title: Tcf7+22kb gp33-Tet+ 8dpi Arm rep2

city: Stanford

first: Andy

last: Chen

address: 240 Pasteur Dr.

institute: Stanford

description: Library name: T4

library_source: genomic single cell

data_processing:
Fastq files were processed with cellranger-atac-2.1.0/cellranger-atac count
Assembly: mm10
Supplementary files format and content: fragment file

source_name: spleen

extract_protocol:
Spleens were mechanically dissociated in MACS buffer (0.1% BSA and 2 mM EDTA), and LCMV-gp33-Tetramer+ CD8+ T cells were isolated by sort purification on 8 days post-infection with LCMV.
scATAC-seq experiments were performed on the 10x Chromium platform

treatment_protocol: Mice were infected with LCMV-Armstrong by intraperitoneal injection with 2 x 105 PFU of virus.

value: spleen

value: CD8+ T cell

value: LCMV-Armstrong


title: D1_E05

status: Public on May 31 2025

type: SRA

city: Cambridge

first: Felix

last: Krueger

address: Granta Park

department: Bioinformatics

country: United Kingdom

institute: Altos Labs

library_source: genomic

data_processing:
All sequencing was carried out on HiSeq instrument. BS-seq libraries were sequenced in 96-plex pools using 125-bp paired-end reads. RNA-seq libraries were pooled as 96-plex or 192-plex pools and sequenced using 75-bp paired-end reads.
Alignments of the single-cell bisulfite sequencing were performed using Bismark as well as subsequent CpG methylation and GpC accessibility calling.
Genome_build: GRCm38
Supplementary_files_format_and_content: The Bismark CpG coverage report is tab-delimited, uses 1-based genomic coordinates for every covered cytosine position in the experiment and is in the following format: <chromosome> <start position> <end position> <methylation percentage> <count methylated> <count non-methylated>

molecule: genomic DNA

organism: Mus musculus

growth_protocol: E14 mESC were cultured in 2i+LIF media consisting out of N2B27 medium, composed out of DMEM/F12 (Life Technologies, 12634010) and Neurobasal (Life Technologies, 21103049) in a volume/volume (v/v) ratio 1:1, 0.1 mM 2-mercaptoethanol, 2 mM L-Glutamine, 1:200 v/v N2 (Life Technologies, 17502048) and 1:100 v/v B27 supplement (Life Technologies, 17504044), supplemented with 3 µM CHIR99021, 1 µM PD0325901 and 20 ng/ml of leukemia inhibitory factor (LIF)  (all Department of Biochemistry, University of Cambridge). mESCs were cultured on tissue culture plastic pre-coated with 0.1 % gelatine in H2O. Cultures were passaged when approaching confluence (every 2 days). All cells were cultured in a humidified incubator at 37 °C in 5 % CO2 and 20 % O2. mESCs were subject to routine mycoplasma testing using the MycoAlert testing kit (Lonza) and tested negative. For induction for 2i-relase experiment, the cell been rinsed with PBS before the culture media was changed to serum + LIF medium consisting of DMEM (Life Technologies, 10566-016), containing 15% Fetal Bovine Serum (FBS) (Gibco, 10270106), 1x non-essential amino acids (NEAA) (Life Technologies, 11140050), 0.1 mM 2-mercaptoethanol (Life Technologies, 31350-010), 2 mM L-Glutamine (Life Technologies, 25030-024), supplemented with 20 ng/ml of LIF (Department of Biochemistry, University of Cambridge).

extract_protocol:
0h, 24h, and 48h after induction of 2i-release  the cells been dissociated into single cells using accutase before flow sorting  (BD Influx) into 96-well plate containing 2.5μl of methylase reaction buffer (1 × M.CviPI Reaction buffer (NEB), 2 U M.CviPI (NEB), 160 μM S-adenosylmethionine (NEB), 1 U μl−1 RNasein (Promega), 0.1% IGEPAL CA-630 (Sigma)). Samples were incubated for 15 min at 37 °C to methylate accessible chromatin before the reaction was stopped with the addition of RLT plus buffer (Qiagen) and samples frozen down and stored at −80 °C before processing.
E14 mESC were cultured in 2i+LIF media. For induction for 2i-relase experiment, the cell been rinsed with PBS before the culture media was changed to serum + LIF medium. 0h, 24h, and 48h after induction of 2i-release single cells were distracted and sc-NMT sequencing was performed.

tag: strain

tag: cell type

value: embryonic stem cell


title: T24 cells, Control 1

city: Xi'an

first: wen

last: liu

address: New temple Road num 1

country: China

institute: Tangdu Hospital of Air Force Medical University

library_source: transcriptomic

data_processing:
Raw data (raw reads) of fastq format were firstly processed through fastp software. In this step, clean data (clean reads) were obtained by removing reads containing adapter
Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5
featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.
Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.20.0).
Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package
Assembly: hg38
Supplementary files format and content: RPKM values for each Sample

molecule: total RNA

organism: Homo sapiens

source_name: cell line

growth_protocol: T24 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
Total RNA was extracted using chloroform, isopropanol and washed with ethanol
RNA libraries for RNA-seq were prepared using NEBNext Ultra RNA Library Prep Kit for illumina following manufacturer's protocols.

treatment_protocol: T24 cells were treated with 3.84μM Caba-780 for 24 hours,then collected cells and dissolved in Trizol

tag: tissue

value: cell line

tag: cell line

value: bladder cancer

tag: genotype

value: WT

tag: treatment


title: Participants 377,control, 12 months

city: Puncak Alam

first: Kalavathy

last: Ramasamy

state: Selangor

address: UiTM Cawangan Selangor Kampus Puncak Alam Bandar Puncak Alam

department: Faculty of Pharmacy

country: Malaysia

institute: Universiti Teknologi MARA (UiTM)

description: Library name: t377

data_processing:
Raw sequencing reads were assessed for quality using FastQC. Adapter sequences and low-quality bases (Phred score <20) were trimmed using Cutadapt. High-quality reads were aligned to the human reference genome (GRCh38) using HISAT2 with default parameters. Gene expression levels were quantified using StringTie, and raw count matrices were generated. Normalization was performed using  Trimmed Mean of M-values (TMM). Differential expression analysis was conducted using DESeq2, and significantly differentially expressed genes (adjusted p-value <0.05) were identified.
Assembly: hg38
Supplementary files format and content: excel file that include raw counts for each samples.

molecule: polyA RNA

extract_protocol:
RNA was prepared using the NucleoSpin RNA Mini Kit (Macherey-Nagel, Düren, Germany) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Messenger RNA was purified from total RNA using poly-T oligo-attachedmagnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNAsynthesis using either dUTP for directional library or dTTP for non-directional library

value: Whole blood

tag: diagnosis

value: Cognitive frailty

tag: age

tag: gender

value: male


title: D1_E04


title: WT gp33-Tet+ 8dpi Arm rep2

country: USA

description: Library name: T2


title: Tcf7+22kb gp33-Tet+ 8dpi Arm rep1

description: Library name: T3


title: Human frontal hair follicles,DHT3,DAY3

city: Guangzhou

first: shizhao

address: guangzhou avenue north

institute: southern medical university

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

source_name: hair follicle

growth_protocol: Hair follicles were maintained in Williams' E medium supplemented with 2 mM L-glutamine, 10 μg/mL insulin, 10 ng/mL hydrocortisone, 10 ng/mL adrenaline, and antibiotics (penicillin and streptomycin).

extract_protocol:
RNA was harvested using Rneasy mini plus kit (Qiagen). 1.3 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols.

treatment_protocol: 5*10^-6 mol/L of DHT was used to organ culture AGA frontal hair follicles.

value: hair follicle

value: DHT


title: Participants 377,control, baseline

description: Library name: b377


title: D1_E02


title: WT gp33-Tet+ 8dpi Arm rep1

description: Library name: T1


title: Human frontal hair follicles,DHT2,DAY3


title: D1_E03


title: Tcf7+22kb gp33-Tet+ 8dpi Arm rep2

description: Library name: T4

library_source: transcriptomic single cell

data_processing:
Fastq files were processed with cellranger-7.0.0/cellranger count
Assembly: mm10
Supplementary files format and content: filtered_feature_bc_matrix

molecule: cytoplasmic RNA

extract_protocol:
Spleens were mechanically dissociated in MACS buffer (0.1% BSA and 2 mM EDTA), and LCMV-gp33-Tetramer+ CD8+ T cells were isolated by sort purification on 8 days post-infection with LCMV.
scRNA-seq libraries were prepared using a 10× 5′ Single Cell Immune Profiling Solution Kit


