 1X MEM non-essential amino acid solution and 0.1 mM β-mercaptoethanol). Then 20μl of cell mixture (500 cells) was seeded onto the cover of petri dish to initiate formation of embryoid bodies (EBs) (D0: day 0). Two days later (D2: day 2), EBs were harvested and plated into differentiation medium (ADFNK medium supplemented with 1 μM all-trans retinoic acid (RA, Sigma) and 0.5μM Sonic Hedgehog Agonist (SAG, Selleck) for caudalization and ventralization. Culture Medium was then changed at D3 (day 3).  For directed hypaxial motor column (HMC) differentiation, 5 μM DAPT was added to the differentiation medium at D4 (day 4) and ADFNK medium at D5 (day 5).

value: Differentiated motor neuron

tag: differentiation stage

tag: antibody


title: Cut&Tag-seq H3K4me3 in D6

description: Library name: Sample22

tag: cell type

value: H3K4me3 (Abcam, #ab8580)


title: Cut&Tag-seq H3K4me3 in D5

description: Library name: Sample21


title: Cut&Tag-seq H3K4me3 in D4H8

description: Library name: Sample20


title: Cut&Tag-seq H3K4me3 in D4

description: Library name: Sample19


title: Cut&Tag-seq H3K4me1 in D4H8

description: Library name: Sample16


title: Cut&Tag-seq H3K4me1 in D4

description: Library name: Sample15


title: Cut&Tag-seq H3K4me1 in D5

description: Library name: Sample17


title: Cut&Tag-seq H3K27ac in D6

description: Library name: Sample14

value: H3K27ac (Abcam, #ab4729)


title: Cut&Tag-seq H3K27ac in D4

description: Library name: Sample11


title: Cut&Tag-seq H3K27me3 in D6

description: Library name: Sample10


title: Cut&Tag-seq H3K27me3 in D4H8

description: Library name: Sample8


title: Cut&Tag-seq H3K27me3 in D5

description: Library name: Sample9


title: Cut&Tag-seq H3K27ac in D4H8

description: Library name: Sample12


title: Cut&Run-seq Neurod1 in D5

description: Library name: Sample5

data_processing:
Fastp was conducted for trimming raw fastq files to remove adaptors and low-quality bases.
The trimmed fastq files were then mapped to the mm10 reference genome utilizing Bowtie2.
Then uniquely mapped reads were obtained using SAMtools.
Picard was used to remove duplicates and For visualization, deepTools was utilized with the “bamCoverage” function to generate normalized RPM bigWig files. Then MACS2 was utilized for peak calling.
Assembly: mm10
Supplementary files format and content: Bigwig files for Cut&Run-seq and Cut&Tag-seq.
Library strategy: CUT&Run

value: Neurod1 (Proteintech, #12081-1-AP)


title: Cut&Run-seq Kdm6b in D6

description: Library name: Sample4

value: Kdm6b (this study)


title: Cut&Run-seq Kdm6b in D5

description: Library name: Sample3


title: AsPC-1 oxaliplatin-treated

type: RNA

city: Heidelberg

first: Dirk

last: Theile

address: Im Neuenheimer Feld 410

institute: Heidelberg University

description: test

data_processing:
Transript level changes of the target genes were displayed as fold change compared to the controls by calculating relative quantities corresponding to the deltadelta Ct method. Gene expression changes are shown as log2-fold changes.
Data processing was performed using GenEx. For normalization, up to five reference genes (ACTB, B2M, GAPDH, GUSB, HPRT1) were evaluated. The optimal set was determined with geNorm and NormFinder: geNorm ranks genes by pairwise stability, while NormFinder uses variance analysis to account for sample treatments and exclude unstable genes. The most suitable reference genes identified by both programs were then used for normalization.

source_name: PDAC cell line

growth_protocol: Cell lines were grown in RPMI-1640, supplemented with 10 % FBS, 100 U/ml penicillin, 100 µg/ml streptomycine sulfate.

