title: Low-input WGBS, 50 ng Arabidopsis leaf DNA, replicate 1

description:
Library name: P50ng_1
low_input protocol, 50 ng input


title: Cell-line Control replicate 2

status: Public on Jul 13 2026

type: SRA

city: Hefei

first: Jun

last: Xu

state: Anhui

address: 17 Lujiang Road

country: China

institute: The First Affiliated Hospital of USTC, Anhui Provincial Hospital

description: Library name: figure6_cell_line_control_rep2

library_source: transcriptomic

data_processing:
Raw sequencing reads were filtered to remove adapters and low-quality reads.
Clean reads were aligned to the human reference genome.
Gene expression values were calculated and reported as FPKM values for all samples.
Assembly: GRCh38
Supplementary files format and content: Fig6_cell_line_FPKM_matrix_GEO.csv is a comma-separated gene-level FPKM matrix for all 12 cell-line RNA-seq samples.

molecule: total RNA

organism: Homo sapiens

source_name: breast

growth_protocol: Human breast cancer cell-line samples were cultured under standard in vitro conditions.

extract_protocol:
Total RNA was extracted from cell-line samples according to the manufacturer's protocol.
RNA-seq libraries were prepared from total RNA and sequenced using paired-end sequencing.

treatment_protocol: Samples were grouped according to LRIG1 and EGFR L747A manipulation status.

tag: tissue

value: breast

tag: cell type

value: epithelial cancer cell

tag: genotype

value: breast cancer

tag: treatment

value: Control


title: HV0045_C_24h_rep2

city: Heidelberg

first: Ralph

last: Grand

address: Im Neunheimer feld 345

country: Germany

institute: Heidelberg University

description: Library name: HV0045_C_24h_rep2_R1.fastq.gz

data_processing:
RNA-seq data were processed using the snakePipes mRNA-seq workflow. Raw sequencing reads in FASTQ format were aligned to the human reference genome  with default settings, including adapter and quality trimming.
Gene-level quantification was performed with featureCounts
Assembly: hg38
Supplementary files format and content: tsv

source_name: HUVEC

growth_protocol: Human umbilical vein endothelial cells (HUVECs) (Promo Cell, C-12203 and Innoprot, #P2020) were cultured in endothelial cell basal medium 2 (ECGM2, PromoCell, #C-22216) with supplement mix (PromoCell, #C-39216) under standard cell culture conditions. HUVECs were split when the confluency reached 80% using TrypLE (Thermo Fisher, 12605028). For experiments cells were passaged at 1:3 or 1:4 into T-75 flasks 2-3 days prior to heat shock treatment. All HUVECs were used between passage four and six.

extract_protocol:
Cells were isolated 0 hours and 24 hours post seeding, snap frozen and stored at -80°C.  RNA was isolated using a RNeasy UCP Micro Kit (Qiagen, RNeasy UCP Micro Kit, #73934). RNA concentration was measured using a NanoDrop 200c.
Sequencing libraries were generated from 100 ng of total RNA using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina

treatment_protocol: Cells in T75 flasks were heat shocked at 42°C for 1h in either a water bath placed inside a tissue culture incubator or a standard water bath, then returned to standard culturing conditions for recovery. Cells recovered between 3 hours in a 37°C cell culture incubator prior to the respective experiments.

tag: cell line

value: HUVEC

value: Entothelial


title: HV0032_HSP_24h_rep1

description: Library name: HV0032_HSP_24h_rep1_R1.fastq.gz

value: Heatshock+Printed


title: Low-input WGBS, 500 pg Arabidopsis leaf DNA, replicate 3

city: München

first: Jie

last: Yang

state: Bavaria

address: Karlsfelder Str. 38a

institute: Technical University of Munich (TUM)

description:
Library name: P500pg_3
low_input protocol, 500 pg input

library_source: genomic

data_processing:
Raw paired-end reads were trimmed with fastp using a two-step procedure to remove adapter contamination and the 10-bp random-primer/Adaptase-derived 5' sequence bias.
Trimmed reads were aligned to the TAIR10 Arabidopsis thaliana reference genome using Bismark (--local mode, non-directional, bowtie2).
Aligned reads were deduplicated with deduplicate_bismark, sorted, and filtered to retain properly paired reads with MAPQ >= 30 using samtools.
Cytosine methylation calls were extracted from filtered alignments using bismark_methylation_extractor (--CX) and converted to per-cytosine CX reports with coverage2cytosine.
Missing cytosine methylation states were imputed from the CX reports using METHimpute (HMM-based binomial test); the complete workflow is implemented in MethylStar (https://github.com/jlab-code/MethylStar).
Assembly: TAIR10
Supplementary files format and content: Tab-separated genome-wide cytosine reports (Bismark coverage2cytosine --CX_context output) for each library. Columns: chromosome (TAIR10); position (TAIR10); strand; count methylated; count unmethylated; methylation context (CpG/CHG/CHH); trinucleotide context. No processed file is provided for P10pg_3 and P50pg_3, which were excluded from downstream methylation analyses due to low bisulfite conversion rate (see Table S1).

molecule: genomic DNA

organism: Arabidopsis thaliana

source_name: leaf

growth_protocol: Arabidopsis thaliana plants were grown at 23°C under long-day conditions (16 h light / 8 h dark).

extract_protocol:
Flash-frozen Arabidopsis thaliana leaf tissue was cryo-milled using a TissueLyser II and lysed with Proteinase K. A serial dilution series of the extracted genomic DNA (5 pg-50 ng) was processed using the EZ DNA Methylation-Direct kit (Zymo Research), which performs bisulfite conversion directly from the digested lysate without a separate DNA purification step.
Bisulfite-converted DNA was processed using a post-bisulfite adapter tagging (PBAT) workflow adapted from the single-cell methyl-seq (snmC-seq) protocol: random priming with a P5L-indexed primer (Klenow Fragment exo-), enzymatic removal of excess primer (Exonuclease I / rSAP), single-stranded adapter ligation to the 3' end using the xGen Adaptase module, and indexing PCR (KAPA HiFi HotStart, 19-21 cycles) to introduce P5/P7 indexes. Standard bulk WGBS libraries (50 ng input, samples S45_1-3) were prepared conventionally for comparison.

treatment_protocol: No treatment was applied.

value: leaf


title: Cell-line Control replicate 1

description: Library name: figure6_cell_line_control_rep1


title: HV0031_HS_24h_rep1

description: Library name: HV0031_HS_24h_rep1_R1.fastq.gz

value: Heatshock


title: HV0030_P_24h_rep1

description: Library name: HV0030_P_24h_rep1_R1.fastq.gz

value: Printed


title: Low-input WGBS, 500 pg Arabidopsis leaf DNA, replicate 2

description:
Library name: P500pg_2
low_input protocol, 500 pg input


title: HV0029_C_24h_rep1

description: Library name: HV0029_C_24h_rep1_R1.fastq.gz


title: HV0052_HSP_1h_rep3

description: Library name: HV0052_HSP_1h_rep3_R1.fastq.gz


title: FFPE resistant sample GZ23090348

description: Library name: GZ23090348

data_processing:
Raw sequencing reads were filtered to remove adapters and low-quality reads.
Clean reads were aligned to the human reference genome.
Gene-level read counts were generated for all samples.
Assembly: GRCh38
Supplementary files format and content: Fig3_FFPE_raw_counts_matrix_GEO.csv is a comma-separated gene-level raw count matrix for all 10 FFPE samples.

source_name: breast tumor tissue

growth_protocol: FFPE tumor tissue samples were collected from clinical specimens.

extract_protocol:
Total RNA was extracted from FFPE tumor tissue samples according to the manufacturer's protocol.
RNA-seq libraries were prepared from FFPE-derived total RNA and sequenced using paired-end sequencing.

treatment_protocol: Samples were grouped according to treatment response status.

value: breast tumor tissue

value: resistant

tag: batch

value: FFPE


title: FFPE resistant sample GZ23094716

description: Library name: GZ23094716


title: Low-input WGBS, 500 pg Arabidopsis leaf DNA, replicate 1

description:
Library name: P500pg_1
low_input protocol, 500 pg input


title: U2OS, scRepli-seq, untreated singleG1 003

city: Kobe

first: Jothivanan

last: Elumalai

state: Hyogo

address: 2-2-3, Minatojima Minamimachi, Chuo-ku

department: Biosystems Dynamics Research (BDR)

country: Japan

institute: RIKEN

data_processing:
We used only one end of the paired-end sequenced reads for our analysis. Raw fastq files were subjected to quality assessment with FastQC v0.11.5 , and the cutadapt program v1.15 was used to eliminate SEQXE and Illumina adapters. Mapping to the hg19 or mm9 genome was carried out using bwa v0.7.17-r1188 (bwa-aln command). The resulting mapped reads were sorted using samtools v1.7, and duplicate reads were flagged using picard-tools v2.8.1.
For single-G1 karyotype analysis: After marking the duplicate reads, copy number analysis was conducted using the AneuFinder package v1.22.0 within R v4.1.3. Initially, duplicate reads, blacklist regions (available at https://github.com/Boyle-Lab/Blacklist) and reads with a mapping quality (MAPQ) below 10 were filtered out. Uniquely mapped reads were counted within each 500-Kb genomic window, adjusted for GC content (GC.BSgenome=BSgenome.Hsapiens.UCSC.hg19 for humans and BSgenome.Mmusculus.UCSC.mm9 for mouse, method="loess"), and copy number segmentation was executed using the "findCNVs" function (method='edivisive', R=50) of the AneuFinder package. For quality control (QC), multivariate clustering was applied, and clusters with a spikiness value exceeding 1 were eliminated.
For Log2percentile RT: We followed our established procedure to generate binarized replication timing (RT) profiles for each of the 14 fractions of S-phase or single-cells under analysis, along with their respective percentage replication scores (Miura et al, Nat. Protoc. 2020). The percentage replication score of each S-phase fraction or single-cells was utilized to compute their raw, non-binarized replication timing profile, termed as "log2repliscore RT".
Assembly: hg19, mm9
Supplementary files format and content: CNV states (.txt)
Supplementary files format and content: log2percentile RT (.txt)
Library strategy: scRepli-seq

source_name: Bone

growth_protocol: Human osteosarcoma cells (U2OS) were cultured in DMEM (Nacalai, 08459-64) supplemented with 10% FBS (Sigma, 172012) and maintained at 37°C with 5% CO2. CBMS1 mouse embryonic stem cells (mESCs) have been described (Murakami et al, Development, 2011) and grown in 2i/LIF medium as described (Hayashi et al, Nat. Protoc. 2013).

extract_protocol:
Single cells were sorted with a Sony SH800 or MA900 cell sorter. Sample preparations were performed as described (Miura et al, Nat. Protoc. 2020). DNA samples were subject to whole-genome amplification (WGA) with a SeqPlex kit (Sigma, SEQXE).
NGS libraries were constructed for amplied DNA using an NGS LTP Library Preparation Kit (KAPA, KK8232) according to the manufacturer’s instructions.

treatment_protocol: Cells were subjected to treatment with or without 0.4 μM aphidicolin for 24 hours and labelled with 10 μM EdU for 60 and 15 minutes before harvesting, respectively.

value: Bone

value: Sarcoma-derived

value: Untreated


title: HV0050_P_1h_rep3

description: Library name: HV0050_P_1h_rep3_R1.fastq.gz


title: HV0051_HS_1h_rep3

description: Library name: HV0051_HS_1h_rep3_R1.fastq.gz


title: Cell-line LRIG1 replicate 1

description: Library name: figure6_cell_line_lrig1_rep1


title: Low-input WGBS, 100 pg Arabidopsis leaf DNA, replicate 3

description:
Library name: P100pg_3
low_input protocol, 100 pg input


title: HV0049_C_1h_rep3

description: Library name: HV0049_C_1h_rep3_R1.fastq.gz


title: FFPE resistant sample GZ23094729

description: Library name: GZ23094729


title: HV0040_HSP_1h_rep2

description: Library name: HV0040_HSP_1h_rep2_R1.fastq.gz


title: Low-input WGBS, 100 pg Arabidopsis leaf DNA, replicate 2

description:
Library name: P100pg_2
low_input protocol, 100 pg input


title: HV0039_HS_1h_rep2

description: Library name: HV0039_HS_1h_rep2_R1.fastq.gz


title: FFPE resistant sample GZ23094731

description: Library name: GZ23094731


title: HV0038_P_1h_rep2

description: Library name: HV0038_P_1h_rep2_R1.fastq.gz


title: HV0037_C_1h_rep2

description: Library name: HV0037_C_1h_rep2_R1.fastq.gz


title: Low-input WGBS, 100 pg Arabidopsis leaf DNA, replicate 1

description:
Library name: P100pg_1
low_input protocol, 100 pg input


title: FFPE resistant sample GZ23094732

description: Library name: GZ23094732


title: Low-input WGBS, 50 pg Arabidopsis leaf DNA, replicate 3

description:
Library name: P50pg_3
low_input protocol, 50 pg input. Excluded from downstream methylation analyses due to low bisulfite conversion rate (Table S1).