title: Human frontal hair follicles,DHT1,DAY3


title: Participants 377,control, 6 months

description: Library name: p377


title: Tcf7+22kb gp33-Tet+ 8dpi Arm rep1

description: Library name: T3


title: Participants 304,control, 12 months

description: Library name: t304

value: female


title: D1_E01


title: Human frontal hair follicles,control3,DAY3


title: Human frontal hair follicles,control2,DAY3


title: WT gp33-Tet+ 8dpi Arm rep2

description: Library name: T2


title: D1_D12


title: Human frontal hair follicles,control1,DAY3


title: Participants 302,control, 12 months

description: Library name: t302


title: D1_D11


title: WT gp33-Tet+ 8dpi Arm rep1

description: Library name: T1


title: Participants 379, intervention, baseline

description: Library name: b379


title: D1_D10


title: Participants 302,control, baseline

description: Library name: b302


title: Participants 285, intervention, 6 months

description: Library name: p285


title: SKMEL28_corin_2

city: Boston

first: Robert

last: Fisher

state: MA

address: 609 Albany St

department: Dermatology

institute: Boston University

data_processing:
Paired-end RNA-seq reads (2x150bp) were quality and adaptor-trimmed using Trimmomatic v.0.36.
Trimmed reads were mapped to the ENSEMBL reference human genome (GRCh38) using STAR v.2.5.2b.
BAM files were generated for downstream splicing and differential gene expression analysis.
Unique gene counts were calculated using featureCounts from Subread v.1.5.2 and differential expression analysis was performed using DESeq2 based on hit counts.
The Wald test was used to produce log2 fold change and p-values between comparisons. A threshold of p < 0.01 and LF > |0.5| was used to call significant changes in gene expression.
Assembly: hg38/Gencodev44
Supplementary files format and content: .txt; hit counts

growth_protocol: Cells were maintained at 37C at 5% CO2 and cultured in DMEM (Invitrogen, ThermoFisher Scientific) supplemented with 1% penicillin/streptomycin, L-glutamine (2mM), and 10% FBS.

extract_protocol:
Total RNA was extracted from melanoma cell lines treated with corin or vehicle control (DMSO) using the Qiagen RNeasy Mini Kit. Samples were quantified using nanodrop and sequenced by Azenta Inc.
Libraries were constructed by Azenta Inc.

treatment_protocol: Cells were treated with 2.5uM corin for 24h (or DMSO))

value: melanoma

value: corin


title: D1_D09


title: SKMEL28_corin_1


title: Participants 285, intervention, baseline

description: Library name: b285


title: CD4Tcells, IL7&IL12&IL18-treated

city: Seoul

first: Jaehyun

last: Choi

address: 50, Yonsei-ro, Seodaemun-gu

department: systembiology

country: South Korea

institute: Yonsei university

description:
Library name: Bys_Th1
10x Genomics

data_processing:
Demultiplexing, barcode processing, gene quantification, and aggregation were performed using Cell Ranger (v8.0.1).
Assembly: mouse mm10
Supplementary files format and content: Matrix Market format(matrix.mtx.gz) containing UMI counts per gene per cell. Accompanied by features.tsv.gz (gene annotation) and barcodes.tsv.gz (cell barcodes). Generated using Cell Ranger (v8.0.1).

source_name: Spleen

extract_protocol:
FACS sorted CD44 high CD4 T cells in spleen
Single-cell RNA libraries were constructed using the 10x Genomics Chromium Single Cell 3' v3 protocol, which employs Gel Beads-in-Emulsion(GEMs) technology to uniquely barcode transcripts from individual cells using ~3,500,000 barcodes. Poly(dT) primers were used to capture polyadenylated mRNAs, and barcoded full-length

value: Spleen

value: CD44 high CD4 T cells

tag: Sex


title: SKMEL28_DMSO_2

value: DMSO


title: CD4Tcells, IL7&IL33-treated

description:
Library name: IL7_IL33
10x Genomics


title: Participants 304,control, baseline

description: Library name: b304


title: D1_D08


title: CD4Tcells, IL7&IL25&IL33-treated

description:
Library name: IL7_IL25_IL33
10x Genomics


title: D1_D06


title: CD4Tcells, IL7&IL25-treated

description:
Library name: IL7_IL25
10x Genomics


title: Participants 245,control, 6 months

description: Library name: p245


title: SKMEL24_corin_1


title: SKMEL28_DMSO_1


title: Participants 245,control, 12 months

description: Library name: t245


title: Participants 245,control, baseline

description: Library name: b245


title: CD4Tcells, IL7

description:
Library name: IL7
10x Genomics


title: D1_D03


title: CD4Tcells, IL7&IL1b&IL23-treated

description:
Library name: Bys_Th17
10x Genomics


title: SKMEL24_DMSO_2


title: Participants 219,control, 6 months

description: Library name: p219


title: CD4Tcells, non-treated

description:
Library name: MP
10x Genomics

value: non-treated


title: SKMEL24_corin_2


title: D1_D07


title: SKMEL5_corin_2


title: Participants 215,control, 6 months

description: Library name: t215


title: Participants 219,control, baseline

description: Library name: b219


title: D1_D01


title: Ischemic hemisphere of wild-type mouse (Ppm1d+/+) post-MCAO

city: Beijing

first: Wei-Na

last: Jin

state: Beijing

address: NO.119, Southwest of the Fourth Ring Road, Fengtai District

department: China National Clinical Research Center for Neurological Diseases

institute: Beijing Tiantan Hospital, Capital Medical University

description:
Library name: Sample 2
Fresh frozen Spatial transcriptome sequencing

data_processing:
Raw FASTQ files and histology images were processed using 10× Genomics Space Ranger with default parameters.
Assembly: mm10
Supplementary files format and content: filtered_feature_bc_matrix.h5：filtered feature barcode matrix in HDF5 format
Supplementary files format and content: scalefactors_json.json: scalfactors in json format
Supplementary files format and content: tissue_hires_image: hi-res image of tissue
Supplementary files format and content: tissue_lowres_image: low-res image of tissue
Supplementary files format and content: tissue_positions_list: list of spatial barcodes and the coordinates specifying spots
Library strategy: Spatial Transcriptomics

source_name: Brain

growth_protocol: Transient ischemia-reperfusion was induced by 30 minutes MCAO in Ppm1d+/+ and Ppm1d-/- mice. 24 hours after MCAO surgery, fresh brain tissues were obtained, frozen and embedded for spatial transcriptomics.

extract_protocol:
Using  magnetic resonance image for lesion localization, a 10 μm frozen tissue section (bregma-1.94 mm) were subjected to spatial gene expression profiling.
Sequencing libraries were prepared following the manufacturer’s protocol using the 10× Genomics Visium Spatial Gene Expression Slide & Reagent Kit.

value: Brain

value: Wild-type

value: middle cerebral artery occlusion (MCAO) model induction

tag: batch

value: Day 1


title: Ischemic hemisphere of Ppm1d-deficient mouse (Ppm1d-/-) post-MCAO

description:
Library name: Sample 1
Fresh frozen Spatial transcriptome sequencing

value: Ppm1d knockdown


title: SKMEL24_DMSO_1


title: Participants 215,control, baseline

description: Library name: b215

value: Whole blood


title: M33_3D_Dy4_5

city: Palo Alto

first: Ishita

last: Jain

address: 3801 Miranda Avenue

institute: Stanford University

description: Library name: M33_3D_Dy4_5

data_processing:
To estimate gene expression levels, the fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM) was quantified for each aligned read. This accounted for both sequencing depth and gene length on counting of fragments.
Assembly: fatsq files for each sample
Supplementary files format and content: FPKM in CSV combined for all samples

source_name: Tissue constructs implanted in vivo

extract_protocol:
All samples were collected and total RNA of each sample was isolated using the GeneJET RNA Isolation Kit (Fisher Scientific). The samples underwent library preparation and bulk RNA sequencing by Novogene Corporation.
CASAVA base recognition (Base Calling) was used to convert original image data files from Illumina sequencing platform to sequenced reads in FASTQ format. A series filtration criteria was applied based on uncertain nucleotides constituting more than 10% of either read (n>10%) and with low-quality nucleotides (base quality less than 5) constituting more than 50 per cent of the read. Next, alignment of raw reads was performed to reference genome using HISAT2. To estimate gene expression levels, the fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM) was quantified for each aligned read. This accounted for both sequencing depth and gene length on counting of fragments.