extract_protocol: To extract total RNA, NucleoSpin® RNA Plus Kit from Machery-Nagel (Düren, Germany) was used. 1 µg of total RNA was reverse transcribed into complementary DNA (cDNA) with the qScript™ cDNA Synthesis Kit (Quanta, Beverly, USA).

treatment_protocol: AsPC-1 and BxPC-3 cells were treated for 4 h with oxaliplatin (AsPC-1 176 µM; BxPC-3 150 µM) or cisplatin (AsPC-1 150 µM; BxPC-3 65 µM) leading to similar intracellular platinum concentrations (662 pg Pt/µg protein), or left untreated (controls).

value: oxaliplatin 150 µM

tag: treatment time

value: 4 h


title: AsPC-1 cisplatin-treated

description: test

value: cisplatin 65 µM


title: AEP female flea 2

description: Library name: Sample AEP.f.2

value: Dog


title: Cut&Tag-seq H3K27ac in D5

description: Library name: Sample13


title: AEE male flea 1

description: Library name: Sample AEE.m.1

value: Male


title: AsPC-1 untreated

description: control

value: untreated control

value: 0 h


title: Cut&Run-seq Kdm6b in D4

description: Library name: Sample1


title: Cut&Run-seq Kdm6b in D4H8

description: Library name: Sample2


title: Cut&Tag-seq H3K27me3 in D4

description: Library name: Sample7


title: BxPC-3  cisplatin-treated

description: test

value: cisplatin 150 µM


title: RNA-seq in D6 GSKJ4 biol rep 3

description:
Library name: Sample43
Column name in allsamples_raw_counts.xls: D6_GSKJ4_biol rep 3

data_processing:
RNA-Seq data were trimmed by Fastp to remove adapters, low-quality bases (Q < 20), and reads shorter than 75 bp.
Trimmed data were aligned to the mm10 reference genome using Hisat2. The aligned reads were sorted and indexed using SAMtools.
HTSeq-count was used to generate count matrices for each sample. We used the DESeq2 in R package to analyze gene expression with count matrices as input.
Assembly: mm10
Supplementary files format and content: Excel files including raw counts and FPKM values

extract_protocol:
For RNA-seq, total RNA was extracted in  TRIzol and mRNA was purified for library construction using poly-T oligo-attached magnetic beads.
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. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Prime and lastly PCR products were purified.

treatment_protocol: mESCs were dissociated with 0.25% trypsin into single cell suspension at 2.5x104 cells/ml in ADFNK medium (Advanced DMEM/F12:Neurobasal (1:1) medium, 10% Knockout Serum Replacement, 100 U/ml penicillin-streptomycin solution, 2 mM L-glutamax, 1X MEM non-essential amino acid solution and 0.1 mM β-mercaptoethanol). Then 20μl of cell mixture (500 cells) was seeded onto the cover of petri dish to initiate formation of embryoid bodies (EBs) (D0: day 0). Two days later (D2: day 2), EBs were harvested and plated into differentiation medium (ADFNK medium supplemented with 1 μM all-trans retinoic acid (RA, Sigma) and 0.5μM Sonic Hedgehog Agonist (SAG, Selleck) for caudalization and ventralization. Culture Medium was then changed at D3 (day 3).  For directed hypaxial motor column (HMC) differentiation, 5 μM DAPT was added to the differentiation medium at D4 (day 4) and ADFNK medium at D5 (day 5). GSKJ4 was treated one day earlier to inhibit endogenous Kdm6b enzyme activity (i.e. D3H8D4H8, D4D5, D5D6).