title: HV0024_HSP_1h_rep1

description: Library name: HV0024_HSP_1h_rep1_R1.fastq.gz


title: HV0023_HS_1h_rep1

description: Library name: HV0023_HS_1h_rep1_R1.fastq.gz


title: FFPE sensitive sample GZ23094709

description: Library name: GZ23094709

value: sensitive


title: HV0022_P_1h_rep1

description: Library name: HV0022_P_1h_rep1_R1.fastq.gz


title: Low-input WGBS, 50 pg Arabidopsis leaf DNA, replicate 2

description:
Library name: P50pg_2
low_input protocol, 50 pg input


title: FFPE sensitive sample GZ23094724

description: Library name: GZ23094724


title: HV0021_C_1h_rep1

description: Library name: HV0021_C_1h_rep1_R1.fastq.gz


title: Liver, P7, scrambled, rep3

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

institute: MPI for heart and lung research

data_processing:
Trimmomatic version 0.39 was employed to trim reads after a quality drop below a mean of Q15 in a window of 5 nucleotides and keeping only filtered reads longer than 15 nucleotides
Reads were aligned versus Ensembl mouse genome version mm10 (GRCm38) with STAR 2.6.1d
Aligned reads were filtered to remove: duplicates with Picard 2.21.7, multi-mapping, ribosomal, or mitochondrial reads.
Gene counts were established with featureCounts 1.6.5. by aggregating reads overlapping exons on the correct strand excluding those overlapping multiple genes
The raw count matrix was normalized with DESeq2 version 1.26.0 (Love et al., Moderated estimation of fold change and dispersion for RNA-Seq data with DESeq2). Contrasts were created with DESeq2 based on the raw count matrix. Genes were classified as significantly differentially expressed at average count > 5, multiple testing adjusted p-value < 0.05, and -0.585 < log2FC > 0.585.
Assembly: mm10
Supplementary files format and content: library size normlaized counts

organism: Mus musculus

source_name: Liver

extract_protocol:
Total RNA was isolated from mouse livers using the miRNeasy® Mini kit (Qiagen). A 3 mm cube (approximately 25mg) of liver tissue was placed into 2 ml tubes containing ceramic beads (Bertin GmbH) and 700 µl QIAzol Lysis Reagent and was homogenized using a Precellys 24-Dual homogenizer (VWR). Ultimately total RNA from liver tissue was resuspended in 50 µl nuclease-free water.
2µg of total RNA was used as input for VAHTS Stranded mRNA-seq V6 Library preparation following manufacture’s protocol (Vazyme).

treatment_protocol: MicroRNA mimics were administered to mice in two different protocols. Mouse litters were randomized to equal-sized litters and distributed to nursing dams. In the first injection protocol, 14 nmol (100 mg; at a dose of 100 mg.kg-1) of either the miR-497a-5p mimic was administered to six mice per group, with the corresponding control scrambled mimic administered to six mice per group; or 1.4 nmol (10 mg; at a dose of 10 mg.kg-1) of the miR-497a-5p mimic was administered to six mice per group, with the corresponding control scrambled mimic administered to six littermates per group. Injections were performed on post-natal day (P)1, defined here as the day of birth, and again on P3. MicroRNA mimics were administered via the intraperitoneal route in a volume of 15 µl.g‑1. Mouse pups were killed as described below at P5, P7, or P14, and the liver was harvested for total RNA isolation.

value: Liver

tag: age

value: inert "scrambled" miR-497a-5p mimic


title: Low-input WGBS, 50 pg Arabidopsis leaf DNA, replicate 1

description:
Library name: P50pg_1
low_input protocol, 50 pg input


title: Liver, P7, scrambled, rep2


title: Liver, P7, scrambled, rep1


title: FFPE sensitive sample GZ23094725

description: Library name: GZ23094725

value: breast cancer


title: Low-input WGBS, 10 pg Arabidopsis leaf DNA, replicate 3

description:
Library name: P10pg_3
low_input protocol, 10 pg input. Excluded from downstream methylation analyses due to low bisulfite conversion rate (Table S1).


title: Liver, P7, mimic, rep3

value: miR-497a-5p mimic


title: FFPE sensitive sample GZ23094741

description: Library name: GZ23094741


title: Liver, P7, mimic, rep2


title: Low-input WGBS, 10 pg Arabidopsis leaf DNA, replicate 2

description:
Library name: P10pg_2
low_input protocol, 10 pg input


title: Liver, P7, mimic, rep1


title: Liver, P5, scrambled, rep3


title: Low-input WGBS, 5 pg Arabidopsis leaf DNA, replicate 3

description:
Library name: P5pg_3
low_input protocol, 5 pg input


title: FFPE sensitive sample GZ23094742

description: Library name: GZ23094742


title: Liver, P5, scrambled, rep2


title: Lenti-shEHMT1-3

city: Wuhan

first: Ding-Sheng

last: Jiang

state: None Selected

address: 1095 Jiefang Ave., Wuhan 430030, China

institute: Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology

description:
Library name: sample6
EH_3

data_processing:
First, the sequenced reads were aligned to the reference genome sequence (downloaded from the genomic website) using HISAT2 v2.0.5, and the annotation file was used to determine the gene name corresponding to each read. Subsequently, featureCounts (1.5.0-p3) was used to calculate the raw read counts for each gene. Based on sequencing depth and transcript length, normalization was performed to obtain FPKM and RPKM values. The raw counts were used to evaluate relative gene expression levels and to calculate the statistical significance of differences between groups
Assembly: GRCh38
Supplementary files format and content: CSV file including gene count for each sample

source_name: HASMCs

extract_protocol:
Human aortic smooth muscle cells (HASMCs) cultured in 10 cm dishes were infected with Lenti-shRNA (control) or Lenti-shEHMT1, with three replicates per group. After culture, cells were harvested using Trizol, and total RNA was extracted by phenol-chloroform extraction. RNA concentration and integrity were precisely analyzed using an Agilent 2100 Bioanalyzer.
Total RNA (≥1 μg) was used for library construction. mRNA with polyA tails was enriched using Oligo(dT) magnetic beads and then randomly fragmented with divalent cations in Fragmentation Buffer.  2) The fragmented mRNA was used as template, and random oligonucleotides as primers, to synthesize first‑strand cDNA in a reverse transcriptase (M‑MuLV) system, followed by mRNA degradation with RNase H.  3) Second‑strand cDNA was synthesized in a DNA polymerase I system using the first‑strand cDNA as template and dNTPs as substrates.  4) The purified double‑stranded cDNA was subjected to end repair, polyA tailing, and adapter ligation. DNA fragments of 370–420 bp were selected using magnetic beads, and the library was amplified by PCR.  5) After library preparation, preliminary quantification was performed using a Qubit 2.0 Fluorometer, followed by quality assessment on an Agilent 2100 Bioanalyzer. Finally, the library was accurately quantified by real‑time PCR to ensure it met the quality requirements for sequencing.  c) Sequencing on the instrument: After passing quality control, the libraries were sequenced according to the required concentration and data output. Sequencing was performed using the principle of sequencing‑by‑synthesis. DNA polymerase, four fluorescently labeled dNTPs, and adapter primers were added to the reaction system for amplification. Because different dNTPs carry distinct fluorescent labels, the fluorescence signals were captured by the sequencer and converted into corresponding base calls by a computer. After sequencing was completed, the reads were assembled to generate fragment information

value: HASMCs


title: Liver, P5, scrambled, rep1


title: Low-input WGBS, 5 pg Arabidopsis leaf DNA, replicate 1

description:
Library name: P5pg_1
low_input protocol, 5 pg input


title: Liver, P5, mimic, rep3


title: Liver, P5, mimic, rep2


title: Liver, P5, mimic, rep1


title: Lenti-shEHMT1-1

description:
Library name: sample4
EH_1


title: M2K_4PIC_R3

city: Houston

first: LI

last: SHEN

state: TX

address: 1515 Holcombe Blvd.

country: USA

institute: MD Anderson Cancer Center

description: Library name: M2K_4PIC_R3

data_processing:
For data processing, raw sequencing reads were processed using the nf-core/rnaseq pipeline v3.14,40 a community-curated workflow built upon the nf-core framework. The workflow was executed with Nextflow v23.10.142 under reproducible software environments provided through Bioconda and BioContainers. Reads were aligned to the GRCm39 reference genome using STAR, and transcript quantification was performed with RSEM to generate gene-level count estimates. Default nf-core parameters were used unless otherwise specified.
Assembly: GRCm39
Supplementary files format and content: tab-delimited text file includes raw counts for each sample

molecule: polyA RNA

source_name: Bone marrow

growth_protocol: Murine bone marrow cells (1.5 × 10^6 cells/mL) from C57BL/6J wild-type, MHC-I KO (B6.129P2-H2-K1b^tm1Bpe H2-D1b^tm1Bpe/DcrJ), MHC-II KO (B6.129S2-H2^dlAb1-Ea/J) were cultured in RPMI 1640 containing 10% heat-inactivated fetal bovine serum (FBS; Atlanta Biologics, Atlanta, GA, USA), 1% penicillin-streptomycin, 1 mM sodium pyruvate, and 50 µM β-mercaptoethanol (complete RPMI), and supplemented with 2 ng/mL murine granulocyte-macrophage colony-stimulating factor (mGM-CSF; PeproTech, Rocky Hill, NJ, USA) and 50 ng/mL human Flt3L (hFlt3L; PeproTech). On day 9, nonadherent cells were collected and replated in fresh complete RPMI at 0.3 × 10^6 cells/mL with 2 ng/mL mGM-CSF and 50 ng/mL hFlt3L. On days 15 to 17, nonadherent cells were collected and cDC1s (CD11c+CD45R−CD24+CD172α−CD103+) were purified by fluorescence-activated cell sorting (FACS) using a FACSAria or FACSAria Fusion cell sorter (BD Biosciences, Palo Alto, CA, USA).

extract_protocol:
Total RNA was isolated from FACS-purified cDC1s using the Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Purified RNA was stored at −80 C until shipment for sequencing.
RNA concentration and purity were evaluated by NanoDrop spectrophotometry, 1% agarose gel electrophoresis, and RNA integrity was assessed using an Agilent 2100 Bioanalyzer to determine RNA integrity numbers, a standardized quantitative measure of RNA degradation based on electrophoresis profiles. Libraries were generated using a poly(A)-enriched mRNA workflow and sequenced on an Illumina NovaSeq X-Plus platform to produce 150-bp paired-end reads.

treatment_protocol: FACS-purified cDC1s were cultured at 2 to 4.5 × 10^6 cells/mL in complete RPMI supplemented with 20 µg/mL poly dI:dC, 20 ng/mL mGM-CSF, and ± 20 µg/mL αCD40 for 16 hours at 37 C or were left untreated.

value: Bone marrow

value: MHC-II KO (B6.129S2-H2^dlAb1-Ea/J)

value: pdI:dC + αCD40


title: Liver, P14, scrambled, rep3


title: U2OS, scRepli-seq, untreated singleG1 028


title: Cell-line sensitive replicate 1

description:
Library name: figure3_cell_line_sensitive_rep1
Column name in Fig3_cell_line_FPKM_matrix_GEO.csv: figure3_cell_line_sensitive_rep1

data_processing:
Raw sequencing reads were filtered to remove adapters and low-quality reads.
Clean reads were aligned to the human reference genome.
Gene expression values were calculated and reported as FPKM values for all samples.
Assembly: GRCh38
Supplementary files format and content: Fig3_cell_line_FPKM_matrix_GEO.csv is a comma-separated gene-level FPKM matrix for all 6 cell-line RNA-seq samples.

treatment_protocol: Samples were grouped according to treatment response status as sensitive or resistant.

value: breast

value: breast cancer cell line

value: epithelial cancer cell

value: cell line


title: M2K_4PIC_R2

description: Library name: M2K_4PIC_R2


title: Liver, P14, scrambled, rep2


title: Lenti-shRNA-3

description:
Library name: sample3
PL_4

value: Lenti-shRNA


title: UTI89/pRRK1_2019 biol rep 1

city: Salt Lake City

first: Matthew

middle: A

last: Mulvey

state: Utah

address: 1390 Presidents' Circle

department: Biology

institute: University of Utah

description:
Library name: Sample 12
all.counts_KO.tsv

data_processing:
Sequences were processed using BBduk v37.10 to trim adapters
Samtools v1.20 was used to read features
Differential gene expression was analyzed using DESeq2 in R v4.4.0
Assembly: Reads were mapped to the UTI89 reference strain GCF_000013265 using bowtie2 v2.5.3
Supplementary files format and content: txt file contains raw counts for each sample
Supplementary files format and content: csv contains l2fc for each gene

organism: Escherichia coli

source_name: whole cell

growth_protocol: Overnight cultures of UTI89/pRRK1, UTI89ΔmicC/pRRK1, and UTI89ΔmicC/pMicC were back diluted 1:100 into 5 mL LB and grown for 3 hours 15 min at 37°C, shaking 225 rpm.