value: Skeletal Muscle

value: 3D 4 Day pre-differentiation


title: SKMEL5_corin_1


title: D1_C12

value: embryonic stem cell


title: Participants 97,control, 12 months

description: Library name: t97

value: Cognitive frailty

value: male


title: D1_D05


title: M32_3D_Dy4_4

description: Library name: M32_3D_Dy4_4


title: SKMEL5_DMSO_1

value: melanoma


title: SKMEL5_DMSO_2


title: M31_3D_Dy4_3

description: Library name: M31_3D_Dy4_3


title: Participants 97,control, 6 months

description: Library name: p97


title: Participants 97,control, baseline

description: Library name: b97


title: WM793_corin_2

value: corin


title: D1_D04


title: M30_3D_Dy4_1

description: Library name: M30_3D_Dy4_1


title: D1_C10


title: D1_C09


title: D1_D02


title: Participants 85, intervention, 12 months

description: Library name: t85

value: female


title: WM793_corin_1


title: M29_3D_Dy2_5

description: Library name: M29_3D_Dy2_5

value: 3D 2 Day pre-differentiation


title: Participants 85, intervention, baseline

description: Library name: b85


title: D1_C06


title: Participants 85, intervention, 6 months

description: Library name: p85


title: WM793_DMSO_1

value: DMSO


title: D1_C07


title: D1_C11


title: 1205Lu_corin_2


title: WM793_DMSO_2


title: D1_C08


title: M26_3D_Dy2_1

description: Library name: M26_3D_Dy2_1


title: M25_3D_Dy0_6

description: Library name: M25_3D_Dy0_6

value: 3D 0 pre-differentiation


title: M28_3D_Dy2_4

description: Library name: M28_3D_Dy2_4


title: D1_C05


title: MO1043, DLL4, rep2

city: Ulm

first: Alena

middle: Sophie

last: Ehrmann

address: Albert-Einstein-Allee 23

department: Internal Medicine III

country: Germany

institute: University Hospital Ulm

description: Library name: P29

data_processing:
Clean data with high quality were obtained by filtering raw data to remove adapter sequences and low-quality reads (The filtering criteria is as follows: 1.Filter out polyA and polyG. 2.Filter out reads with complexity less than 10 3.Filter out reads with average quality score less than 20. 4.Filter out reads with a length less than 100 bases after removing the adapters. 5.Filter out reads with more than 50% of bases having a quality score below 10.). The clean reads were mapped to the human reference genome (Homo_sapiens.GRCh38_release95.genome.fa) using HISAT2. Mapping statistics showed high mapping ratios ranging from 94.27% to 99.25%. StringTie was used for transcript assembly and gene expression quantification in Fragments Per Kilobase of transcript per Million fragments mapped (FPKM).
Assembly: Homo_sapiens.GRCh38_release95.genome.fa
Supplementary files format and content: processed data are provided as FPKM values in a tsv format

growth_protocol: Cell lines were incubated at 37°C and normoxia with 5% CO2 and cultured in 90% RPMI 1640 + 10% heat inactivated (h.i.) FBS. For treatment cells were seeded with a density of 1x10^6 cells/ml in 2 ml medium.

extract_protocol:
Total RNA from the different cell lines (HG3, PGA-1, Granta-519, MEC-1 and MO1043) was extracted using the mRNeasy Mini Kit (Qiagen) and quality control was performed using NanoDrop, Qubit 2.0, and Agilent 2100
Libraries were constructed using mRNA isolated by poly-T oligo-attached magnetic beads followed by random fragmentation and cDNA synthesis with random hexamers. Double-stranded cDNAs were end-repaired, adenylated, and ligated to adapters before PCR amplification

treatment_protocol: cells were treated with 1 µM Nirogacestat, 4µg/ml recombinant Human DLL4 Fc Chimera Protein or DMSO


title: M27_3D_Dy2_3

description: Library name: M27_3D_Dy2_3


title: MO1043, Nirogacestat, rep2

description: Library name: P30


title: MO1043, DMSO, rep2

description: Library name: P28


title: M24_3D_Dy0_5

description: Library name: M24_3D_Dy0_5


title: MEC-1, DLL4, rep2

description: Library name: P26


title: 1205Lu_corin_1


title: M23_3D_Dy0_4

description: Library name: M23_3D_Dy0_4


title: D1_C03


title: 1205Lu_DMSO_2


title: D1_C02


title: D1_C04


title: MEC-1, Nirogacestat, rep2

description: Library name: P27


title: 451Lu_corin_2


title: 1205Lu_DMSO_1


title: D1_C01


title: MEC-1, DMSO, rep2

description: Library name: P25


title: 451Lu_corin_1


title: M22_3D_Dy0_3

description: Library name: M22_3D_Dy0_3


title: M21_3D_Dy0_1

description: Library name: M21_3D_Dy0_1


title: 451Lu_DMSO_2


title: Granta-519, Nirogacestat, rep2

description: Library name: P24


title: M20_2D_Dy5_5

description: Library name: M20_2D_Dy5_5

source_name: Cells cultured on 2D surface

value: 2D Day 5 differentiation control


title: D1_B12


title: M19_2D_Dy5_4

description: Library name: M19_2D_Dy5_4


title: Granta-519, DLL4, rep2

description: Library name: P23


title: Granta-519, DMSO, rep2

description: Library name: P22


title: Rat N6 intravitreal injection of 2ul 20mM NMDA

city: Hangzhou

first: Da

last: Qian

state: Zhejiang

address: No. 158 Shangtang Road

department: Breast Surgery

institute: Zhejiang Provincial People's Hospital

data_processing:
Data filtering The sequencing data was filtered with SOAPnuke  by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 15) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterwards clean reads were obtained and stored in FASTQ format. The subsequent analysis and data mining were performed on Dr. Tom Multi-omics Data mining system (https://biosys.bgi.com).
RNA identification Bowtie2 was applied to align the clean reads to the gene set, in which known and novel, coding and noncoding transcripts were included.
Gene Quantification Differential Expression Analysis Expression level of gene was calculated by RSEM (v1.3.1) . The heatmap was drawn by pheatmap (v1.0.8)  according to the gene expression difference in different samples. Essentially, differential expression analysis was performed using the DESeq2(v1.4.5)  (or DEGseq or PoissonDis)with Q value ≤ 0.05 (or FDR ≤ 0.001).
Gene Annotation To take insight to the change of phenotype, GO (http://www.geneontology.org/) and KEGG (https://www.kegg.jp/) enrichment analysis of annotated different expression gene was performed by Phyper based on Hypergeometric test. The significant levels of terms and pathways were corrected by Q value with a rigorous threshold (Q value ≤ 0.05) .
Assembly: GRCr8
Supplementary files format and content: tab-delimited text files include FPKM values and gene count for each Sample

organism: Rattus norvegicus

source_name: retina

extract_protocol:
1. Experimental Procedures 1.1 Sample Lysis 1.1.1 Transfer appropriate tissue sample into corresponding tissue grinding tube (tissue≤30 mg, cell≤1×107). 1.1.2 Add 600 μL Buffer RLT Plus. Homogenize the lysate for using a rotor–stator homogenizer, stay at room temperature for 5 min. 1.1.3 Centrifuge at 12,000×g for 3 min in room temperature, transfer the supernatant to AllPrep DNA spin column. 1.1.4 Centrifuge 12,000×g for 30 s in room temperature, transfer the solution from the collection tube for the RNA purification. 1.1.5 Transfer the DNA spin column to the new collection tube for DNA purification. 1.2 DNA Purification 1.2.1 Add 500μL Buffer AW1 to AllPrep DNA spin column, centrifuge at 10,000×g for 30 s and discard the filtrate. 1.2.2 Add 500μL Buffer AW2 to AllPrep DNA spin column, centrifuge at 10,000×g for 30 s and discard the filtrate. 1.2.3 Centrifuge at 16,000×g for 2 min, transfer the AllPrep DNA spin column to 1.5mL centrifuge tube. 1.2.4 Add 50μL Buffer EB to AllPrep DNA spin column, stay at room temperature for 1 min. 1.2.5 Centrifuge at 16,000×g for 2 min and collect the DNA. 1.3 RNA Purification 1.3.1 Add the equal volume of 70% ethanol to the collection tube, mix by pipetting. 1.3.2 Transfer 700 μL mixed solution, to a RNeasy spin column placed in a 2 mL collection tube. 2.3.3 Centrifuge at 10,000×g for 30 s and allow it to empty by gravity flow. 1.3.4 Add 700 μL Buffer RW1 to RNeasy spin column. Centrifuge at 10,000×g for 30 s and discard the filtrate. 1.3.5 Add 500 μL Buffer RPE to RNeasy spin column. Centrifuge at 10,000×g for 30 s and discard the filtrate. 1.3.6 Add 500 μL Buffer RPE to RNeasy spin column. Centrifuge at 10,000×g for 30 s and discard the filtrate. 1.3.7 Centrifuge at 16,000×g for 2 min and transfer the RNeasy spin column to the 1.5 mL centrifuge tube. 1.3.8 Add 35 μL RNase-free water to RNeasy spin column. Stay at room temperature for 1min. 1.3.9 Centrifuge at 16,000×g for 2 min at room temperature to collect RNA.
After total RNA was extracted, mRNA was purified from total RNA (5ug) using Dynabeads Oligo (dT) with two rounds of purification. Following purification, the mRNA was fragmented into short fragments using divalent cations under elevated temperature (Magnesium RNA Fragmentation Module under 94℃ 5-7min). Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase, which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I, RNase H and dUTP Solution. An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA librarys were 300±50 bp. At last, we performed the 2×150bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.