tag: differentiation stage_and_treatment


title: Cut&Run-seq Cux1/2 in D6

description: Library name: Sample6


title: RNA-seq in D6 GSKJ4 biol rep 2

description:
Library name: Sample42
Column name in allsamples_raw_counts.xls: D6_GSKJ4_biol rep 2


title: BxPC-3  oxaliplatin-treated

description: test

value: oxaliplatin 176 µM


title: RNA-seq in D6 GSKJ4 biol rep 1

description:
Library name: Sample41
Column name in allsamples_raw_counts.xls: D6_GSKJ4_biol rep 1

value: WT


title: BxPC-3 untreated

description: control


title: RNA-seq in D6 biol rep 3

description:
Library name: Sample40
Column name in allsamples_raw_counts.xls: D6_biol rep 3


title: RNA-seq in D6 biol rep 2

description:
Library name: Sample39
Column name in allsamples_raw_counts.xls: D6_biol rep 2


title: RNA-seq in D5 GSKJ4 biol rep 3

description:
Library name: Sample37
Column name in allsamples_raw_counts.xls: D5_GSKJ4_biol rep 3


title: RNA-seq in D5 GSKJ4 biol rep 2

description:
Library name: Sample36
Column name in allsamples_raw_counts.xls: D5_GSKJ4_biol rep 2


title: RNA-seq in D5 GSKJ4 biol rep 1

description:
Library name: Sample35
Column name in allsamples_raw_counts.xls: D5_GSKJ4_biol rep 1


title: wt-like, Sppl3 KO, rep3

first: Nathan

middle: E.

last: Lewis

address: 9500 Gilman Dr. IBRFII, 5220E

department: Bioengineering; Pediatrics

institute: University of California, San Diego

description:
Library name: WT_R3
Clone1_R3

data_processing:
The samples were quality controlled using FastQC and the adapter sequences trimmed using Trimmomatic
The sequences were demultiplexd with bcl2fastq v2.20 Conversion Software.
Reads were mapped to the CHO GCF_003668045.1_CriGri-PICRR genome, aligned and quantified with Kallisto.
Assembly: CHO GCF_003668045.1_CriGri-PICRR
Supplementary files format and content: csv files includes raw counts for sample
Supplementary files format and content: cvs files include TPM values for each sample

organism: Cricetulus griseus

source_name: CHO-S

growth_protocol: The geCHO cells were grown in 125 ml Erlenmeyer shaker flasks with baffled bottom (Fisher Scientific, USA) at 130 rpm in 37°C at 5% CO2 with maintenance media containing CD CHO Medium (Gibco, USA) supplemented with 8 mM L-Glutamine (Gibco) and 1x anti-clumping agent (Gibco). The geCHO cells expressing E1E2 and Furin were grown in media containing 1 mg/ml Geneticin Selective Antibiotic (G418 sulfate; Gibco) and 0.75 mg/ml Hygromycin B (Gibco) for selection.

extract_protocol:
RNA was harvested and extracted using the RNAeasy Kit (Qiagen)
RNA quality was analyzed using the Agilent Tapestation 4200, and only samples with an RNA Integrity Number (RIN) exceeding 8.0 were selected for library construction. Libraries were prepared using the Illumina Stranded mRNA Sample Prep Kit with Illumina RNA UD Indexes (Illumina, San Diego, CA), following the manufacturer's guidelines.

treatment_protocol: The geCHO cells were initially transfected with the furin plasmid and cloned. The cloned furin cell lines were then transfected with sE1E2.SZ-H445P plasmid and cloned again.

value: CHO-S

value: Chinese Hamster Ovaries

value: Sppl3 KO


title: wt-like, Sppl3 KO, rep2

description:
Library name: WT_R2
Clone1_R2


title: wt-like, Sppl3 KO, rep1

description:
Library name: WT_R1
Clone1_R1


title: wt-like, Sppl3 KO, E1E2, Furin,clone1_3, rep3

description:
Library name: WT_3_R3
Clone1_3_R3

value: Sppl3 KO, E1E2, Furin


title: wt-like, Sppl3 KO, E1E2, Furin,clone1_3, rep2

description:
Library name: WT_3_R2
Clone1_3_R2


title: wt-like, Sppl3 KO, E1E2, Furin,clone1_3, rep1

description:
Library name: WT_3_R1
Clone1_3_R1


title: wt-like, Sppl3 KO, E1E2, Furin,clone1_1, rep3

description:
Library name: WT_1_R3
Clone1_1_R3


title: RNA-seq in D5 biol rep 2

description:
Library name: Sample33
Column name in allsamples_raw_counts.xls: D5_biol rep 2