extract_protocol:
RNA was extracted using a PureLinkTM RNA Mini Kit (Invitrogen)
Libraries constructed using the Nugen Ovation Prokaryotic RNA-seq kit as per manufacturer instructions

treatment_protocol: At the two-hour growth mark IPTG was added to each culture to a final concentration of 0.1 mM.

value: whole cell

tag: strain

value: Gram negative rod, bacteria

value: WT with empty vector


title: U2OS, scRepli-seq, untreated singleG1 025

value: Bone

value: Sarcoma-derived

value: Untreated


title: Liver, P14, scrambled, rep1

value: Liver

value: inert "scrambled" miR-497a-5p mimic


title: Liver, P14, mimic, rep3

value: miR-497a-5p mimic


title: M2K_4PIC_R1

description: Library name: M2K_4PIC_R1

value: Bone marrow


title: Lenti-shRNA-2

description:
Library name: sample2
PL_3

value: HASMCs


title: M2K_PIC_R3

description: Library name: M2K_PIC_R3

value: MHC-II KO (B6.129S2-H2^dlAb1-Ea/J)


title: Liver, P14, mimic, rep1


title: Liver, P14, mimic, rep2


title: Lenti-shRNA-1

description:
Library name: sample1
PL_2


title: M2K_PIC_R2

description: Library name: M2K_PIC_R2


title: U2af38RNAi sorted spermatocytes, biological replicate 2

city: Farmington

first: Mayu

last: Inaba

state: CT

address: 263 Farmington Ave

institute: UConn Health

description: Library name: U2afRNAi_sorted2

data_processing:
Adapter sequences and low-quality bases were trimmed using TrimGalore v0.6.7 with a quality threshold of Q20 and a minimum read length of 36 bp
For genes: Trimmed reads were aligned to the Drosophila melanogaster reference genome (release 6.65) using STAR v2.7.11b. STAR was run in two-pass mode with a maximum of 1 multi-mapping location per read (--outFilterMultimapNmax 1). STAR indices were generated using the genome FASTA and gene annotation GTF (FlyBase r6.65).
For genes: Gene-level read counts were quantified using featureCounts (Subread v2.0.3) in paired-end mode with reverse strandedness (-s 2) to match the library preparation protocol. Read pairs were assigned to genes based on overlap with exonic features defined in the FlyBase r6.65 GTF annotation.
For TEs: trimmed reads were aligned to the D. melanogaster r6.65 genome using STAR v2.7.11b with parameters optimized for multi-mapping reads: --outFilterMultimapNmax 150 and --winAnchorMultimapNmax 150
For TEs: Telescope was run with the following parameters: --attribute gene_id for locus-level quantification, --reassign_mode conf for confidence-based probabilistic reassignment, --conf_prob 0.95 to retain only alignments with ≥95% posterior probability, --overlap_threshold 0.2 requiring reads to overlap at least 20% of their length with a TE annotation.
For identifying TE divergence: we subjected Telescope locus-level counts to a post-hoc divergence-based filter. Per-locus percent divergence from the TE consensus sequence was retrieved from the divergence attribute field of the Telescope TE annotation GTF, which records the Kimura 2-parameter divergence estimated by RepeatMasker for each annotated TE insertion. Loci whose divergence from consensus exceeded 5% were excluded from downstream quantification
For genes and TEs: Repeated alignment and quantification of transcripts using kallisto/0.50.1.To enable parallel quantification of gene transcripts and transposable element families within a single “competitive” framework, we constructed a combined pseudo-alignment reference by concatenating the Drosophila melanogaster FlyBase transcript sequences (dmel-all-transcript-r6.65.fasta) with the Bergman laboratory curated TE consensus sequence library. A kallisto index was built from the combined reference using kallisto index. Transcript-level abundance estimation was performed for each sample using kallisto quant in paired-end mode with 100 bootstrap resampling iterations (-b 100). Gene-level count estimates were subsequently aggregated from transcript-level output using tximport 35 with a transcript-to-gene mapping derived from the FlyBase r6.65 GTF.
Assembly: Drosophila melanogaster (Flybase r6.65) and D. melanogaster TE consensus sequences from the Bergman laboratory curated library (https://github.com/bergmanlab/drosophila-transposons)
Supplementary files format and content: text file of raw counts for genes in each sample (featureCounts)
Supplementary files format and content: text file of raw counts for all TE families in each sample (telescope)
Supplementary files format and content: text file of raw counts for all TE loci in each sample (telescope)
Supplementary files format and content: text file of raw counts for TE families with ≤5% divergence of consensus in each sample (telescope)
Supplementary files format and content: text file of raw counts for TE loci with ≤5% divergence of consensus in each sample (telescope)
Supplementary files format and content: tsv files of gene and TE counts in each sample (kallisto)

organism: Drosophila melanogaster

source_name: testes

extract_protocol:
RNA was extracted using the RNeasy Plus Micro Kit (Qiagen)
rRNA depletion was performed by Ampseq

value: testes

value: spermatocyte

value: blanks-gfp, bamgal4>u2af38RNAi


title: U2af38RNAi sorted spermatocytes, biological replicate 1

description: Library name: U2afRNAi_sorted1


title: GM40

city: New York

first: Edridge

middle: Kevin

last: D'Souza

state: NY

address: 1130 St Nicholas Ave

department: Hematology/Oncology

institute: Columbia University

description:
Library name: GM40
Single nucleus RNA sequencing

library_source: transcriptomic single cell

data_processing:
Raw snRNA data were analyzed by aligning demultiplexed FASTQ files to HRCh38-2020-A human transcriptome using CellRanger v7.1.0
Ambient RNA was removed using CellBender v0.2.2 and doublets were flagged for downstream removal using Scrublet (Wolock et al., 2019)
Expression matrices were processed in R v4.2.0 using Seurat v4.3.0.1. Barcodes were filtered to keep cells with 200-12000 genes and <15% mitochondrial gene load, and to retain predicted singlets as well as predicted doublets in the G2M/S phase.
Downstream analysis followed the Seurat NormalizeData pipeline, and sample integration used the Seurat CCA Integration pipeline.
Assembly: GRCh38-2020-A transcriptome
Supplementary files format and content: Gene expression matrix (raw output from CellRanger)

molecule: nuclear RNA

source_name: Peripancreatic lymph node metastasis

extract_protocol:
We obtained fresh frozen tissue specimens from the New York Presbyterian/Columbia University Medical Center under IRB approval (#AAAT6521, AAAS9513) and prepared them as previously described for sequencing (Wang, Y. et al., Nat. Genet. 2023; Luthria, K. et al., Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 2024). We embedded samples in Tissue-Tek Optimal Cutting Temperature (OCT) Compound (Sakura Finetek; #4583) and obtained 4-5 sections of 20 µm thickness per sample using a Leica CM1950 cryostat (Leica Biosystems, Wetzlar, Germany). We placed the samples in 5 mL tubes (Eppendorf, Hamburg, Germany), washed with ice cold PBS (Thermo Fisher, #10010023), centrifuged for 2 minutes at 400g, then discarded the supernatant and resuspended in 1 mL ST buffer [146 mM NaCl, 10 mM Tris-HCl pH7.5, 1 mM CaCl2, and 21 mM MgCl2 in ultrapure water] with 0.03% Tween-20 (Sigma Aldrich, #P7949) and 0.1% BSA (New England Biolabs, #B9000S) plus or minus 40 U/mL RNAse OUT inhibitor (Thermo Fisher, #10777019). We pipetted each suspension 15 times with a 1 mL pipette to mechanically dissociate tissue, then incubated for 5 minutes on ice before repeating the pipetting and finally quenching with 4 mL ST buffer plus or minus 40 U/mL RNAse OUT (Thermo Fisher, #10777019). We pre-wetted sequential 70-micron nylon mesh filters (Fisher Scientific, #NC0446099), then filtered samples into a 50 mL conical tube and washed the filter with 5 mL ST buffer. We centrifuged for 5 minutes at 500g and discarded supernatant to isolate the dissociated nuclei, which were resuspended in 100-400 µl ST buffer, filtered with a 40 µm mesh filter (Fisher Scientific), stained with 50 µg/ml Hoechst 33342 (Thermo Fisher, #H3570), and then counted with a Neubauer counting chamber (Bulldog Bio, Inc., Portsmouth, NH).
We loaded 0.9-1.5e3 nuclei in ST buffer without RNAse inhibitor using a Chromium controller and 5’ chemistry v2 capture (10X Genomics, #1000263). We prepared cDNA libraries according to manufacturer instructions for reverse transcription and cleanup, and performed an extra cycle of cDNA amplification to compensate for the reduced total RNA content when analyzing single nuclei compared to single cells. We prepared final sequencing libraries with the 10X Library Construction Kit (#1000190) and Dual Index Kit TT Set A (#1000215) and quantified them using D1000 and D5000 reagents (Agilent, #5067-5583 and #5067-5589) on an Agilent 2200 TapeStation system. We then mixed samples and sequenced to a target depth of at least 20,000 reads per droplet using NovaSeq S4 (Illumina, San Diego, CA) with at least 2x100bp read length.

value: Peripancreatic lymph node metastasis

value: Tumor (nuclei)

tag: treatment stage

tag: patient id

tag: response

value: PR


title: M2K_PIC_R1

description: Library name: M2K_PIC_R1


title: control sorted spermatocytes, biological replicate 2

description: Library name: BlanksGFP_sorted2

value: blanks-gfp


title: GM39

description:
Library name: GM39
Single nucleus RNA sequencing

source_name: Hepatic metastasis

value: Hepatic metastasis


title: control sorted spermatocytes, biological replicate 1

description: Library name: BlanksGFP_sorted1


title: M2K_NS_R3

description: Library name: M2K_NS_R3

value: No stim


title: Gast_125_CTRL_Nanopore

city: Schlieren

first: Ataman

last: Sendoel

address: Wagistrasse 12

department: Institute of Regenerative Medicine

country: Switzerland

institute: University of Zurich

description:
Library name: Gast_125_CTRL_Nanopore
Nanopore direct RNA sequencing from control 72h gastruloids, Replicate 2