value: retina


title: 451Lu_DMSO_1


title: M18_2D_Dy5_3

description: Library name: M18_2D_Dy5_3


title: D1_B09


title: PGA-1, Nirogacestat, rep2

description: Library name: P21


title: D1_B10


title: D1_B11


title: Rat N5 intravitreal injection of 2ul 20mM NMDA


title: M17_2D_Dy5_2

description: Library name: M17_2D_Dy5_2


title: PGA-1, DLL4, rep2

description: Library name: P20


title: D1_B08


title: PGA-1, DMSO, rep2

description: Library name: P19


title: M16_2D_Dy5_1

description: Library name: M16_2D_Dy5_1


title: Rat N3 intravitreal injection of 2ul 20mM NMDA


title: D1_B06


title: Rat N4 intravitreal injection of 2ul 20mM NMDA


title: D1_B07


title: M15_2D_Dy4_5

description: Library name: M15_2D_Dy4_5

value: 2D Day 4 differentiation control


title: Rat N2 intravitreal injection of 2ul 20mM NMDA


title: M14_2D_Dy4_4

description: Library name: M14_2D_Dy4_4


title: M13_2D_Dy4_3

description: Library name: M13_2D_Dy4_3


title: HG3, DLL4, rep2

description: Library name: P17


title: Rat C6 injected with 2uL PBS via intravitreal injection


title: HG3, Nirogacestat, rep2

description: Library name: P18


title: Rat N1 intravitreal injection of 2ul 20mM NMDA


title: M12_2D_Dy4_2

description: Library name: M12_2D_Dy4_2


title: MO1043, DLL4, rep1

description: Library name: P14


title: Rat C5 injected with 2uL PBS via intravitreal injection


title: MO1043, Nirogacestat, rep1

description: Library name: P15


title: M11_2D_4Dy_1

description: Library name: M11_2D_4Dy_1


title: M10_2D_Dy2_6

description: Library name: M10_2D_Dy2_6

value: 2D Day 2 differentiation control


title: Rat C4 injected with 2uL PBS via intravitreal injection


title: D1_B02


title: D1_B01


title: MO1043, DMSO, rep1

description: Library name: P13


title: MEC-1, Nirogacestat, rep1

description: Library name: P12


title: D1_A12


title: M8_2D_Dy2_4

description: Library name: M8_2D_Dy2_4


title: Rat C2 injected with 2uL PBS via intravitreal injection


title: D1_A11


title: MEC-1, DLL4, rep1

description: Library name: P11


title: HG3, DMSO, rep2

description: Library name: P16


title: Rat C3 injected with 2uL PBS via intravitreal injection


title: D1_B05


title: M7_2D_2Dy_3

description: Library name: M7_2D_2Dy_3


title: D1_B04


title: Rat C1 injected with 2uL PBS via intravitreal injection


title: M6_2D_Dy2_2

description: Library name: M6_2D_Dy2_2


title: Granta-519, Nirogacestat, rep1

description: Library name: P9


title: MEC-1, DMSO, rep1

description: Library name: P10


title: D1_A10


title: D1_B03


title: D1_A09


title: M5_2D_Dy2_1

description: Library name: M5_2D_Dy2_1


title: Mice model of SLE treated with DC-517-8

city: Hefei

first: ming

last: Li

state: Anhui

address: No.117 Meishan Road, Shushan District

department: Department of Rheumatology

institute: The First Affiliated Hospital of Anhui University of Chinese Medicine

description: Library name: DC517-8

data_processing:
FLASH (Fast Length Adjustment of SHort reads) is an accurate and fast tool， which was used to merge paired-end reads. Fastq to fasta format step was then  processed using the Fastx toolkit . Reads less than 10-fold were removed and overall sequencing depth for each sample were evaluated.
Reads with fasta format were mapped to targeted Bisulfite Genome (mm10) by Blast. Unmapped reads were filtered and mapped reads with coverage greater than 90% and identity greater than 95% were kept as effective reads and were used for following statistics. Sequencing depth for each amplicon per sample was calculated   by blasting the effective reads against the targeted genome region.
Methylation were analyzed using Perl script. Statistics were performed by t-test and ANOVA.
Assembly: mm10
Supplementary files format and content: The text file contains the methylation levels of all CpG sites in all samples

growth_protocol: A total of 48 SPF-grade female MRL/lpr mice and 8 SPF-grade female C57BL/6 mice (6 weeks old) were purchased from Shanghai Slac Laboratory Animal Co., Ltd. (Animal license number: SCXK [Hu] 2022-0004). All mice were housed in the animal facility of the First Affiliated Hospital of Anhui University of Chinese Medicine, where the temperature was maintained at 23±2 °C, humidity was kept between 40–60%, and a 12-hour light/dark cycle was followed. Adaptive feeding was conducted for 2 weeks before the start of the experiments.

extract_protocol:
DNA was extracted from CD4+ T cells using a TIANamp Genomic DNA Kit (Tiangen Biotech Co. Ltd., Beijing, China, cat. DP304)
The methylation level of the Cytosine-phosphate-guanine (CpG) site within the transcription start site region of the Foxp3 gene was evaluated utilizing the MethylTarget™ assays (targeted bisulfite sequencing) developed by Genesky BioTech (Shanghai, China). Genomic DNA was extracted and subsequently subjected to methylation using the EZ DNA Methylation Gold Sulfurous Acid Kit (ZYMO, CA, USA) according to the manufacturer’s instructions. Primers for the bisulfite-converted DNA samples were designed using FastTarget V4.1 (Genesky, Shanghai, China). The specific primer sequences (5′-3′) were as follows: Foxp3-(Treg-specific demethylated regions) TSDR: forward: TTGGGTTTTTTTGGTATTTAAGA, reverse: AAATCTACATCTAAACCCTATTATCACAA. Following the bisulfite conversion of genomic DNA, PCR was conducted to amplify the target DNA sequences. The amplified products were sequenced using an Illumina HiSeq sequencer (Illumina, CA, USA).

treatment_protocol: 48 MRL/lpr mice were randomly divided into six groups with eight mice: model group, low-dose JPZS group (6.305 g/kg, JPZS-L), medium-dose JPZS group (12.61 g/Kg, JPZS-M), high-dose JPZS group (25.22 g/kg, JPZS-H), 5-aza-CdR inhibitor group, and DC_517 inhibitor group, with eight mice in each group. eight female C57BL/6 mice were used as controls.

value: spleen

value: T cell


title: Mice model of SLE treated with DC-517-7

description: Library name: DC517-7


title: Granta-519, DLL4, rep1

description: Library name: P8


title: D1_A08


title: M4_2D_dy0_4

description: Library name: M4_2D_dy0_4

value: Skeletal Muscle

value: 2D Day 0 differentiation control


title: Granta-519, DMSO, rep1

description: Library name: P7


title: M9_2D_Dy2_5

description: Library name: M9_2D_Dy2_5


title: Mice model of SLE treated with DC-517-5

description: Library name: DC517-5


title: M3_2D_dy0_3

description: Library name: M3_2D_dy0_3


title: D1_A06

value: embryonic stem cell


title: M2_2D_dy0_2

description: Library name: M2_2D_dy0_2


title: M1_2D_dy0_1

description: Library name: M1_2D_dy0_1


title: D1_A05


title: Mice model of SLE treated with DC-517-4

description: Library name: DC517-4


title: D1_A04


title: Mice model of SLE treated with DC-517-2

description: Library name: DC517-2


title: Mice model of SLE treated with DC-517-3

description: Library name: DC517-3


title: Mice model of SLE treated with DC-517-6

description: Library name: DC517-6


title: D1_A07


title: PGA-1, DLL4, rep1

description: Library name: P5


title: HG3, Nirogacestat, rep1

description: Library name: P3


title: Mice model of SLE treated with DC-517-1

description: Library name: DC517-1


title: D1_A02


title: Tg, Male, sham, 7d - 1

city: Atlanta

first: Karen

middle: N

last: McFarland

state: GA

address: 100 Woodruff Circle

department: Pharmacology

institute: Emory University

description: Library name: CRND8_NM2

data_processing:
Cutadapt was used to remove reads with adaptor contamination, low quality bases and undetermined bases. FastQC was used to verify sequence quality
STAR v2.7.9a was used to align to mouse genome GRCm39/mm39
DESeq2 (Love 2014) was used to extract counts and fpkm values from protein-coding genes
Assembly: GRCm39/mm39
Supplementary files format and content: csv file with raw counts for each sample
Supplementary files format and content: csv file with FPKM values for each sample

source_name: forebrain

extract_protocol:
Frozen pulverized forebrain tissue was incubated in TRIzol reagent (Invitrogen) followed by phase separation and RNA isolation with ethanol precipitation. RNA was further purified with PureLink RNA Mini kit with on-column DNase treatment (Invitrogen). RNA quality and quantity were measured with the NanoDrop Microvolume spectrophotometers (Thermo) and on the Agilent Bioanalyzer 2100 with the Eurkaryote Total RNA Nano chip.
Poly(A) RNA sequencing libraries were prepared following Illumina's TruSeq-stranded mRNA sample preparation protocol. Quality control analysis and quantification of the sequencing library was performed using Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA chip. Paired-end sequencing was performed on Illumina's NoveSeq6000 sequencing system.