value: Differentiated motor neuron


title: RNA-seq in D4H8 GSKJ4 biol rep 3

description:
Library name: Sample31
Column name in allsamples_raw_counts.xls: D4H8_GSKJ4_biol rep 3


title: RNA-seq in D5 biol rep 1

description:
Library name: Sample32
Column name in allsamples_raw_counts.xls: D5_biol rep 1


title: RNA-seq in D5 biol rep 3

description:
Library name: Sample34
Column name in allsamples_raw_counts.xls: D5_biol rep 3


title: wt-like, Sppl3 KO, E1E2, Furin,clone1_1, rep1

description:
Library name: WT_1_R1
Clone1_1_R1


title: RNA-seq in D4H8 GSKJ4 biol rep 2

description:
Library name: Sample30
Column name in allsamples_raw_counts.xls: D4H8_GSKJ4_biol rep 2


title: RNA-seq in D4H8 GSKJ4 biol rep 1

description:
Library name: Sample29
Column name in allsamples_raw_counts.xls: D4H8_GSKJ4_biol rep 1


title: Cell-line 1, Sppl3/Mgat2/St3gal3/4/6/B3gnt2 KO, cell-line1, rep2

description:
Library name: CL1_R2
B12_R2

value: Sppl3/Mgat2/St3gal3/4/6/B3gnt2 KO


title: wt-like, Sppl3 KO, E1E2, Furin,clone1_1, rep2

description:
Library name: WT_1_R2
Clone1_1_R2


title: Cell-line 1, Sppl3/Mgat2/St3gal3/4/6/B3gnt2 KO, cell-line1, rep3

description:
Library name: CL1_R3
B12_R3


title: RNA-seq in D4H8 biol rep 3

description:
Library name: Sample28
Column name in allsamples_raw_counts.xls: D4H8_biol rep 3


title: RNA-seq in D4H8 biol rep 2

description:
Library name: Sample27
Column name in allsamples_raw_counts.xls: D4H8_biol rep 2


title: sample liver from HF diet mice vs. MET diet mice

city: guadalajara

first: angel

middle: omar

last: vazquez

address: SIERRA MOJADA 950

country: Mexico

institute: IBMMTG

hyb_protocol: Samples were hybridized on an M. musculus microarray following the manufacturer's protocol.

scan_protocol: Fluorescence detection was performed using a confocal microarray scanner. The microarrays were scanned using [Agilent G2565CA Microarray Scanner] at a resolution of [5 µm].

data_processing:
Image quantification was analyzed with the genArise microarray analysis tool version 2.0
Basically, the background value is subtracted from the signal value, the channel where the control sample is located is set, and the values obtained for the experimental sample are normalized (for this normalization, complex functions such as Loess and/or Lowess are used (Quackenbush 2002)). Once the values are normalized, a benchmark test must be used to identify those genes that deviate from the normalization. A simple and functional test is the Z-score (Cheadle et al. 2003), which uses the standard deviation of the signal intensity calculated for each individual in relation to that of their neighbors.
zi = (Ri – mean(R)) / sd(R)
Where zi is the Z-score for each individual, Ri is the logarithm of the ratio of the signal intensity value obtained for the experiment to the control value, and sd(R) is the standard deviation of the logarithm of that ratio. With this function, genes with a Z-score greater than 2 are considered to show a statistically significant change between the experimental condition and the control (genes with higher expression), and genes with values lower than -2 (genes with lower expression).'
Adjusted P values of <0.05 and Z score values of >1.5 and <1.5 will be considered significant.
M22K_15_11_all.txt = normalized “Cy3” and “Cy5” data used for the analysis of the project.