data_processing:
Bioinformatic analysis of single-cell sequencing data: Raw sequencing data in the form of BCL files, were demultiplexed using Illumina bcl2fastq v2.20 with default settings. Up to one mismatch in the sample barcode sequences was allowed. Resulting fastq files were processed using the BD Rhapsody Pipeline (version 1.9.1) hosted at the Seven Bridges’ cloud platform (https://www.sevenbridges.com/). A custom mouse reference genome, based on Gencode vM25 (GRCm38.p6), was generated and combined with the 669 sgRNAs sequences. Based on STAR aligner, a custom STAR reference genome was created and the Refined Putative Cell Calling option was disabled during the pipeline execution. For downstream analysis, unique molecular identifier (UMI) count data was corrected by BD Genomics Recursive Substitution Error Correction (RSEC).  Bioinformatic analysis of dial-out data: PCR dial-out data was processed using an in-house custom Python script as previously described. Three 9-nucleotide cell barcodes were extracted from the 97 possible barcodes from Read 1. Up to one mismatch was allowed for each barcode (Hamming distance of 1). The script retrieved the 8-nucleotide UMI sequences from Read 1 as per the BD manual. For valid barcodes, sgRNAs were identified in Read 2 by performing an exact match search for the 20-nucleotide sgRNA sequence. Following UMI deduplication sgRNA UMI counts per cell were computed. In case of multiple sgRNAs detected for an individual cell, specific sgRNA assignment was only performed if the cells UMI count exceeded the 99th percentile.  Processing of single-cell RNA sequencing data: The Scrublet Python package was used for duplet detection and removal on each individual sample. Expected doublet rates were adjusted based on estimates by the BD Rhapsody scanner. Genes expressed at least 5 cells were retained. Following filtering, 4 datasets from 2 different screen replicates were combined in Seurat using the Harmony package. Data from Cnot8 KO, Cnot7 KO and Control organoids comes from a single cartridge/whole transcriptome assembly, with no need for integration. Only cells containing detected sgRNAs and classified as singlets were selected for further analysis. Cells were filtered based on the following criteria: UMI counts > 500, UMI counts lower than the 0.99 quantile (to remove outliers) and mitochondrial gene content < 20%. Filtered datasets were then processed using Seurat’s standard pipeline.
Bulk RNA sequencing data analysis: Paired-end RNA-seq data was processed using the Nextflow RNA-seq pipeline (https://github.com/nf-core/rnaseq) version 3.14.0 as previously described. In short, the FastQC program was used for quality control, followed by quality and adapter trimming using TrimGalore. Read alignment was performed to the Gencode vM25 (GRCm38.p6) reference by STAR. Reads were quantified using Salmon. Quality control of analysis pipelines was done by MultiQC.    Ribosome profiling data analysis: UMI- tools in combination with a custom “regular expression pattern” were used to extract ribosome footprints from fastq files. Footprints above minimal length cutoff of 20 nt were extracted, along with sample barcode 5 nt in length. For further processing adapter sequences (AGATCGGAAG) were moved to the 7 nt UMI sequence of the fastq header. Removal of reads aligning to rRNAs and tRNAs was performed using bowtie2 v2.2.5. All remaining reads were aligned to the Gencode vM25 (GRCm38.p6) reference genome by STAR v2.7.11a allowing up to one mismatch (--outFilterMismatchNmax 1) and a maximum of 20 multimappers (--outFilterMultimapNmax 20). UMI deduplication of the aligned bam files was performed using the UMI-tools dedup command. Reads mapping to the CDS regions of the genome in forward strand were counted by featureCounts program.
Nanopore data bioinformatic analysis and poly(A) length estimation: Raw nanopore reads in .pod5 format were basecalled and mapped to the reference mouse genome GRCm38 (primary assembly, obtained from the GENCODE website) using the Dorado software [https://github.com/nanoporetech/dorado] version 0.8.3 with the model rna004_130bps_sup@v5.1.0. Dorado is the software package developed and supported by Oxford Nanopore Technologies for the processing of raw nanopore data obtained on their platform. Dorado uses a deep learning-based algorithm for basecalling (transforming nanopore current data to nucleotide sequence), minimap2 aligner [PMID: 29750242] for read alignment to the genome, and a separate heuristic algorithm for estimating poly(A) tail length in each read. Importantly, successfully estimated tail lengths are stored as the “pt:i” tags in the output .bam files, while the basecalled sequences of reads contain only short fragments of poly(A) tails which are not informative for actual length of the poly(A) tails. Identification of the poly(A) tails in the reads was configured to tolerate non-adenine gaps up to 3 nt in length: tail_interrupt_length = 3. We further retained only uniquely mapped reads with mapping quality above or equal to one (in minimap2 output, quality=0 indicates that a read is a multimapper that aligns equally well to more than one genomic loci) and present “pt:i” tag. In total, ~8.1 mln reads were retrieved from four samples (two bioreplicates for each of the two conditions: Cnot8 KO and Control), with ~1.7 to ~2.5 mln reads per sample. Read counting was performed by featureCounts.
For a subset of analyses, the WTA reads were aligned and quantified with STARsolo (STAR v2.7.11b) against the Gencode vM25 (GRCm38.p6) genome, using the BD Rhapsody bead cell-barcode and UMI configuration (CB_UMI_Complex mode with single-mismatch whitelist matching).
Assembly: Gencode Mouse vM25 (GRCm38.p6)
Supplementary files format and content: Comma-separated value files and matrix files with count numbers, *_counts.csv.gz files contain Cell Barcode, Gene Symbol and RSEC adjusted counts.
Supplementary files format and content: Some single-cell WTA gene-expression matrices are provided in the standard STARsolo output format - three files per sample: *_matrix.mtx.gz (genes x cells, integer UMI counts) with *_barcodes.tsv.gz (composite cell barcodes) and _features.tsv.gz (Ensembl gene ID, gene symbol, feature type). Dial-out sgRNA files (_DO.csv.gz) give the per-cell sgRNA assignment with columns: cell_barcode, cell_index, sgRNA, umi_count, sgrna_detected.

source_name: Gastruloids

growth_protocol: Mouse embryonic stem cell culture: The dual T/Brachyury-mCherry, SOX1-GFP reporter (SBR) mouse ES cell line was cultured in in tissue culture treated 6 well plates (Thermo Scientific, 140675), in DMEM with Glutamax (Gibco, 7001569) supplemented with 10% FCS (XXX), Penicillin-Streptomycin (100U/ml, 100µg/ml respectively), non-essential amino acids (0.1 mM, Gibco, 11140050), sodium pyruvate (1mM, Thermo Scientific, 11360070), , beta-mercaptoethanol (0.11 mM, Gibco, 21985023) , 3 µM CHIR99021 (SML1046), 1 µM PD0325901 (S1036), 100u/mL LIF (produced in house). Cells were split every 48-72h, by accutase treatment followed by centrifugation at 200 g, 4min and seeding between 75 000 and 150 000 cells per well.

extract_protocol:
Gastruloids were generated as described previously. In short, murine embryonic stem cells (mESCs) are washed twice with PBS, followed by a detachment using Accutase (AT-104) for 4 min at 37C, 5% CO2. Cells were collected in 4.5 ml of embryonic stem cell medium with 10% fetal calf serum. Cells were centrifuged 200xg, 4min, 4°C and then resuspended in 10 ml PBS, followed by a second spin and PBS wash. Finally, cells were resuspended in 1 ml of N2B27 and counted using a hemocytometer. 40 µl of N2B27 with 300 cells per well were seeded in low adhesion 96 well plates (CLS7007-24EA) and incubated for 48h at 37C, 5% CO2. After 48 hours gastruloids were pulsed with 150uL of N2B27 supplemented with 3uM CHIR99021 (SML1046-5MG). Medium was refreshed using N2B27 every 24 hours for the extend of culture (between 120-144h). 120h gastruloids were harvested using P1000 cut-tips and subjected to single cell sequencing. In short gastruloids were collected in 15 ml tubes, washed once with PBS and incubated with accutase for 15 mins at 37C. Cell suspensions were resuspended using low adhesion tips and centrifuged 200xg, 4min, 4C. For sorting, cells were resuspended in FACS buffer (2% FCS and 0.5 ng/ul DAPI in PBS), passed through a 35 µm filter cap into 5 ml FACS tubes and sorted for GFP+ populations using a BD FACSAria III cell sorter using a 70 µM nozzel. Sorted cells were then spun down 15 min at 400xg, 4°C and then resuspended in BD sample buffer. Unsorted gastruloids were harvested and processed the same way, resuspended in sample buffer and then passed through 35 µm cell filters 3 times. Single cell suspensions were prepared for sequencing using the BD Rhapsody Single-Cell Analysis System with the BD Rhapsody Cartridge Kit (633733). Bead based reverse transcription, amplification and sequencing library production was performed following manufacturer’s instructions (BD Biosciences, doc ID: 210967 rev. 2.0).                                                                   Samples for Nanopore direct RNA-seq: Samples for Nanopore direct RNA-sequencing were harvested at 72 h of gastruloid development. Gastruloids were harvested using cut P1000 tips, collected in 15 ml tubes and washed 2 times in PBS+CHX. Supernatant was removed, and organoids were flash frozen in liquid nitrogen. Frozen organoids were then resuspended in ribosome lysis buffer (1x mammalian polysome buffer, 1mM DTT, 1% TritonX-100, 0.5%NP40, 25U/µL Turbo DNase, 100µg/mL CHX), incubated for 5 min on ice, followed by 5 min spin at 20 000 g at 4°C. Supernatant was collected and flash frozen in liquid nitrogen, before being stored at -80 °C before downstream processing.                    Samples for ribosome profiling: Ribosome profiling experiments were performed as previously described, with minor modifications for gastruloids. In short, gastruloids were harvested at indicated time points using cut P1000 tips and collected into 15 ml tubes. Gastruloids were washed 2 times in PBS+CHX, supernatant was removed, and organoids were flash frozen in liquid nitrogen. Frozen organoids were then resuspended in ribosome lysis buffer (1x mammalian polysome buffer, 1mM DTT, 1% TritonX-100, 0.5%NP40, 25U/µL Turbo DNase, 100µg/mL CHX), incubated for 5 min on ice, followed by 5 min spin at 20 000 g at 4°C. Supernatant was collected and flash frozen in liquid nitrogen, before being stored at -80 °C before downstream processing. Samples for RNA degradation: Gastruloids at 96 h of development were treated with 10 µM Actinomycin D (ThermoScientific, 11805017) in N2B27 and gastruloids were harvested after 0, 2 and 4 h of treatment. Gastruloids were harvested at indicated time points using cut P1000 tips and flash frozen. Frozen organoids were then resuspended in ribosome lysis buffer without cyclohexamide (1x mammalian polysome buffer, 1mM DTT, 1% TritonX-100, 0.5%NP40, 25U/µL Turbo DNase), incubated for 5 min on ice, followed by 5 min spin at 20 000 g at 4°C. Supernatant was collected and RNA was purified using the DirectZol RNA kit. Purified RNA was submitted for library preparation and sequencing to Novogene.
Reverse transcription from beads and sequencing library production was carried out according to the manufacturer’s instructions (BD Biosciences, doc ID: 210967 rev. 1.0). Library construction protocol for RNA-seq: RNA was isolated from lysates gastruloids at indicated time points as described in the Ribosome profiling section using TRIzol®  LS Reagent (Invitrogen, 10296028) and column purified with a Direct-zol RNA MiniPrep kit (Zymo Research, R2052). Libraries were prepared and sequenced at Novogene. Library construction protocol for Other:  As previously described, the sgRNA-containing region was amplified from the BD Rhapsody beads using a nested KAPA Polymerase reaction. In the first polymerase reaction (PCR1), the Rhapsody beads from each separate cartridges were resuspended and distributed into 4 separate 50 µL reactions  (100 µl KAPA HiFi HotStart ReadyMix (Roche 07958935001), 6 µl forward primer (5′-ACACGACGCTCTTCCGATCT-3′, 10 μM), 6 µl reverse primer (5′-TCTTGTGGAAAGGACGA-3′, 10 μM), 12 μl Bead RT/PCR Enhancer reagent from BD Biosciences and 72 μl nuclease-free water for a total volume of 200 μl). PCR1 conditions were as follows: initial denaturation at 95 °C for 5 min, followed by 25 cycles of denaturation 95 °C for 30 s, annealing 53 °C for 30 s, extension 72 °C for 20 s and a final extension of 10 min at 72 °C. The 4 x 50 µl PCR1 product was pooled, and beads were removed using a magnet. Amplicons were cleaned up using Agencourt AMPure XP beads (Beckman Coulter, A63881) following manufacturer’s instructions. 3 μl of cleaned up PCR1 was used as template for PCR 2 (25 µL KAPA HiFi HotStart Ready Mix, 2 μl forward primer (5′-ACACGACGCTCTTCCGATCT-3′, 10 μM), 2 μl reverse primer (CAGACGTGTGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA*C*C*G-3′, 10 μM), 18 μl nuclease-free water) for a total volume of 50 μl. PCR2 conditions were as follows: initial denaturation at 95 °C for 3 min, followed by 10 cycles of denaturation 95 °C for 30 s, annealing 60 °C for 3 min, extension 72 °C for 60 s, and a final extension of 5 min at 72 °C. Agencourt AMPure XP beads were used to clean up PCR2 product according to the manufacturer’s instructions  before the indexing PCR. Indexing PCR was performed according to BD Biosciences ‘mRNA Targeted Library Preparation’ protocol (doc ID: 210968 rev. 3.0). Library construction protocol for Ribo-seq: Ribosome profiling was conducted with some adjustments of the original method (64, 65). For RNA digestion, cell lysates were incubated with 1 U/µg of RNase 1 (Epicentre/Lucigen, 10U/µL) for 45 minutes at room temperature. Reaction was stopped by adding 4 µL of SUPERase Inhibitor (20 U/µL, SUPERase-InTM RNase Inhibitor Thermo Scientific AM2694). To eliminate free nucleotides and isolate ribosome-protected fragments, the RNase I-treated samples were processed using Amersham MicroSpin S-400 HR columns (Cytiva, 27514001). RNA extraction was performed utilizing TRIzol® LS Reagent (Invitrogen 10296028) in combination with the Direct-zolTM RNA MiniPrep kit (Zymo Research, R2052). Total RNA concentration was measured using QubitTM RNA broad range kit (Invitrogen, Q10210). Ribosome footprints were excised and extracted from a 15% TBE-UREA gel (Invitrogen, EC68852BOX) using the 20nt, 22nt, 27nt, 30nt and 60nt oligonucleotides mix as markers (1 µL of 10 µM dilution in 19µL RNase-free H2O). Extraction was performed overnight with RNA extraction buffer (300mM NaOAc pH=5.5, 1mM EDTA, 0.25% v/v SDS). T4 PNK (NEB, M0201S) end-healing reaction was performed on the ribosome footprints for 1 hour at 37˚C. Linker ligation reaction followed using T4 RnI truncated K227Q (NEB, M0351L) in combination with 20 µM of preadenylated linker for 3 hours at 22˚C (50% w/v PEG-8000, 10X T4 RNA ligase, preadenylated linker, T4 RnI truncated K227Q). Ribosomal RNA was depleted using biotinylated rRNA depletion oligos (2 µM) by mixing 10 µL of sample with 10 µL of oligo mix and incubated for 2 minutes at 80˚C, followed by a gradual decrease in temperature to 25˚C. MyOne Streptavidin dynabeads (Life Technologies, 65001) were used to recover the samples. Reverse transcription was performed on the Ribosomal RNA-free samples using SuperScriptTM IV Reverse Transcriptase (Invitrogen, 18090050). Footprints were excised and extracted from a 15% TBE-UREA gel. Extraction was performed overnight with DNA extraction buffer (10mM Tris-HCl pH=8, 300mM NaCl, 1mM EDTA). cDNA was circularized using CircLigase II (100 U/µL, Biosearch, CL4115K) for 1 hour at 60˚C. cDNA was amplified using Phusion High-Fidelity DNA polymerase (Thermo Scientific, F530S) using an individual index per sample. 6-12 cycle amplicons were visualized using an 8% TBE non-denaturing polyacrylamide gel to determine optimal cycle number. PCR was carried out with 7-9 cycles and analyzed on an 8% TBE non-denaturing polyacrylamide gel (Invitrogen, EC61252BOX). DNA was excised from gel and extracted using DNA extraction buffer by incubating overnight with gentle shaking. DNA was recovered and precipitated in 10 mM Tris-HCl pH=8. DNA was quantified using Qubit TM 1X dsDNA High Sensitivity Assay (Invitrogen, Q33230) and quality was verified using the Bioanalyzer High Sensitivity DNA kit (Agilent, 5067-4626). Samples were pooled and prepared for sequencing.               Nanopore direct RNA-seq: RNA was isolated from ribosome profiling lysate buffer lysates using Direct-zol RNA Kit, according to manufacturer’s instruction. Nanopore direct RNA-sequencing library preparation was performed using the Direct RNA Sequencing Kit (SQK-RNA004) according to manufacturer’s instructions. Samples were loaded onto MinION flow cells (FLO-MIN114).