treatment_protocol: TgCRND8 and wild-type controls were subjected to the surgical sepsis protocol (cecal ligation puncture with daily chronic stress) and were euthanized either 7 days or 3 months post-sepsis induction. Mice were euthanized with carbon dioxide inhalation followed by cervical dislocation. Brains were extracted and dissect at the midline. The right hemisphere was immediately flash frozen and stored at -80 degrees Celsius until further use. Prior to RNA extraction, brains were pulverized under liquid nitrogen.

value: forebrain

value: TG

value: sham, 7d

value: Male


title: D1_A01


title: PGA-1, Nirogacestat, rep1

description: Library name: P6


title: HG3, DMSO, rep1

description: Library name: P1


title: Tg, Male, sham, 3 mon - 3

description: Library name: N_113022_NTg_M3

value: sham, 3 mon


title: Mice model of SLE treated with 5-Aza-CdR-7

description: Library name: 5aza-7


title: H6_TS3_day2

description: Library name: H6_TS3_day2

value: Primary Human Muscle

value: Human 3D 2 day predifferentiation


title: D0_H12


title: D0_H11


title: Mice model of SLE treated with 5-Aza-CdR-5

description: Library name: 5aza-5


title: H4_TS1_day2

description: Library name: H4_TS1_day2


title: Tg, Male, sham, 3 mon - 2

description: Library name: PC19


title: Mice model of SLE treated with 5-Aza-CdR-4

description: Library name: 5aza-4


title: Tg, Male, sham, 3 mon - 1

description: Library name: PC17


title: Tg, Male, sepsis, 7d - 3

description: Library name: CRND8_S7_M12

value: sepsis, 7d


title: H3_sus3_day2

description: Library name: H3_sus3_day2

source_name: Suspension Cells implanted in vivo

value: Human Suspension Control 2 day Prediff


title: Mice model of SLE treated with 5-Aza-CdR-3

description: Library name: 5aza-3


title: D0_H10


title: Tg, Male, sepsis, 7d - 2

description: Library name: CRND8_S7_M11


title: PGA-1, DMSO, rep1

description: Library name: P4


title: Tg, Male, sham, 7d - 3

description: Library name: CRND8_NM7


title: Tg, Male, sham, 7d - 2

description: Library name: CRND8_NM3


title: HG3, DLL4, rep1

description: Library name: P2


title: D0_H09


title: Mice model of SLE treated with 5-Aza-CdR-2

description: Library name: 5aza-2


title: Mice model of SLE treated with 5-Aza-CdR-8

description: Library name: 5aza-8


title: H2_sus2_day2

description: Library name: H2_sus2_day2


title: Tg, Male, sepsis, 7d - 1

description: Library name: CRND8_S7_M10


title: H5_TS2_day2

description: Library name: H5_TS2_day2


title: H1_sus1_day2

description: Library name: H1_sus1_day2


title: Tg, Male, sepsis, 3 mon - 3

description: Library name: PC30

value: sepsis, 3 mon


title: Mice model of SLE treated with 5-Aza-CdR-6

description: Library name: 5aza-6


title: D0_H06


title: D0_H07


title: Mice model of SLE treated with JPZS-8

description: Library name: JPZS-8


title: JAK2 PMF 5

city: Salt Lake City

first: Marco

last: Marchetti

state: Utah

department: Human Genetics

institute: University of Utah

description:
Library name: 37
JAK2 PMF 5

data_processing:
Adaptor sequences and low-quality reads were removed from raw reads using Trim Galore! 0.6.10
STAR 2.7.11a was used to map trimmed reads to the GRCh38 genome and to generate stranded gene read counts
Assembly: GRCh38
Supplementary files format and content: Tab-delimited text file includes stranded raw gene counts for each sample

source_name: Peripheral blood mononuclear cells

extract_protocol:
RNA was extracted following the standard protocols recommended by the RNeasy Mini Kit (Qiagen)
The library for RNA-Seq was constructed using TruSEQ stranded mRNA kit (Illumina) following manufacturer’s protocol