source_name: Liver sample

label_protocol: Labeling of the RNA sample was performed using the protocol provided by the Microarray Facility at UNAM. Briefly, total RNA was reverse transcribed to generate complementary DNA (cDNA), which was then labeled with fluorescent dyes (Cy3 or Cy5). The labeled cDNA was purified to remove unincorporated dyes and any other contaminants. The specific protocol details can be found at Microarray Facility UNAM.

growth_protocol: Male C57BL/6J mice were housed under controlled conditions with a 12-hour light/dark cycle, constant temperature (22±2°C), and humidity (50-60%). Mice were allowed ad libitum access to water and standard chow until they reached the defined weight of 20-25g. Following this, they underwent a two-week adaptation period. After adaptation, the mice were assigned to their respective dietary groups and subjected to their specific diets for 18 weeks.

extract_protocol: The animals were euthanized using an excess of the anesthetic Zoletil 100 (tiletamine 250 mg, zolazepam 250 mg). A representative fragment of liver tissue was extracted and weighed at the time of collection. The samples were immediately snap-frozen in liquid nitrogen and stored at -80ºC for cryopreservation. Total RNA was extracted from mouse liver samples using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The tissue was homogenized in Trizol, followed by phase separation with chloroform. After centrifugation, the aqueous phase was collected, and RNA was precipitated with isopropanol. The RNA pellet was washed with 75% ethanol, air-dried, and resuspended in RNase-free water. The concentration and purity of RNA were assessed using spectrophotometry.

treatment_protocol: The treatment group receiving a Hihg fat and high sugar diet for 10 weeks followed by 8 weeks of supplementation with methionine, choline, betaine, folate, cobalamin, and ZnSO4.

value: liver

value: HF (high-fat and high-sugar diet)

value: MET (high-fat and high-sugar + methyl donnor diet)


title: Cell-line 1, Sppl3/Mgat2/St3gal3/4/6/B3gnt2 KO, E1E2, Furin,cell-line1_21, rep3

description:
Library name: CL1_21_R3
B12_21_R3


title: RNA-seq in D6 biol rep 1

description:
Library name: Sample38
Column name in allsamples_raw_counts.xls: D6_biol rep 1


title: Cell-line 1, Sppl3/Mgat2/St3gal3/4/6/B3gnt2 KO, E1E2, Furin,cell-line1_21, rep2

description:
Library name: CL1_21_R2
B12_21_R2


title: RNA-seq in D4 biol rep 3

description:
Library name: Sample25
Column name in allsamples_raw_counts.xls: D4_biol rep 3


title: RNA-seq in D4H8 biol rep 1

description:
Library name: Sample26
Column name in allsamples_raw_counts.xls: D4H8_biol rep 1


title: Cell-line 1, Sppl3/Mgat2/St3gal3/4/6/B3gnt2 KO, E1E2, Furin,cell-line1_21, rep1

description:
Library name: CL1_21_R1
B12_21_R1


title: RNA-seq in D4 biol rep 2

description:
Library name: Sample24
Column name in allsamples_raw_counts.xls: D4_biol rep 2


title: Cell-line 1, Sppl3/Mgat2/St3gal3/4/6/B3gnt2 KO, E1E2, Furin,cell-line1_17, rep2

description:
Library name: CL1_17_R2
B12_17_R2


title: RNA-seq in D4 biol rep 1

description:
Library name: Sample23
Column name in allsamples_raw_counts.xls: D4_biol rep 1


title: Cell-line 1, Sppl3/Mgat2/St3gal3/4/6/B3gnt2 KO, E1E2, Furin,cell-line1_17, rep1