value: Gastruloids

value: SBR

value: mESCs/gastruloid cells

value: Control


title: Gast_123_CTRL_Nanopore

description:
Library name: Gast_123_CTRL_Nanopore
Nanopore direct RNA sequencing from control 72h gastruloids, Replicate 1


title: GM38

description:
Library name: GM38
Single nucleus RNA sequencing

value: SD


title: M2K_NS_R2

description: Library name: M2K_NS_R2

value: Bone marrow


title: Gast_125_KO_Nanopore

description:
Library name: Gast_125_KO_Nanopore
Nanopore direct RNA sequencing from Cnot 8 KO 72h gastruloids, Replicate 2

value: Cnot8 KO


title: Gast_123_KO_Nanopore

description:
Library name: Gast_123_KO_Nanopore
Nanopore direct RNA sequencing from Cnot 8 KO 72h gastruloids, Replicate 1


title: GM37

description:
Library name: GM37
Single nucleus RNA sequencing


title: LIM1215-SRSF3, uninfected rep 4

city: Melbourne

first: Jamie

last: Gearing

address: 27-31 Wright St

country: Australia

institute: Hudson Institute of Medical Research

description: Library name: 4_SRSF3_uninf_R

data_processing:
Base calling was performed using Dragen BCLConvert (v3.7.4).
RNA sequencing read alignment was performed in R (v4.1.2) using the Rsubread package (v2.6.4). A genome index was built using the custom combined human and EMCV genome using the buildindex function and alignment was performed using sunjunc, with default settings.
Exon- and intron-level differential gene expression analyses followed the methods of the index package (v1.0)
A custom GTF file consisting of human and EMCV data was used for mapping. This GTF file was processed using the Annotations scripts from the Intron-reads repository (https://github.com/charitylaw/Intron-reads) in order to define exon and gene body regions. Counts were obtained using featureCounts from Rsubread (v2.8.1), with isPairedEnd and useMetaFeatures set to TRUE, allowMultiOverlap set to FALSE and strandSpecific. Intron counts were obtained by subtracting exon counts from gene body counts.
Assembly: Combined hg38 and EMCV (DQ288856.1) genomes
Supplementary files format and content: CSV file containing raw read counts mapped to introns or exons

growth_protocol: LIM1215 cells were maintained in RPMI (Sigma Aldrich) supplemented with 10% v/v foetal calf serum (FCS) and 2 mM L-glutamine (Gibco).

extract_protocol:
Cytoplasmic RNA was extracted using Cytoplasmic & Nuclear RNA Purification Kit (Norgen) according to manufacturer’s instructions.
Total RNA libraries were prepared, starting with 500 ng of total RNA, according to the Illumina Tru-Seq Stranded Total RNA-Seq protocol (protocol 1000000040499) with RiboZero depletion, using unique dual indexes. 150 base paired-end sequencing was performed on a NextSeq 2000 P3 chip, loading pooled libraries at a concentration of 1,000 pM (protocol 1000000109376). Approximately 100 million reads were obtained per sample.

treatment_protocol: Cells were infected with EMCV (MOI = 0.01) for 48 hr, or treated with IFN for 6 hr.

tag: biological replicate

value: uninfected


title: M2K_NS_R1

description: Library name: M2K_NS_R1

value: MHC-II KO (B6.129S2-H2^dlAb1-Ea/J)


title: LIM1215-SRSF3, EMCV infected rep 4

description: Library name: 4_SRSF3_EMCV_R

value: EMCV


title: GM36

description:
Library name: GM36
Single nucleus RNA sequencing

source_name: Lung metastasis

value: Lung metastasis


title: LIM1215-GFP, uninfected rep 4

description: Library name: 4_GFP_uninf_R

value: GFP


title: M1K_4PIC_R3

description: Library name: M1K_4PIC_R3

value: MHC-I KO (B6.129P2-H2-K1b^tm1Bpe H2-D1b^tm1Bpe/DcrJ)

value: pdI:dC + αCD40


title: LIM1215-GFP, EMCV infected rep 4

description: Library name: 4_GFP_EMCV_R


title: GM35

description:
Library name: GM35
Single nucleus RNA sequencing

value: Tumor (nuclei)

value: PR


title: LIM1215-SRSF3, uninfected rep 3

description: Library name: 3_SRSF3_uninf_R


title: M1K_4PIC_R2

description: Library name: M1K_4PIC_R2


title: LIM1215-SRSF3 IFN treated rep 3

description: Library name: 3_SRSF3_IFN_R

value: IFN


title: GM34

description:
Library name: GM34
Single nucleus RNA sequencing

value: Hepatic metastasis


title: LIM1215-SRSF3, EMCV infected rep 3

description: Library name: 3_SRSF3_EMCV_R


title: M1K_4PIC_R1

description: Library name: M1K_4PIC_R1


title: LIM1215-GFP, uninfected rep 3

description: Library name: 3_GFP_uninf_R


title: GM33

description:
Library name: GM33
Single nucleus RNA sequencing

source_name: Primary pancreatic tumor

value: Primary pancreatic tumor


title: M1K_PIC_R3

description: Library name: M1K_PIC_R3


title: LIM1215-GFP, IFN treated rep 3

description: Library name: 3_GFP_IFN_R


title: LIM1215-GFP, EMCV infected rep 3

description: Library name: 3_GFP_EMCV_R


title: GM32

description:
Library name: GM32
Single nucleus RNA sequencing


title: LIM1215-SRSF3, uninfected rep 2

description: Library name: 2_SRSF3_uninf_R


title: GM31

description:
Library name: GM31
Single nucleus RNA sequencing


title: LIM1215-SRSF3 IFN treated rep 2

description: Library name: 2_SRSF3_IFN_R


title: M1K_PIC_R1

description: Library name: M1K_PIC_R1


title: LIM1215-SRSF3, EMCV infected rep 2

description: Library name: 2_SRSF3_EMCV_R


title: GM30

description:
Library name: GM30
Single nucleus RNA sequencing

value: PD


title: LIM1215-GFP, uninfected rep 2

description: Library name: 2_GFP_uninf_R


title: LIM1215-GFP, IFN treated rep 2

description: Library name: 2_GFP_IFN_R


title: M1K_NS_R3

description: Library name: M1K_NS_R3

value: No stim


title: LIM1215-GFP, EMCV infected rep 2

description: Library name: 2_GFP_EMCV_R


title: GM29

description:
Library name: GM29
Single nucleus RNA sequencing


title: M1K_NS_R2

description: Library name: M1K_NS_R2


title: LIM1215-SRSF3 IFN treated rep 1

description: Library name: 1_SRSF3_IFN_R


title: GM28

description:
Library name: GM28
Single nucleus RNA sequencing

value: SD


title: LIM1215-SRSF3, EMCV infected rep 1

description: Library name: 1_SRSF3_EMCV_R


title: M1K_NS_R1

description: Library name: M1K_NS_R1

value: Bone marrow


title: LIM1215-GFP, IFN treated rep 1

description: Library name: 1_GFP_IFN_R


title: WT_4PIC_R3

description: Library name: WT_4PIC_R3

value: C57BL/6J wild-type


title: LIM1215-GFP, EMCV infected rep 1

description: Library name: 1_GFP_EMCV_R


title: GM26

description:
Library name: GM26
Single nucleus RNA sequencing

value: PRE


title: WT_4PIC_R2

description: Library name: WT_4PIC_R2


title: COVID-19 case 3

city: San Cristobal de La Laguna

first: Malena

last: Gajate Arenas

address: Astrofisico Sanchez Avenue

country: Spain

institute: Universidad de La Laguna

description: Library name: COV-3

data_processing:
The number of known miRNAs was calculated by counting data against relevant entries in the miRBase v22 database (http://mirbase.org). The miRNA expression was represented as Tags Per Million (TPM).
Supplementary files format and content: tab-delimited text file includes raw counts for each sample.
Supplementary files format and content: tab-delimited text file includes p-value, FDR and LogFC.

source_name: Oropharyngeal saliva

extract_protocol:
RNA was isolated from saliva samples using the miRNeasy Serum/Plasma Advanced Kit (Qiagen, Hilden, Germany).
RNA libraries for RNA-seq were prepared using QIAseq miRNA Library Kit (Qiagen, Hilden, Germany).

value: Oropharyngeal saliva

tag: agent

value: SARS-CoV-2 infection


title: COVID-19 case 4

description: Library name: COV-4


title: COVID-19 case 2

description: Library name: COV-2


title: GM24

description:
Library name: GM24
Single nucleus RNA sequencing


title: U2OS, scRepli-seq, aphidicolin singleG1 015

value: Bone

value: Sarcoma-derived

value: Aphidicolin


title: WT_4PIC_R1

description: Library name: WT_4PIC_R1

value: pdI:dC + αCD40


title: COVID-19 case 1

description: Library name: COV-1


title: GM23

description:
Library name: GM23
Single nucleus RNA sequencing

value: Tumor (nuclei)


title: control subject 4

description: Library name: CN-4

value: No infection


title: control subject 3

description: Library name: CN-3


title: GM22

description:
Library name: GM22
Single nucleus RNA sequencing

value: PR


title: M1K_PIC_R2

description: Library name: M1K_PIC_R2

value: MHC-I KO (B6.129P2-H2-K1b^tm1Bpe H2-D1b^tm1Bpe/DcrJ)


title: control subject 2

description: Library name: CN-2


title: WT_PIC_R2

description: Library name: WT_PIC_R2


title: WT_PIC_R3

description: Library name: WT_PIC_R3


title: control subject 1

description: Library name: CN-1


title: GM21

description:
Library name: GM21
Single nucleus RNA sequencing

value: Hepatic metastasis


title: mixed 'Patient_bone_femoral_head_biopsy' and 'scaffold_populated_with_patient_PBMC or _BMMNC'

city: Lübeck

first: Kristian

last: Händler

address: Ratzeburger Allee 160

department: Human Genetics

institute: UKSH

description:
Library name: mixed sample
10X Genomics Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit

library_source: other

data_processing:
Samples were demultiplexed and count tables were generated using Cellranger 7.0.0 and the supplied config.csv file
QC filtering and anlysis were performed using Seurat4 and standard R packages
Integration of samples was performed using harmony
Assembly: hg38 (refdata-gex-GRCh38-2020-A by 10X Genomics)
Supplementary files format and content: config.csv for demultiplexing the fastq files into individual count data of 3 samples using Cellranger 7 and higher
Supplementary files format and content: Seurat 4 object .rds containing complete integrated, filtered and annotated dataset