value: Peripheral blood mononuclear cells

value: JAK2 V617F


title: JAK2 PMF 4

description:
Library name: 36
JAK2 PMF 4


title: D0_H08


title: Mice model of SLE treated with JPZS-7

description: Library name: JPZS-7

value: spleen


title: Tg, Male, sepsis, 3 mon - 2

description: Library name: PC29


title: D0_H05


title: JAK2 PMF 3

description:
Library name: 35
JAK2 PMF 3


title: Mice model of SLE treated with JPZS-6

description: Library name: JPZS-6

value: T cell


title: Tg, Male, sepsis, 3 mon - 1

description: Library name: PC23


title: D0_H03


title: D0_H04


title: Tg, Female, sham, 7d - 2

description: Library name: PC11

value: Female


title: JAK2 PMF 1

description:
Library name: 33
JAK2 PMF 1


title: Tg, Female, sham, 7d - 3

description: Library name: PC15


title: JAK2 PMF 2

description:
Library name: 34
JAK2 PMF 2


title: Mice model of SLE treated with JPZS-5

description: Library name: JPZS-5


title: Mice model of SLE treated with 5-Aza-CdR-1

description: Library name: 5aza-1


title: D1_A03


title: Tg, Female, sham, 7d - 1

description: Library name: PC10


title: D0_H01


title: JAK2 ET 5

description:
Library name: 32
JAK2 ET 5


title: Mice model of SLE treated with JPZS-3

description: Library name: JPZS-3


title: JAK2 ET 4

description:
Library name: 31
JAK2 ET 4


title: D0_H02


title: Mice model of SLE treated with JPZS-2

description: Library name: JPZS-2


title: Tg, Female, sham, 3 mon - 2

description: Library name: PC12


title: Tg, Female, sham, 3 mon - 3

description: Library name: PC14


title: Mice model of SLE treated with JPZS-1

description: Library name: JPZS-1


title: Tg, Female, sham, 3 mon - 1

description: Library name: PC18


title: JAK2 ET 3

description:
Library name: 30
JAK2 ET 3


title: Mice model of SLE treated with JPZS-4

description: Library name: JPZS-4


title: JAK2 ET 2

description:
Library name: 29
JAK2 ET 2


title: Tg, Female, sepsis, 7d - 2

description: Library name: PC3


title: Systemic lupus erythematosus mice model-8

description: Library name: Model-8


title: Tg, Female, sepsis, 7d - 3

description: Library name: PC6


title: Systemic lupus erythematosus mice model-7

description: Library name: Model-7


title: D0_G09


title: Type 2 PMF 2

description:
Library name: 27
Type 2 PMF 2


title: D0_G10


title: JAK2 ET 1

description:
Library name: 28
JAK2 ET 1


title: D0_G08


title: Systemic lupus erythematosus mice model-6

description: Library name: Model-6


title: Tg, Female, sepsis, 7d - 1

description: Library name: PC1


title: Type 2 PMF 1

description:
Library name: 26
Type 2 PMF 1


title: D0_G07


title: Tg, Female, sepsis, 3 mon - 3

description: Library name: PC27


title: Systemic lupus erythematosus mice model-5

description: Library name: Model-5


title: Type 2 ET 8

description:
Library name: 25
Type 2 ET 8


title: Systemic lupus erythematosus mice model-4

description: Library name: Model-4


title: D0_G11


title: D0_G12


title: D0_G05


title: Systemic lupus erythematosus mice model-3

description: Library name: Model-3


title: Tg, Female, sepsis, 3 mon - 2

description: Library name: PC26


title: D0_G06


title: Type 2 ET 7

description:
Library name: 24
Type 2 ET 7


title: Type 2 ET 6

description:
Library name: 23
Type 2 ET 6


title: Tg, Female, sepsis, 3 mon - 1

description: Library name: PC24


title: Systemic lupus erythematosus mice model-2

description: Library name: Model-2


title: D0_G04


title: NTg, Male, sham, 7d - 3

description: Library name: CRND8_NM6

value: NTG


title: Type 2 ET 5

description:
Library name: 22
Type 2 ET 5


title: D0_G02

value: embryonic stem cell


title: Systemic lupus erythematosus mice model-1

description: Library name: Model-1


title: NTg, Male, sham, 7d - 2

description: Library name: CRND8_NM5

value: forebrain


title: NTg, Male, sham, 7d - 1

description: Library name: CRND8_NM4


title: Type 2 ET 3

description:
Library name: 20
Type 2 ET 3


title: D0_G01


title: Healthy mice-7

description: Library name: Controls-7


title: D0_F12


title: Type 2 ET 2

description:
Library name: 19
Type 2 ET 2


title: Healthy mice-8

description: Library name: Controls-8


title: NTg, Male, sham, 3 mon - 1

description: Library name: PC13


title: Type 2 ET 1

description:
Library name: 18
Type 2 ET 1


title: D0_F11


title: Healthy mice-5

description: Library name: Controls-5


title: NTg, Male, sham, 3 mon - 3

description: Library name: N_113022_NTg_M2


title: NTg, Male, sham, 3 mon - 2

description: Library name: N_113022_NTg_M1


title: Type 1 PMF 5

description:
Library name: 17
Type 1 PMF 5


title: D0_F10


title: Healthy mice-4

description: Library name: Controls-4


title: Healthy mice-6

description: Library name: Controls-6


title: D0_F09


title: Type 1 PMF 4

description:
Library name: 16
Type 1 PMF 4


title: NTg, Male, sepsis, 7d - 3

description: Library name: CRND8_S7_M8


title: NTg, Male, sepsis, 7d - 2

description: Library name: CRND8_S7_M7


title: D0_G03


title: D0_F08


title: D0_F07


title: Type 2 ET 4

description:
Library name: 21
Type 2 ET 4


title: Type 1 PMF 3

description:
Library name: 15
Type 1 PMF 3


title: Healthy mice-2

description: Library name: Controls-2


title: Healthy mice-3

description: Library name: Controls-3


title: Healthy mice-1

description: Library name: Controls-1


title: NTg, Male, sepsis, 7d - 1

description: Library name: CRND8_S7_M6


title: Type 1 PMF 2

description:
Library name: 14
Type 1 PMF 2


title: NTg, Male, sepsis, 3 mon - 3

description: Library name: S3m_112922_NTg_M3


title: Type 1 PMF 1

description:
Library name: 13
Type 1 PMF 1


title: Equi_hypo4

city: Groningen

first: Hans

last: van Veen

address: Nijenborgh 7

department: GELIFES

country: Netherlands

institute: University of Groningen

data_processing:
Kallisto read allignment, edgeR differenital expression
Assembly: Marchantia polymorpha v3.1 (Phytozome genome ID: 320); Physcomitrium patens v3.3 (Phytozome genome ID: 318); Selaginella moellendorffii v1.0 (Phytozome genome ID: 91); Pteris vittata (TSA); Equisetum hyemale (TSA)
Supplementary files format and content: processed_Ehym.txt
Supplementary files format and content: processed_Mpol.txt
Supplementary files format and content: processed_Ppat.txt
Supplementary files format and content: processed_Pvit.txt
Supplementary files format and content: processed_Smoe.txt

organism: Equisetum hyemale

source_name: shoot

extract_protocol:
Thermo Scientific™ GeneJET Plant RNA Purification Kit (Pteris, Marcha, Physco, Sella); Sigma Spectrum RNA extraction (Equi)
mRNA stranded library preparation (poly A enrichment)

value: shoot


title: D0_F05


title: NTg, Male, sepsis, 3 mon - 2

description: Library name: S3m_112922_NTg_M2


title: Equi_hypo3


title: Type 1 ET 5

description:
Library name: 12
Type 1 ET 5


title: NTg, Male, sepsis, 3 mon - 1

description: Library name: S3m_112922_NTg_M1


title: Equi_hypo1


title: Type 1 ET 4

description:
Library name: 11
Type 1 ET 4


title: Type 1 ET 3

description:
Library name: 10
Type 1 ET 3


title: D0_F02


title: D0_F03


title: Equi_air4

value: aerobic conditions (21% O2)


title: NTg, Female, sham, 7d - 1

description: Library name: PC7

value: sham, 7d


title: NTg, Female, sham, 7d - 3

description: Library name: PC9


title: D0_F04


title: NTg, Female, sham, 7d - 2

description: Library name: PC8


title: Equi_air1


title: D0_F01


title: Type 1 ET 2

description:
Library name: 9
Type 1 ET 2


title: NTg, Female, sham, 3 mon - 3

description: Library name: PC22

value: sham, 3 mon


title: Healthy Control J

description:
Library name: 7
Healthy Control J


title: Type 1 ET 1

description:
Library name: 8
Type 1 ET 1


title: D0_E12


title: Roots, P0, rep4

city: Gembloux

first: Said

last: Khourchi

address: rue de déportes

country: Belgium

institute: University of Liège

description: Triticum turgidum subsp. durum (Desf.) Husn., 1914

data_processing:
Low-quality raw RNA-seq reads were filtered using SOAPnuke
The filtered reads were mapped onto the reference genome of durum wheat (Triticum turgidum subsp. durum, Svevo.v1) using HISAT2 (Version: v2.0.4), followed by novel gene prediction
Assembly: Transcript assembly based on reference genome for each library was performed using StringTie
Supplementary files format and content: Files include raw data
Supplementary files format and content: Files include processing data (FKPM, DEseq2)

organism: Triticum turgidum

source_name: Root tissue

growth_protocol: Grains of wheat were grown in P-deficient soil supplied with PolyP and OthoP fertilizers under controlled conditions

extract_protocol:
The RNA was extracted from each sample using NucleoSpin RNA Plant (Macherey-Nagel, Germany),
RNA sequencing and library construction were carried out at Beijing Genomics Institute (BGI), China, at a sequencing depth of 20 million paired end reads.

treatment_protocol: The phosphorus was applied as PolyC (long chain potassium PolyP), PolyB (short chain sodium PolyP) and OrthoP at rate of 60 Kg P ha–1.

value: Root tissue


title: D0_F06


title: D0_E11


title: Healthy Control N

description:
Library name: 6
Healthy Control N


title: NTg, Female, sham, 3 mon - 1

description: Library name: PC16


title: D0_E10


title: Healthy Control M

description:
Library name: 5
Healthy Control M


title: NTg, Female, sepsis, 7d - 3

description: Library name: PC5


title: Roots, P0, rep2

description: Triticum turgidum subsp. durum (Desf.) Husn., 1912


title: Equi_air3


title: Healthy Control D

description:
Library name: 4
Healthy control D


title: NTg, Female, sham, 3 mon - 2

description: Library name: PC21


title: NTg, Female, sepsis, 7d - 2

description: Library name: PC4


title: Roots, P0, rep1

description: Triticum turgidum subsp. durum (Desf.) Husn., 1911


title: Healthy Control H

description:
Library name: 3
Healthy control H


title: D0_E08


title: NTg, Female, sepsis, 7d - 1

description: Library name: PC2


title: Roots, OrthoP, rep4

description: Triticum turgidum subsp. durum (Desf.) Husn., 1910

value: OrthoP


title: Healthy Control S

description:
Library name: 2
Healthy control S


title: D0_E07


title: NTg, Female, sepsis, 3 mon - 3

description: Library name: PC28


title: Healthy Control B

description:
Library name: 1
Healthy control B


title: Roots, OrthoP, rep2

description: Triticum turgidum subsp. durum (Desf.) Husn., 1908


title: Roots, OrthoP, rep3

description: Triticum turgidum subsp. durum (Desf.) Husn., 1909


title: H4K20me3 rep2

city: hangzhou

first: Danya

last: Wu

state: zhejiang

address: xihu

institute: zhejiang unvercitt

description: Library name: Sample3

data_processing:
Quality control for raw reads was performed by FastQC v0.11.8
First 10 bp and last 90 bp of both paired-end reads were trimmed using cutadapt v2.9
Reads were aligned to the human reference genome GRCh38.97 using STAR v2.7.0e
Reads with a maximum of 1000 multiple mapped sites and no more than 3 mismatches were retained, with only the best hit kept (--outFilterMultimapNmax 1000, --outFilterMismatchNmax 3, --outSAMmultNmax 1).
ChIP-seq peaks were identified using the callpeaks function in MACS2 v2.2.7.1
Normalized BigWig tracks were generated using deeptools v3.4.3 (Ramírez et al., 2014) with RPKM normalization and a bin size of 10 bp.
Assembly: GRCh38.97
Supplementary files format and content: bigwig

growth_protocol: The primed hESC line H9 was cultured in mTeSR (Stem cell technologies, 85,851) on Matrigel-coated plates (Corning, 354,277) in a 37 ℃, 5% CO2 incubator.

extract_protocol:
Cells were fixed with 1% formaldehyde for 10mins. After quenching and cell lysis,chromatin was sonicated into short fragments with an average size of 500 bp
Purified DNA was prepared for library construction using the KAPA HyperPrep Kit (Roche, KK8504) following the manufacturer's instructions. Sequencing was performed on the Illumina Novaseq 6000 platform with paired-end 150-bp reads.