description:
Library name: CL1_17_R1
B12_17_R1


title: Carotid Artery, CDH5cre positive Cx43fl/fl, 18hr post-injury

city: Roanoke

first: Mark

middle: Colin

last: Renton

state: VA

address: 4 Riverside Circle, Room 2208

department: Fralin Biomedical Research Institute

institute: Virginia Tech

description:
Library name: CDH5pos
Novogene 10X single cell 3' RNA Analysis

data_processing:
Raw sequencing data in FASTQ format were pre-processed with Cell Ranger (v8.0.1) using default parameters, and subsequent reads were aligned to the mouse genome using the STAR aligner within Cell Ranger.
Assembly: Mouse mm10 (Cell Ranger mm10-2020-A)
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Carotid Artery

extract_protocol:
Both EC-Cx43 KO (CDH5cre positive) and EC-WT (CDH5cre negative)  mice were euthanized by CO2 overdose and cervical dislocation 18 hr post carotid EC injury and the injured carotid was exposed without perfusion to minimize time and disruption to the endothelial layer. The carotid was then removed from the mouse and dipped in a solution of heparin saline followed by a saline-only solution. Before digestion, carotids were stored in ice-cold EC media (Vascular Cell Basal Medium; ATCC #PCS-100-030) supplemented with all components of an Endothelial Cell Growth Kit (ATCC #PCS-100-041) excluding fetal bovine serum (FBS) while subsequent carotids were removed. To enrich for EC while collecting enough total cells for processing and sequencing, carotids were cut open lengthwise prior to removal to expose the EC layer for digestion. Carotids were kept whole and a short 20 min digest (described below) was performed.  Up to two carotids were digested in a 1 mL digestion solution of 0.2 mg/ml liberase TM (Roche #5401119001) and 60 U/ml DNase1 in supplemented EC media. Carotids were incubated at 37°C and 400 rpm in a benchtop orbital shaking heat block for 20 min, pipetting up and down with a 1 mL pipette every 5 min. Digestion was stopped with the addition of 1 mL stopping solution (2% FBS, 1mM EDTA, in supplemented EC media), after which samples of the same condition were combined (up to 5 at a time to reduce time to digestion). A 100 μm cell strainer was primed by flushing through 2 mL of supplemented EC media with 2% FBS. Cell solutions were filtered through the strainer and rinsed with 3 mL of supplemented EC media with 2% FBS. The flow through was centrifuged at 1000 rpm, 4°C for 10 min, followed by removal of the supernatant and resuspension of the cell pellet in 2 mL RBC lysis buffer (Invitrogen 00-4333-57) at room temperature for 5 min. RBC lysis was stopped using 10 ml supplemented EC media with 2% FBS, samples were centrifuged at 1000 rpm, 4°C for 10 min and the cell pellet was resuspended in 10% DMSO, 10% FBS in cold supplemented EC media. Single cell suspensions were slowly frozen at -80°C overnight in a cell freezer container, then transferred to liquid nitrogen until sequencing. A total of 15 carotids for EC-WT and 20 carotids for EC-Cx43 KO were pooled for use for scRNAseq by Novogene.
Novogene scRNAseq Methods. Cell viability analysis, RNA integrity analysis, library preparation, sequencing, raw read count generation, and reference genome alignment were performed commercially by Novogene (Sacremento, CA, USA) using their Plant and Animal 10X Single Cell Transcriptome Sequencing pipeline. Full parameters, protocols, and quality control reports are available on request, but are explained briefly below:  Sample and Library Preparation. Cryopreserved cell samples were thawed and processed by Novogene. Cell count and viability were assessed using a TC20 Automated Cell Counter (Bio-Rad, USA) with both samples achieving approximately 60,000 cells with an overall cell viability of >75%. Libraries were then constructed using the 10x Chromium Single Cell 3’ Kit with a target of 10,000 cells.  Sequencing and Data Preprocessing. Sequencing was performed on the NovaSeq X Plus Series (Illumina, USA) platform with 150 bp paired end reads with a minimum of 150 G raw data per sample.

value: Carotid Artery

value: CDH5 Cre+ Cx43fl/fl

value: 18 hr ligation injury recovery