molecule: other

source_name: bone/ bone marrow

growth_protocol: For the cultivation of the bone chips, the HUMIMIC Chip2 system from TissUse GmbH (Berlin, Germany) was used. The microfluidic culture setup is based on the work of Sieber et al., 201814 and was modified for the purposes of this study. Cultivation was carried out over a total of six weeks with a dynamic flow of 0.5 Hz at +/- 500 mbar, generating an unidirectional pulsatile fluid flow of 5.2 µl/min. Each compartment held a volume of 400 µL medium. A half medium change was performed every 2-3 days during the first two weeks, followed by a half medium change every 3-4 days until the end of the culture. A human decellularized cancellous bone cylinder (height: 10 mm, width: 6mm) from the tissue bank of the Charité Universitätsmedizin was used as a scaffold. First, BM monocytes or PB monocytes were seeded at a density of 1.5x 106 cells/scaffold after magnetic isolation as previously described. These were differentiated into OCs within 14 days according to the protocol described above with increased cytokine concentrations of rhM-CSF and rhsRANKL to 150 ng/mL. After two weeks, 2.25x 105 MSCs and 0.75x 105 OBs were mixed per scaffold, seeded onto the OC-containing scaffold and osteogenically differentiated under dynamic conditions for an additional 14 days, as previously described. In the last step, 2x 106 BMMNCs or PBMCs were added per scaffold. These scaffolds were cultured for a further 14 days in 50 % α-MEM Eagle (PAN-Biotech, Aidenbach, Germany)/ 50 % X-vivo15 (Lonza, Basel, Switzerland) with 10 % hAB and 1 % P/S (Bio&SELL, Feucht, Germany) and 1 % Glutamax (Gibco/Thermo Fisher Scientific, Massachusetts, USA). Supernatants were collected at each medium change to monitor LDH, BALP and CTX-I. In addition, the scaffold was scanned using µCT for co-registration before and after culture.

extract_protocol:
Single-nuclei RNA sequencing was performed in single replicates of two primary BOAC systems colonized with BMMNCs (BM-BOAC), or with PBMCs (PB-BOAC), as well as a biopsy from the same donor serving as a reference. Single nuclei were extracted from fixed (using 10X Genomics tissue fixation protocol), frozen sample by a combination of physical disruption into small pieces and incubation at 37°C for 10 min in dissociation buffer (1 mg/mL Liberase TH, Merck, Darmstadt, Germany) in RPMI 1640 (Gibco/Thermo Fisher Scientific, MA, USA) before filtering through a 10 µm pluriStrainer (pluriSelect Life Science, Leipzig, Germany). Single-nuclei suspensions were subsequently pelleted by centrifugation (850 x g, 5 min, RT) the supernatant was discarded and nuclei pellets were resuspended in quenching buffer (10X Genomics, Pleasanton, CA, USA).
library construction was performed according to manufacturer's instructions (10X Genomics Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit). The samples were incubated with differently barcoded Chromium Human Transcriptome Probe Sets (10X Genomics, Pleasanton, CA, USA) for multiplexing and a single-nuclei RNA seq library was generated using the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit on a Chromium X device (10X Genomics, Pleasanton, CA, USA). Libraries were quantified and library fragment size was determined using a Qubit dsDNA HS assay (Thermo Fisher Scientific, Waltham, MA, USA) and a HS DNA Kit assay on a 2100 Bioanalyzer device (Agilent).

value: bone/ bone marrow

value: ex vivo bone and bone marrow; bone and immune cells


title: WT_PIC_R1

description: Library name: WT_PIC_R1


title: Knockout T5

city: Boston

first: Boston University

last: Microarray and Sequencing Resource

state: MA

address: 72 East Concord Street, E631

department: Microarray and Sequencing Resource

institute: Boston University

description: MDA-MB-231 cells with DOT1L knockout allele.

data_processing:
FASTQ files were aligned to human genome build hg38 using STAR (Dobin et al., 2013) (version 2.6.0c).
Ensembl-Gene-level counts for non-mitochondrial genes were generated using featureCounts (Subread package, version 1.6.2) and Ensembl annotation build 100 (uniquely aligned proper pairs, same strand).
FASTQ quality was assessed using FastQC (version 0.11.7), and alignment quality was assessed using RSeQC (version 3.0.0).
Variance-stabilizing transformation (VST) was accomplished using the varianceStabilizingTransformation function in the DESeq2 R package (version 1.23.10) (Love et al., 2014).
Assembly: hg38
Supplementary files format and content: counts.txt: 60646 x 8 matrix of counts produced by Subread featureCounts
Supplementary files format and content: varianceStabilizingTransformation.txt: 60646 x 8 matrix of variance-stabilizing-transformed (VST) expression values

growth_protocol: Cells were grown in DMEM supplemented with 5% FBS in 10 cm plates.

extract_protocol:
RNA was extracted using the Direct-zol RNA miniprep kit (Zymo Research).
Libraries were prepared from total RNA depleted for ribosomal RNA using Illumina Ribo-Zero Plus with Total RNA Stranded Library Preparation Kit and sequenced on an Illumina NextSeq 2000 instrument at 750 pM.

treatment_protocol: DOT1L/+ KO MDA-MB-231 cells were generated using gRNA targeting the first exon of DOT1L. WT and DOT1L/+ KO MDA-MB-231 cells were plated at around 50% confluency and collected the next day for RNA extraction.

value: DOT1L knockout


title: GM20

description:
Library name: GM20
Single nucleus RNA sequencing


title: Knockout T3


title: WT_NS_R3

description: Library name: WT_NS_R3

value: Bone marrow

value: No stim


title: Knockout T2


title: GM19

description:
Library name: GM19
Single nucleus RNA sequencing

value: PRE

value: PD


title: Knockout T1


title: WT_NS_R2

description: Library name: WT_NS_R2

value: C57BL/6J wild-type


title: GM18

description:
Library name: GM18
Single nucleus RNA sequencing


title: Wild-type T5

description: MDA-MB-231 cells with wild-type DOT1L allele.

value: wild-type


title: Wild-type T3


title: WT_NS_R1

description: Library name: WT_NS_R1


title: Wild-type T2


title: SUDHL5 cells, IgG CUT&RUN, bio rep 2

city: Stanford

first: Sai

last: Gourisankar

state: CA

address: 240 Pasteur Dr, Rm 4700

department: Chemical Systems Biology

institute: Stanford

description: Library name: DHL5_IgG_S2

data_processing:
The data quality was checked using fastq (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
The raw reads were trimmed from adapters with cutadapt (parameters: -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCA -A AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT).
Raw reads were aligned to hg38 human genome assembly using bowtie2 (parameters: --local --maxins 1000).
Low quality reads, duplicated reads and reads with multiple alignments were removed using samtools and Picard (https://broadinstitute.github.io/picard/).
macs2 was used to map position of peaks with FDR cutoff of 0.05.
Bedtools was used to find a consensus set of peaks by merging peaks across multiple conditions (bedtools merge), count number of reads in peaks (bedtools intersect -c) and generate genome coverage (bedtools genomecov -bga).
deepTools was used to generate coverage densities across multiple experimental conditions (deeptools computeMatrix and deeptools plotProfile) and to generate bigwig files (deeptools bamCoverage), where reads mapping to ENCODE blacklist regions were excluded.
Assembly: hg38
Supplementary files format and content: bigWig, narrowPeak (except for Input sample)
Library strategy: CUT&Run

extract_protocol:
Briefly, cells were collected and washed by PBS. After final wash, nuclei were isolated using nuclei extraction buffer (20mM HEPES pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% glycerol, 1x cOmplete protease inhibitors (Roche 11836153001), 0.5mM spermidine) and then washed twice with wash buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.5mM Spermidine, 1x cOmplete protease inhibitor). Five-hundred thousand nuclei were counted and incubated with activated Concanavalin A coated beads (EpiCypher 21-1411) and subsequently mixed with BCL6 antibodies (Cell Signaling D65C10) in antibody buffer (wash buffer + 0.01% digitonin + 2 mM EDTA) for overnight incubation on nutator in a cold room. IgG controls (normal rabbit IgG, Cell Signaling 2729) were set in parallel for antibody enrichment and specificity validation. Nuclei were then washed three times with digitonin buffer (wash buffer + 0.01% digitonin) and incubated with pAG-MNase (EpiCypher 15-1116) for 10 minutes at room temperature. After 10 minutes of incubation, nuclei were washed three times again with digitonin buffer and resuspended with prechilled low-salt, high-Ca2+ buffer (20 mM HEPES pH 7.5, 0.5mM spermidine, 1x cOmplete protease inhibitor, 10mM CaCl2) to initiate 1-hour digestion at 0°C using an ice block. The reaction was terminated by switching into stop buffer (340 mM NaCl, 20mM EDTA, 4 mM EGTA, 50 µg/mL RNaseA, 50 µg/mL glycogen) and incubating at 37°C for 10 minutes to release digested DNA from the nuclei. DNA was then purified with Ampure beads (Beckman A63882), subjected to two-sided size selection(0.5X to remove large fragment and 2X to recover desired nucleosomal DNA fragments).
Sequencing libraries were generated with KAPA Hyper Prep Kits (Roche KK8502) using 14 cycles of PCR amplification. Libraires were validated on a Tapestation 4200 (Agilent G2991BA), quantified by Qubit High Sensitivity dsDNA Kit (Life Technologies Q32854)

value: DLBCL

tag: chip antibody

value: normal rabbit IgG


title: Wild-type T1


title: S_894_26, Col4a5, Fisetin

city: Bar Harbor

first: Courtney

last: Willey

state: Maine

address: 600 Main Street

institute: The Jackson Laboratory

description:
Library name: S_894_26
Counts_S89426_GT26-02370; TPM_S89426_GT26-02370; FPKM_S89426_GT26-02370

data_processing:
JAX RNA-seq Nextflow Pipeline.
Sequence reads were trimmed and filtered using fastp, including adapter removal, low-quality base trimming, and filtering of reads with >10% N contenct or Phred scores <5.
Trimmed sequence reads were mapped to GrCM38/mm10 using STAR.
Read counts were extracted using featureCounts, and normalization using DESeq2.
Assembly: mm10
Supplementary files format and content: Tab delimited text file includes raw counts, tpm, and fpkm for each sample.

source_name: Kidney

extract_protocol:
RNA was harvested using NucleoMag RNA Kit (Macherey-Nagel). Samples with RIN >8  was used for library preparation.
RNA libraries for for RNA-seq were prepared using KAPA mRNA HyperPrep Kit (Roche) following manufacturer's protocols.

value: Kidney

value: HET


title: S4

city: Nanjing

first: Xuan

last: Wang

address: Guangzhou Road

institute: Nanjing medical university

description:
Library name: Control 3
S4

data_processing:
The raw reads were filtered by Seqtk before mapping to genome using Hisat2 (version:2.0.4). The fragments of genes were counted using stringtie(v1.3.3b) followed by TMM (trimmed mean of M values) normalization. Significant differential expressed genes (DEGs) were identified as those with a False Discovery Rate (FDR) value above the threshold (Q< 0.05) and fold-change >2 using edgeR software. n = 3 biologically independent samples.
Assembly: Hisat2 (version:2.0.4).
Supplementary files format and content: xls files include FPKM values, p values and gene list, etc, for each Sample

organism: Oryctolagus cuniculus

source_name: trachea

extract_protocol:
Total RNA (50 ng) was extracted from the tracheal mucosa in the control and high cuff pressure groups.
Libraries preparation according  to DNBSEQ standard instruction (VAHTS Universal V6 RNA-seq Library Prep Kit for BGI®). Agilent 4200 bioanalyzer was employed to evaluate the concentration and size distribution of cDNA library before sequencing with DNBSEQ-T7. The protocol of high-throughput sequencing was fully according to the manufacturer's instructions (T7).