treatment_protocol: No treatment

value: WT

value: no treatment

tag: chip antibody

value: H4K20me3 (Abcam,ab9053)


title: D0_E05

data_processing:
All sequencing was carried out on HiSeq instrument. BS-seq libraries were sequenced in 96-plex pools using 125-bp paired-end reads. RNA-seq libraries were pooled as 96-plex or 192-plex pools and sequenced using 75-bp paired-end reads.
Alignments of the single-cell bisulfite sequencing were performed using Bismark as well as subsequent CpG methylation and GpC accessibility calling.
Genome_build: GRCm38
Supplementary_files_format_and_content: The Bismark CpG coverage report is tab-delimited, uses 1-based genomic coordinates for every covered cytosine position in the experiment and is in the following format: <chromosome> <start position> <end position> <methylation percentage> <count methylated> <count non-methylated>

extract_protocol:
0h, 24h, and 48h after induction of 2i-release  the cells been dissociated into single cells using accutase before flow sorting  (BD Influx) into 96-well plate containing 2.5μl of methylase reaction buffer (1 × M.CviPI Reaction buffer (NEB), 2 U M.CviPI (NEB), 160 μM S-adenosylmethionine (NEB), 1 U μl−1 RNasein (Promega), 0.1% IGEPAL CA-630 (Sigma)). Samples were incubated for 15 min at 37 °C to methylate accessible chromatin before the reaction was stopped with the addition of RLT plus buffer (Qiagen) and samples frozen down and stored at −80 °C before processing.
E14 mESC were cultured in 2i+LIF media. For induction for 2i-relase experiment, the cell been rinsed with PBS before the culture media was changed to serum + LIF medium. 0h, 24h, and 48h after induction of 2i-release single cells were distracted and sc-NMT sequencing was performed.


title: Roots, P0, rep3

description: Triticum turgidum subsp. durum (Desf.) Husn., 1913


title: Roots, OrthoP, rep1

description: Triticum turgidum subsp. durum (Desf.) Husn., 1907


title: H4K20me3 rep1

description: Library name: Sample2


title: NTg, Female, sepsis, 3 mon - 1

description: Library name: PC20

value: sepsis, 3 mon


title: Roots, PolyC, rep4

description: Triticum turgidum subsp. durum (Desf.) Husn., 1906

value: PolyC


title: Roots, PolyC, rep3

description: Triticum turgidum subsp. durum (Desf.) Husn., 1905


title: HGG_3

city: Columbus

first: Alessandro

last: Canella

state: OH

address: 700 Children’s Drive

department: Institute for Genomic Medicine

institute: Nationwide Children's Hospital

description:
Library name: 11
60-80M read pairs

data_processing:
Low-quality reads (q<10), and adaptor sequences were eliminated from raw reads using bbduk version 37.64
Features were identified from the GFF file that came with the assembly from Gencode (V28).
Feature coverage counts were calculated using featureCounts
Normalized (TPM) quantification for each sample was done using salmon counts
Assembly: GRCm38.p6 assembly of the Mus musculus reference from NCBI
Supplementary files format and content: comma separated file that includes the aggregated normalized salmon TPM counts for all samples

source_name: brain

growth_protocol: DF1-PDGFb and DF1-CRE cells were injected intracranially in the right hemisphere of RCAS mice at the age of 2 days.

extract_protocol:
Total RNA from tumor-infiltrating immune cells was isolated using the Qiagen miRNeasy Micro kit (Qiagen) and eluted with 14uL H2O. The quality and quantity of RNA was checked with Bioanalyzer.
Total RNA-seq by Illumina NovaSeq6000

value: brain

value: Tumor infiltrating immune cells


title: Input

description: Library name: Sample1


title: HGG_4

description:
Library name: 12
60-80M read pairs


title: D0_E09

city: Cambridge

first: Felix

institute: Altos Labs


title: D0_E04


title: D0_E03


title: D0_E06


title: D0_E01


title: A196PlaB_rep1

description: Library name: Sample11

data_processing:
Quality control for raw reads was performed by FastQC v0.11.8
Sequence reads were trimmed for adaptor sequence/low-quality sequence using cutadapt v2.9
Trimmed sequence reads were mapped to GRCm38/mm10 using STAR,Reads with maximal 1000 multiple mapped sites and no more than 3 mismatches were retained, and only the best hit was kept (parameters- --outFilterMultimapNmax 1000, --outFilterMismatchNmax 3, --outSAMmultNmax 1).
Quantification was calculated by FeatureCounts v2.0.0(parameters- -p -B -P -C)
Differentially expressed genes (DEGs) and transposable elements (DE-TEs) were generated by R package EdgeR v3.30.3
Assembly: GRCh38.97
Supplementary files format and content: bigwig

extract_protocol:
RNA was harvested using trizol. 2 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using NEB Next R UltraTM RNA Library Prep Kit (strand-unspecific, ployA-specific) following manufacturer's protocols.

treatment_protocol: DUX4 over-expression or PlaB treatment was performed following a 72-hour treatment with 10 µM A196.

value: A196 and PlaB treatment


title: Roots, PolyC, rep1

description: Triticum turgidum subsp. durum (Desf.) Husn., 1903


title: D0_D12


title: NTg, Female, sepsis, 3 mon - 2

description: Library name: PC25

value: NTG

value: Female


title: HGG_2

description:
Library name: 10
60-80M read pairs


title: Roots, PolyB, rep4

description: Triticum turgidum subsp. durum (Desf.) Husn., 1902

value: PolyB


title: Roots, PolyC, rep2

description: Triticum turgidum subsp. durum (Desf.) Husn., 1904


title: CTRL_HGG_3

description:
Library name: 8
60-80M read pairs


title: HGG_1

description:
Library name: 9
60-80M read pairs


title: D0_E02

value: embryonic stem cell


title: PlaB_rep1

description: Library name: Sample9

value: PlaB treatment


title: CTRL_HGG_2

description:
Library name: 7
60-80M read pairs


title: Roots, PolyB, rep3

description: Triticum turgidum subsp. durum (Desf.) Husn., 1901


title: A196DUX4OE_rep2

description: Library name: Sample8

value: A196 treatment and DUX4 over-expression


title: PlaB_rep2

description: Library name: Sample10


title: D0_D11


title: Roots, PolyB, rep2

description: Triticum turgidum subsp. durum (Desf.) Husn., 1900


title: D0_D10


title: D0_D09


title: A196PlaB_rep2

description: Library name: Sample12


title: CTRL_HGG_1

description:
Library name: 6
60-80M read pairs


title: A196DUX4OE_rep1

description: Library name: Sample7


title: Roots, PolyB, rep1

description: Triticum turgidum subsp. durum (Desf.) Husn., 1899


title: D0_D08


title: MS1 YT cells doxycycline treament day 3 (Rep 4)

city: West Lafayette

first: Majid

last: Kazemian

state: IN

address: 201 S. University Steet

department: Biochemistry and Computer Science

institute: Purdue University

description: Library name: YT4

data_processing:
Raw paired-end sequencing data were processed, and gene expression was quantified using RSEM v1.3.1 with default parameters, with Bowtie2 as the aligner.
The required Bowtie2 reference was prepared using 'rsem-prepare-reference' for the GRCm38 transcriptome with RefSeq genes, downloaded from UCSC.
Differentially expressed gene (DEG) analysis was performed using DESeq2.
Assembly: mm10
Supplementary files format and content: Tab delimited textcontaining gene level expression estimates for each sample (RSEM output).

growth_protocol: MS1 cells were were maintained in DMEM with 10% FBS, 1× antibiotic‐antimycotic penicillin, streptomycin, and amphotericin B (PSA) and incubated at 37 °C in 5% CO2.

extract_protocol:
Total RNA was prepared using the Quick-RNA Miniprep Kit (Zymo)
cDNA

treatment_protocol: RNA was extracted 3 days post doxycycline treatment (1µg/mL)

value: cell line

value: epithelioid hemangioendothelioma

value: YT

value: doxycycline


title: DUX4OE_rep2

description: Library name: Sample6

value: WT

value: DUX4 over-expression


title: MS1 YT cells doxycycline treament day 3 (Rep 3)

description: Library name: YT3


title: D0_D07

last: Krueger

address: Granta Park


title: rhd6-Tre-3

city: Qingdao

first: Zhong-Bao

last: Yang

state: Shandong

address: 72# Binhai Road

department: School of Life Science

institute: Shandong University (Qingdao)

data_processing:
The sequence reads with were mapped to Arabidopsis genome reference sequences and  then the ownstream analysis including gene expression, Gene Ontology GO) enrichment analysis, KEGG pathway enrichment nalysis, cluster analysis, etc. was performed.
The gene expression levels were calculated using RSEM according to Li and Dewey (2011), and the NOISeq method (Tarazona et al., 2011) was used to screen the differentially expressed genes (DEGs) within samples.
Assembly: The sequence reads with were mapped to Arabidopsis genome reference sequences
Supplementary files format and content: Excel files include RPKM values for each Sample

organism: Arabidopsis thaliana

source_name: root

extract_protocol:
The total RNA was isolated using the RNeasy PlantMini Kit (Qiagen) following the manufacturer’s protocol. RNAseq analysis was performed by BGI Tech (Shenzhen, China) via BGISEQ-500 RNA-Seq platform according to Zhu et al. (2022)
RNAseq analysis was performed by BGI Tech (Shenzhen, China) via BGISEQ-500 RNA-Seq   platform.