value: trachea

value: primary cell

value: epithelial cell

value: 3 months old

value: total RNA

value: batch 1


title: SUDHL5 cells, IgG CUT&RUN, bio rep 1

description: Library name: DHL5_IgG_S1


title: S2

description:
Library name: Control 2
S2


title: S_893_26, Col4a5, Control

description:
Library name: S_893_26
Counts_S89326_GT26-02364; TPM_S89326_GT26-02364; FPKM_S89326_GT26-02364


title: single-cell RNA-seq of Arabidopsis thaliana: XPPCs transitioning to LRFCs, 6 h

city: Shanghai

first: Haotian

last: Shi

state: Shanghai

address: 800 Dongchuan RD. Minhang District

institute: Shanghai Jiaotong University

description: Library name: WT, 6h

data_processing:
The 10x Genomics Cell Ranger pipeline (version 5.0.0) was used for demultiplexing and alignment to the Arabidopsis Information Resource (TAIR 10) reference genome, generating feature-barcode unique molecular identifier (UMI) count matrices. Seurat (version 3.1.1) was used for downstream analysis. Cells with UMI or gene counts exceeding mean ±2 standard deviations were removed to filter low-quality and potential multiplets. Cells with >10% mitochondrial genes expression were also excluded. A total of 19,013 high-quality pericycle cells were retained for further analyses.  Gene expression normalization was performed using Seurat’s NormalizeData function, and variable genes were identified using the FindVariableGenes function. Batch effects were corrected using the mutual nearest neighbors (MNN) method (46) implemented in the batchelor (version 1.3.4) package.
Assembly: TAIR10
Supplementary files format and content: an RDS of aggregated sc-RNA sequencing data

source_name: Root

extract_protocol:
Five-day-old J0121 seedlings germinated on 1/2 MS medium supplemented with 10 μM N-1-naphthylphthalamic acid (NPA, auxin transport inhibitor) were transferred to fresh plates containing 10 μM 1-naphthaleneacetic acid (NAA, synthetic auxin) for 0, 1, 3, or 6 h. Root segments (~ 5 mm) were excised and digested in enzyme solution [0.4 M mannitol (Aladdin, 69-65-8), 10 mM CaCl2 (Yuanye, S24109-500g), 20 mM KCl (Sinopharm Chemical Reagent Co., Ltd, 7447-40-7), 20 mM MES (Aladdin, 145224948) PH5.7 and 0.05% BSA (Sigma, 9048-46-8), 1% cellulase R10 (Yakult, 9012-54-8), 1% cellulase RS (Yakult, 9012-54-8), 1% macerozyme R10 (Yakult, 9032-75-1), 1% hemicelluase (Sigma, 9025-56-3), 0.5% pectolyase (Yakult, 9033-35-6)] at room temperature for 2 h.Following digestion, protoplast suspensions were passed through a 40-μm cell strainer (Sorfa, 251100), washed three times with resuspension buffer [0.4 M mannitol, 20 mM KCl, 20 mM MES PH5.7 and 0.05% BSA], and kept on ice. Fluorescence-activated cell sorting (FACS; BD FACS Melody, BD-65745) was performed using wild-type root cells as autofluorescence controls. Sorting gate were adjusted based on fluorescence intensity and forward/side scatter properties, with purity mode enabled. GFP-positive cells undergoing LRFC specification and commitment were isolated following NAA treatment for 0, 1, 3, or 6 hours. Cell viability was assessed using Trypan blue staining (>85% viable), and concentration were determined with a hemocytometer.
Protoplasts were processed using the 10x Genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 following the manufacturer’s protocol. cDNA libraries were sequenced using the Illumina NovaSeq platform.

value: Root


title: SUDHL5 cells, BCL6 CUT&RUN, bio rep 2

description: Library name: DHL5_BCL6_S2

value: BCL6 (Cell Signaling D65C10)


title: S_888_26, Col4a5, Control

description:
Library name: S_888_26
Counts_S88826_GT26-02365; TPM_S88826_GT26-02365; FPKM_S88826_GT26-02365


title: S1

description:
Library name: Control 1
S1


title: single-cell RNA-seq of Arabidopsis thaliana: XPPCs transitioning to LRFCs, 3 h

description: Library name: WT, 3h


title: SUDHL5 cells, BCL6 CUT&RUN, bio rep 1

description: Library name: DHL5_BCL6_S1


title: H3

description:
Library name: Model 3
H3


title: S_884_26, Col4a5, Control

description:
Library name: S_884_26
Counts_S88426_GT26-02366; TPM_S88426_GT26-02366; FPKM_S88426_GT26-02366


title: single-cell RNA-seq of Arabidopsis thaliana: XPPCs transitioning to LRFCs, 1 h

description: Library name: WT, 1h


title: H2

description:
Library name: Model 2
H2


title: Human Neuromuscular Organoid, FATE

city: Seoul

first: Eun-Ji

last: Kwon

address: 1, Gwanak-ro

country: South Korea

institute: Seoul National University

description:
Library name: Sample 3
10X Genomics

data_processing:
Raw sequencing base call (BCL) files were demultiplexed into FASTQ files using the cellranger mkfastq pipeline. The resulting FASTQ files were processed with cellranger (10x Genomics, v-8.0.1 )count to align reads to a GRCm38 reference genome and generate a feature-barcode count matrix. This matrix, containing UMI counts per gene for each cell, served as the input for all subsequent analyses. All processing was performed using a consistent version of the Cell Ranger software to ensure reproducibility.
Assembly: Human GRCh38
Supplementary files format and content: Tab-seperated values files and matix files

source_name: Organoid

extract_protocol:
For single-cell RNA sequencing (scRNA-seq), 6 of day 70 organoids (HGPS-NMOs, Edit-NMOs, and FATE-NMOs) were washed with DPBS and transferred to 1.5 mL microcentrifuge tubes. Organoids were then incubated with 1 mL of Liberase™ TL Research Grade (Roche) at 37°C for 30 minutes, following the manufacturer’s protocol. After enzymatic digestion, mechanical dissociation was performed by pipetting until a single-cell suspension was achieved. The suspension was passed through a 40 μm cell strainer (Falcon®) to eliminate cell aggregates and debris. The resulting cell suspension was washed with DPBS and resuspended in HypoThermosol® FRS (STEMCELL Technologies), followed by quality assessment using the Celinius single-cell QC platform.
Single cells were partitioned into Gel Beads-in-emulsion (GEMs) using a Chromium controller Within each GEM, mRNA was captured and barcoded. scRNA-seq libraries were subsequently generated following the manufacturer's protocol (10x genomics, CG000315), which includes reverse transcription, cDNA amplification, and library construction Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics, PN1000268) The quality and size distribution of the resulting cDNA and final libraries were assessed, and concentrations were quantified. Libraries were sequenced on a compatible platform using NovaSeq 6000 platform(Illumina) and a paired-end configuration, with read lengths structured to capture the cell barcode, Unique Molecular Identifier (UMI), and the transcript sequence.

value: Organoid

value: derived from FATE-treated iPSCs


title: S_872_26, Col4a5, Fisetin

description:
Library name: S_872_26
Counts_S87226_GT26-02371; TPM_S87226_GT26-02371; FPKM_S87226_GT26-02371


title: single-cell RNA-seq of Arabidopsis thaliana: XPPCs transitioning to LRFCs, 0 h

description: Library name: WT, 0h


title: S_867_26, Col4a5, Fisetin

description:
Library name: S_867_26
Counts_S86726_GT26-02372; TPM_S86726_GT26-02372; FPKM_S86726_GT26-02372


title: H1

description:
Library name: Model 1
H1


title: DO_Gast146

description:
Library name: DO_Gast146
dial-out nested PCR for sgRNA amplification for 120h gastruloids, Cnot7 KO, Cnot8 KO, control

value: Gastruloids

value: SBR

value: mESCs/gastruloid cells


title: Human Neuromuscular Organoid, HGPS

description:
Library name: Sample 1
10X Genomics

value: derived from HGPS iPSCs


title: RA2_PFC

first: Julia

last: Belk

state: California

address: 450 Serra Mall

institute: Stanford University

description: Library name: RA2_PFC

library_source: genomic single cell

data_processing:
Read trimming, deduplication, and alignment was performed using the cellranger-atac count pipeline from 10X Genomics (v2.1.0). We used a previously described custom hg38 reference genome which has been hard masked to prevent spurious alignments of mtDNA to the nuclear genome, which is standard practice for mtDNA variant calling. We then imported fragments files from cellranger-atac count into ArchR (v1.0.3) for downstream clustering and analysis.
Assembly: hg38
Supplementary files format and content: 10X fragments file

source_name: brain

extract_protocol:
Each brain region was cut into 2g pieces, minced, and placed into individual GenteMACS C tubes. Enzymatic digest was performed according to the Neural Tissue Dissociation Kit (P) (Cat# 130-092-628; Miltenyi Biotec). Briefly, an enzyme cocktail consisting of 250µl Enzyme P, 50µl Enzyme A, 9500µl Buffer X, 100µl Buffer Y was added to each tube. The mixture was run through a GentleMACS Octo Dissociator (37C_NTDK_1) for 22 minutes. 10ml HBSS (with Calcium and Magnesium) was added to the suspension and filtered through a 100µm filter into a new 50ml tube, and an additional 10ml of HBSS (with Calcium and Magnesium) was used to rinse the filter. Each sample was centrifuged at 300 x g for 10 min at 4 °C. Upon removing the supernatant, the pellet was resuspended with 12.4 ml ice-cold PBS. 3.6 mL Debris Removal Solution was then added and mixed well. Finally, 8mL ice-cold PBS was overlayed on top of the mixture. Each gradient was centrifuged at 3000x g for 10min at 4 °C. The supernatant containing the removed myelin debris was aspirated. The remaining pellets were resuspended and pooled with a total volume of 3mL of 1x RBC Lysis and incubated for 5min. The RBC Lysis was quenched with 7mL PBS and centrifuged at 300 x g for 5 min at 4 °C. The supernatant was aspirated, and the pellet was resuspended with 1mL of PBS for staining.
Cells were pelleted at 500 × g for 5 min at 4 °C, resuspended in PBS, and fixed by adding 16% formaldehyde to a final concentration of 1% for 10 min at room temperature with occasional mixing. The reaction was quenched with 2 M glycine (0.125 M final), followed by centrifugation and washes with ice-cold PBS and wash buffer. Fixed cells were lysed in chilled lysis buffer, washed, and counted using trypan blue staining. Cell concentrations were adjusted according to the 10x Genomics recommendations, and samples were processed using the Chromium Single Cell ATAC workflow for transposition, barcoding, and library preparation.

value: brain

tag: sort

value: live, CD66b- cells

tag: Sex

value: M


title: Human Neuromuscular Organoid, EDIT

description:
Library name: Sample 2
10X Genomics

value: derived from genome-edited iPSCs


title: S_866_26, Col4a5, Control

description:
Library name: S_866_26
Counts_S86626_GT26-02367; TPM_S86626_GT26-02367; FPKM_S86626_GT26-02367

value: Kidney

value: HET


title: WTA_Gast146

description:
Library name: WTA_Gast146
scRNA-seq WTA of 120h gastruloids, Cnot7 KO, Cnot8 KO, control


title: RA2_Ce

description: Library name: RA2_Ce


title: Sample 5 - Endometrial Cells

city: Herston

first: Sally

last: Mortlock

state: Queensland

address: AWaGHR Centre, School of Public Health, The University of Queensland

department: School of Public Health

institute: University of Queensland

description:
Library name: Sanmple 5
10x Genomics

data_processing:
Raw sequencing data was processed to fastq files using the mkfastq function in Cell Ranger (v6.0.1). Reads were aligned to the hg38 human reference, filtered for valid cell barcodes and unique molecular identifiers (UMIs) and counted using the count function.
Assembly: hg38 human reference
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Endometrium

extract_protocol:
Endometrial samples were either fixed in 4% PFA overnight at 4C (n=3) or placed into cold DMEM:F12 collection medium supplemented with 5% newborn calf serum and antibiotics/antimycotic and immediately transported at 4C to the laboratory for processing (n=5). Endometrial tissue was digested to single cells within 18 hours. The functionalis was removed by scraping the tissue off the surface of the myometrium and finely minced, then the full basalis was recovered by dissecting 1-2 mm myometrium with attached endometrium (visible microscopically) and finely dicing the tissue.The minced tissue underwent a 2-step enzymatic/mechanical dissociation with vigorous trituration every 5 min at 37C in PBS (Gibco) containing collagenase type I (5 mg/mL; Worthington), DNAse I (0.08 mg/mL; Worthington) and glucose (5mM; Merck), then strained (40 µM sieve, Falcon) to separate  stromal cells from epithelial gland fragments. Red blood cells in the stromal fraction were removed by Ficoll-paque (Sigma-Aldrich) density gradient separation. Epithelial cells underwent a second digestion with a similar cocktail using collagenase type II (0.8 mg/ml, Worthington) and DNAse I 0.16 mg/mL, then epithelial cells were strained using a 40 µM sieve. The single cell fractions were counted and combined for FACS staining. Single cell suspensions were blocked with Fc Receptor blocker (FcR; 2 µL/106 cells, Miltenyi Biotec) and Rat IgG (4.4 µg/106 cells; Jackson ImmunoResearch) as described (Wyatt et al., 2021). Cells were then distributed in tubes as unstained, viability and Fluorescence minus 1 (FMO) controls (up to 5X105 cells in 100 µL) and the full antibody panel (remaining cells at 5X105/100µL at the same final antibody concentration) and incubated with conjugated antibodies, PE-CD325 (N-cadherin) (10 µg/mL), BV421-CD15 (SSEA1) (1.25 µg/mL; BioLegend), APC-SUSD2 (1.25 µg/mL; BioLegend,), BB515-CD326 (EpCAM) (0.25 µL/5X105cells, BD Biosciences) and CD45 (pan leukocyte exclusion marker) PECy7-CD45 (0.25 µL/5X105cells, BD Biosciences), for 25 min at 4ºC. Propidium iodide (PI; 62.5 ng/mL; BD Biosciences) was added 10 min before sorting (Wyatt et al., 2021). Compensation Plus Particle Set beads (60 µL/test; BD Biosciences) were stained as single staining protocols with the above antibodies following manufacturer’s instructions. Labelled endometrial cells underwent FACSorting of endometrial stem/progenitor cell subpopulations and their differentiated progeny (epithelial (EpCAM+) progenitors N-cadherin+/SSEA1-; N-cadherin+/SSEA1+; N-cadherin-/SSEA1+; mature epithelial cells, EpCAM+/N-cadherin-/SSEA1-) (Nguyen et al., 2017) and eMSC (SUSD2+; SUSD2- fibroblasts) (Masuda et al., 2012) using an Influx Cytopeia-BD 5 laser sorter and BD FACS™ Sortware software (Monash University FlowCore).
The 6 cell fractions were tagged using TotalSeq A anti-human hashtags. The stem/progenitor cell populations were pooled and mature epithelial and stromal cells added for scRNAseq for each of the 5 biological samples to generate barcoded single-cell 3′ gene expression libraries with the Chromium Single-cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics) and their matched HTO library to resolve the hashtags (BioLegend). The quality of the libraries was assessed by Agilent Bioanalyzer High Sensitivity DNA chip. Libraries were sequenced to an average depth of 384,676 reads/cell on the Illumina NovaSeq6000 at UQ. Sequence quality checks revealed that HTO library construction had failed; and were excluded from subsequent analyses.

value: Endometrium


title: S_862_26, Col4a5, Fisetin

description:
Library name: S_862_26
Counts_S86226_GT26-02373; TPM_S86226_GT26-02373; FPKM_S86226_GT26-02373


title: Gast_125_Ribo

description:
Library name: Gast_125_Ribo
Ribosome Profiling from Cnot7 KO, Cnot8 KO and control organoids, Replicate 3

data_processing:
Bioinformatic analysis of single-cell sequencing data: Raw sequencing data in the form of BCL files, were demultiplexed using Illumina bcl2fastq v2.20 with default settings. Up to one mismatch in the sample barcode sequences was allowed. Resulting fastq files were processed using the BD Rhapsody Pipeline (version 1.9.1) hosted at the Seven Bridges’ cloud platform (https://www.sevenbridges.com/). A custom mouse reference genome, based on Gencode vM25 (GRCm38.p6), was generated and combined with the 669 sgRNAs sequences. Based on STAR aligner, a custom STAR reference genome was created and the Refined Putative Cell Calling option was disabled during the pipeline execution. For downstream analysis, unique molecular identifier (UMI) count data was corrected by BD Genomics Recursive Substitution Error Correction (RSEC).  Bioinformatic analysis of dial-out data: PCR dial-out data was processed using an in-house custom Python script as previously described. Three 9-nucleotide cell barcodes were extracted from the 97 possible barcodes from Read 1. Up to one mismatch was allowed for each barcode (Hamming distance of 1). The script retrieved the 8-nucleotide UMI sequences from Read 1 as per the BD manual. For valid barcodes, sgRNAs were identified in Read 2 by performing an exact match search for the 20-nucleotide sgRNA sequence. Following UMI deduplication sgRNA UMI counts per cell were computed. In case of multiple sgRNAs detected for an individual cell, specific sgRNA assignment was only performed if the cells UMI count exceeded the 99th percentile.  Processing of single-cell RNA sequencing data: The Scrublet Python package was used for duplet detection and removal on each individual sample. Expected doublet rates were adjusted based on estimates by the BD Rhapsody scanner. Genes expressed at least 5 cells were retained. Following filtering, 4 datasets from 2 different screen replicates were combined in Seurat using the Harmony package. Data from Cnot8 KO, Cnot7 KO and Control organoids comes from a single cartridge/whole transcriptome assembly, with no need for integration. Only cells containing detected sgRNAs and classified as singlets were selected for further analysis. Cells were filtered based on the following criteria: UMI counts > 500, UMI counts lower than the 0.99 quantile (to remove outliers) and mitochondrial gene content < 20%. Filtered datasets were then processed using Seurat’s standard pipeline.
Bulk RNA sequencing data analysis: Paired-end RNA-seq data was processed using the Nextflow RNA-seq pipeline (https://github.com/nf-core/rnaseq) version 3.14.0 as previously described. In short, the FastQC program was used for quality control, followed by quality and adapter trimming using TrimGalore. Read alignment was performed to the Gencode vM25 (GRCm38.p6) reference by STAR. Reads were quantified using Salmon. Quality control of analysis pipelines was done by MultiQC.    Ribosome profiling data analysis: UMI- tools in combination with a custom “regular expression pattern” were used to extract ribosome footprints from fastq files. Footprints above minimal length cutoff of 20 nt were extracted, along with sample barcode 5 nt in length. For further processing adapter sequences (AGATCGGAAG) were moved to the 7 nt UMI sequence of the fastq header. Removal of reads aligning to rRNAs and tRNAs was performed using bowtie2 v2.2.5. All remaining reads were aligned to the Gencode vM25 (GRCm38.p6) reference genome by STAR v2.7.11a allowing up to one mismatch (--outFilterMismatchNmax 1) and a maximum of 20 multimappers (--outFilterMultimapNmax 20). UMI deduplication of the aligned bam files was performed using the UMI-tools dedup command. Reads mapping to the CDS regions of the genome in forward strand were counted by featureCounts program.
Nanopore data bioinformatic analysis and poly(A) length estimation: Raw nanopore reads in .pod5 format were basecalled and mapped to the reference mouse genome GRCm38 (primary assembly, obtained from the GENCODE website) using the Dorado software [https://github.com/nanoporetech/dorado] version 0.8.3 with the model rna004_130bps_sup@v5.1.0. Dorado is the software package developed and supported by Oxford Nanopore Technologies for the processing of raw nanopore data obtained on their platform. Dorado uses a deep learning-based algorithm for basecalling (transforming nanopore current data to nucleotide sequence), minimap2 aligner [PMID: 29750242] for read alignment to the genome, and a separate heuristic algorithm for estimating poly(A) tail length in each read. Importantly, successfully estimated tail lengths are stored as the “pt:i” tags in the output .bam files, while the basecalled sequences of reads contain only short fragments of poly(A) tails which are not informative for actual length of the poly(A) tails. Identification of the poly(A) tails in the reads was configured to tolerate non-adenine gaps up to 3 nt in length: tail_interrupt_length = 3. We further retained only uniquely mapped reads with mapping quality above or equal to one (in minimap2 output, quality=0 indicates that a read is a multimapper that aligns equally well to more than one genomic loci) and present “pt:i” tag. In total, ~8.1 mln reads were retrieved from four samples (two bioreplicates for each of the two conditions: Cnot8 KO and Control), with ~1.7 to ~2.5 mln reads per sample. Read counting was performed by featureCounts.
For a subset of analyses, the WTA reads were aligned and quantified with STARsolo (STAR v2.7.11b) against the Gencode vM25 (GRCm38.p6) genome, using the BD Rhapsody bead cell-barcode and UMI configuration (CB_UMI_Complex mode with single-mismatch whitelist matching).
Assembly: Gencode Mouse vM25 (GRCm38.p6)
Supplementary files format and content: Comma-separated value files and matrix files with count numbers, *_counts.csv.gz files contain Cell Barcode, Gene Symbol and RSEC adjusted counts.
Supplementary files format and content: Some single-cell WTA gene-expression matrices are provided in the standard STARsolo output format - three files per sample: *_matrix.mtx.gz (genes x cells, integer UMI counts) with *_barcodes.tsv.gz (composite cell barcodes) and _features.tsv.gz (Ensembl gene ID, gene symbol, feature type). Dial-out sgRNA files (_DO.csv.gz) give the per-cell sgRNA assignment with columns: cell_barcode, cell_index, sgRNA, umi_count, sgrna_detected.
Library strategy: Ribo-Seq

value: Cnot7 KO, Cnot8 KO, control


title: LIM1215-SRSF3, uninfected rep 1

description: Library name: 1_SRSF3_uninf_R

value: uninfected


title: RA2_BM_2

description: Library name: RA2_BM_2

source_name: bone marrow

value: bone marrow


title: Sample 4 - Endometrial Cells

description:
Library name: Sample 4
10x Genomics


title: S_861_26, Col4a5, Control

description:
Library name: S_861_26
Counts_S86126_GT26-02368; TPM_S86126_GT26-02368; FPKM_S86126_GT26-02368


title: Gast_124_Ribo

description:
Library name: Gast_124_Ribo
Ribosome Profiling from Cnot7 KO, Cnot8 KO and control organoids, Replicate 2


title: Sample 3 - Endometrial Cells

description:
Library name: Sample 3
10x Genomics


title: RA2_BM_1

description: Library name: RA2_BM_1


title: S_857_26, Col4a5, Fisetin

description:
Library name: S_857_26
Counts_S85726_GT26-02374; TPM_S85726_GT26-02374; FPKM_S85726_GT26-02374


title: Gast_123_Ribo

description:
Library name: Gast_123_Ribo
Ribosome Profiling from Cnot7 KO, Cnot8 KO and control organoids, Replicate 1


title: Sample 2 - Endometrial Cells

description:
Library name: Sample 2
10x Genomics


title: RA1_Pu

description: Library name: RA1_Pu

value: F


title: S_856_26, Col4a5, Control

description:
Library name: S_856_26
Counts_S85626_GT26-02369; TPM_S85626_GT26-02369; FPKM_S85626_GT26-02369


title: Pseudomonas syringae pv. porri CFBP 1770, infected with phage KIL5; control

city: Heverlee

first: Rob

last: Lavigne

address: Kasteelpark Arenberg 20

department: Biosystems

country: Belgium

institute: KU Leuven

description: Library name: Sample 4

data_processing:
Real time basecalling and demultiplexing by the Guppy software (v3.2.10) in high accuracy mode.
read processing, mapping and identification of the viral transcription start sites (TSS) and termination sites (TTS) was performed according to the ONT-cappable-seq2 automated data-analysis workflow (https://github.com/LoGT-KULeuven/ONT-cappable-seq2)
Assembly: KIL3b (KU130131), KIL5 (KU130130), pphageB21 (MZ868714.1), f2 (NC_013638), KNP (NC_047827)
Supplementary files format and content: .txt files contain information on the location and efficiency of transcription start and termination sites of the phages

organism:
Pseudomonas coronafaciens pv. porri
Pseudomonas phage vB_PsyM_KIL5

source_name: bacterial cells infected with phage

growth_protocol: An overnight culture of the bacterial host was diluted in 30 ml of fresh culture medium and grown to an OD600 of 0.3. At this point phages were added  and samples were taken throughout the entire infection cycle of the phage

extract_protocol:
All samples were pooled together and homogenized. The resulting sample was subjected to hot phenol treatment and ethanol precipitation to extract and purify the RNA
Total RNA was enriched for primary transcripts using the ONT-cappable-seq enrichment protocol. In parallel, control samples were subjected to similar environmental conditions. The RNA was reverse transcribed, PCR amplified and barcoded accoriding to Oxford Nanopore Technology cDNA-PCR protocol (SQK-PCB109)

treatment_protocol: Total RNA was extracted using hot-phenol extraction, followed by DNAse treatment. The enriched samples were subjected to an enrichment procedure based on streptavidin beads to enrich for primary transcripts. For the control samples the enrichment steps were omitted.

value: bacterial cell

value: no enrichment

tag: infection stage

value: infected with phage KIL5


title: Sample 1 - Endometrial Cells

description:
Library name: Sample 1
10x Genomics

tag: tissue


title: S_852_26, Col4a5, Fisetin

description:
Library name: S_852_26
Counts_S85226_GT26-02375; TPM_S85226_GT26-02375; FPKM_S85226_GT26-02375


title: Pseudomonas syringae pv. porri CFBP 1770, infected with phage KIL5; enriched

description: Library name: Sample 3

tag: cell type

value: enriched for primary transcripts


title: RA1_Ce

description: Library name: RA1_Ce


title: RA1_Co

description: Library name: RA1_Co