value: root

value: with Al

tag: time

value: 6 h


title: DUX4OE_rep1

description: Library name: Sample5


title: MS1 YT cells doxycycline treament day 3 (Rep 2)

description: Library name: YT2


title: rhd6-Tre-2


title: MS1 YT cells doxycycline treament day 3 (Rep 1)

description: Library name: YT1


title: rhd6-Tre-1


title: A196_rep2

description: Library name: Sample4

value: A196 treatment


title: A196_rep1

description: Library name: Sample3


title: rhd6-Con-3

value: without Al


title: D0_D06


title: D0_D04


title: MS1 RFP cells doxycycline treament day 3 (Rep 3)

description: Library name: RPF3

value: endothelial

value: RFP


title: D0_D05


title: rhd6-Con-1


title: MS1 RFP cells doxycycline treament day 3 (Rep 2)

description: Library name: RPF2


title: D0_D03


title: DMSO_rep2

description: Library name: Sample2

value: no treatment


title: DMSO_rep1

description: Library name: Sample1

value: embryonic stem cell


title: rhd6-Con-2


title: TSLC

city: Kyoto-shi

first: Masatoshi

last: Ohgushi

state: Kyoto

address: 53 Shogoin Kawahara-cho

country: Japan

institute: Kyoto University

description:
Library name: Sample 1
10x Genomics

data_processing:
Sequence reads were aligned against GRCh38 genome assembly and a count matrix of unique molecular identifiers (UMIs) was generated using using Cell Ranger (version 4.0.0)
Assembly: GRCh38 genome assembly
Supplementary files format and content: cvs file includes read counts of each cell.

source_name: TS-like cells

growth_protocol: TSLCs were cultured in TSC medium (DMEM/F12 supplemented with 0.1mM 2-mercaptoethanol, 0.2% FBS, 0.3% BSA, 1% ITS-X supplement, 1.5 mg/ml L-ascorbic acid, 50 ng/ml EGF, 2 µM CHIR99021, 0.5 µM A83-01, 1 µM SB431542, 0.8 µM VPA and 5 µM Y27632) in humidified atmosphere with 5% Co2 at 37C.

extract_protocol:
The ESC-derived TSLCs (passage number 18) were dissociated by TrypLE select into single cells and suspended in PBS containing DRAQ-7. DRAQ-7-negative live cells were isolated as single cells using a Flow cytometer. From these cells, total RNAs were extracted using A Chromium Single Cell 3’ Library & Gel Beads Kit v3.1 (10x Genomics) following the manufacturer's protocol.
Sequencing libraries were constructed using A Chromium Single Cell 3’ Library & Gel Beads Kit v3.1 (10x Genomics) following the manufacturer's protocol.

value: TS-like cells


title: WT-Tre-3


title: D0_D02


title: Kira8+Stattic-3

city: Albuquerque

first: Dandan

state: NM

address: 915 CAMINO DE SALUD  NE room 360

department: Molecular Genetics and Microbiology

institute: University of New Mexico

data_processing:
Sequence and filtering of Clean Reads
Alignment with Reference Genome
Quantification of Gene Abundance
Differentially expressed genes (DEGs) Analysis
GO Enrichment Analysis
Assembly: mm10
Supplementary files format and content: tab-delimited text file includes gene counts for each sample

source_name: lymph nodes

growth_protocol: TH17 cell were maintained in RPMI supplemented with 10% fetal bovine serum (FBS) and antibiotics in humified atmosphere with 5% CO2 at 37℃

extract_protocol:
Total RNA was extracted using Trizol reagent (thermofisher, 15596018) following the manufacturer's procedure
The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent,CA, USA, 5067-1511) , high-quality RNA samples with RIN number > 7.0 were used to construct sequencing

treatment_protocol: Th17 cells treated with Kira8, Stattic and Kira8+Stattic inhibitor 4 hours.

value: lymph nodes

value: T cell

value: Naive T differentiation


title: MS1 RFP cells doxycycline treament day 3 (Rep 1)

description: Library name: RPF1

value: cell line


title: Corin_2

city: Boston

first: Robert

last: Fisher

address: 609 Albany St

institute: Boston University

data_processing:
All custom scripts described herein are available on the AdelmanLab GitHub (https://github.com/AdelmanLab/NIH_scripts). Dual, 6nt Unique Molecular Identifiers (UMIs) were extracted from read pairs using UMI-tools [10.1101/gr.209601.116]. Read pairs were trimmed using cutadapt 1.14 to remove adapter sequences (-O 1 --match-read-wildcards -m {20,26}). The UMI length was trimmed off the end of both reads to prevent read-through into the mate’s UMI, which will happen for shorter fragments. An additional nucleotide was removed from the end of read 1 (R1), using seqtk trimfq (https://github.com/lh3/seqtk), to preserve a single mate orientation during alignment. The paired end reads were then mapped to a combined genome index, including both the spike (dm6) and primary (hg38) genomes, using bowtie2 [10.1038/nmeth.1923]. Properly paired reads were retained. These read pairs were then separated based on the genome (i.e. spike-in vs primary) to which they mapped, and both these spike and primary reads were independently deduplicated, again using UMI-tools. Paired-end RNA-seq reads were mapped to the hg38 reference genome via HISAT2 v2.2.1 (--known-splicesite-infile). To select gene-level features for differential expression analysis, and for pairing with PRO-seq data, we assigned a single, dominant TSS and transcription end site (TES) to each active gene. This was accomplished using a custom script, get_gene_annotations.sh (available at  https://github.com/AdelmanLab/GetGeneAnnotation_GGA), which uses RNA-seq read abundance and PRO-seq R2 reads (RNA 5’ ends) to identify dominant TSSs, and RNA-seq profiles to define most commonly used TESs. RNA-seq and PRO-seq data from all conditions were used for this analysis, to comprehensively capture gene activity in these samples.Reads were summed within the TSS to TES window for each active gene using the make_heatmap script (https://github.com/AdelmanLab/NIH_scripts), which counts each read one time, at the exact 3’ end location of the nascent RNA.  DEseq2, using the Wald test, was used to determine statistically significant differentially expressed genes. Unless otherwise noted, the default size factors determined by DEseq2 were used.
Assembly: hg38
Library strategy: PRO-seq

extract_protocol:
Aliquots of frozen (-80°C) permeabilized cells were thawed on ice and pipetted gently to fully resuspend. Aliquots were removed and permeabilized cells were counted using a Luna II, Logos Biosystems instrument. For each sample, 1 million permeabilized cells were used for nuclear run-on, with 50,000 permeabilized Drosophila S2 cells added to each sample for normalization. Nuclear run on assays and library preparation were performed essentially as described in Reimer et al. [K. A. Reimer, C. A. Mimoso, K. Adelman, K. M. Neugebauer, Molecular Cell (2021)] with modifications noted: 2X nuclear run-on buffer consisted of (10 mM Tris (pH 8), 10 mM MgCl2, 1 mM DTT, 300mM KCl, 20uM/ea biotin-11-NTPs (Perkin Elmer), 0.8U/uL SuperaseIN (Thermo), 1% sarkosyl).  Run-on reactions were performed at 37°C.
Random hexamer extensions (UMIs) were added to the 3' end of the 5' adapter and 5' end of the 3' adapter.  Adenylated 3' adapter was prepared using the 5' DNA adenylation kit (NEB) and ligated using T4 RNA ligase 2, truncated KQ (NEB, per manufacturer's instructions with 15% PEG-8000 final) and incubated at 16°C overnight.  180uL of betaine buffer (1.42g of betaine brought to 10mL) was mixed with ligations and incubated 5 min at 65°C and 2 min on ice prior to addition of streptavidin beads.  After T4 polynucleotide kinase (NEB) treatment, beads were washed once each with high salt, low salt, and 0.25X T4 RNA ligase buffer (NEB) and resuspended in 5' adapter mix (10 pmol 5' adapter, 30 pmol blocking oligo, water).  5' adapter ligation was per Reimer but with 15% PEG-8000 final. Eluted cDNA was amplified 5-cycles (NEBNext Ultra II Q5 master mix (NEB) with Illumina TruSeq PCR primers RP-1 and RPI-X) following the manufacturer's suggested cycling protocol for library construction. A portion of preCR was serially diluted and for test amplification to determine optimal amplification of final libraries. Pooled libraries were sequenced using the Illumina NovaSeq platform

value: Corin


title: Kira8+Stattic-2

value: WT


title: WT-Tre-2


title: D0_C12


title: D0_D01


title: DMSO_2

value: DMSO


title: WT-Tre-1

value: root


title: Corin_1


title: Stattic-3


title: WT-Con-3


title: D0_C10


title: Stattic-2


title: Kira8+Stattic-1


title: D0_C11


title: D0_C09


title: DMSO_1


title: control 3

city: Hsinchu

first: Tingwen

last: Chen

address: No. 75, Boat Street, East District

country: Taiwan

institute: National Yang Ming Chiao Tung University

data_processing:
HISAT, featureCounts
Assembly: GRCh38
Supplementary files format and content: .txt

extract_protocol:
mRNA was purified from total RNA using magnetic oligo-dT beads
KAPA mRNA HyperPrep Kit

value: control


title: WT-Con-2

value: 6 h


title: Stattic-1


title: WT-Con-1


title: control 2


title: control 1


title: Kira8-3


title: HCT116-UPF1,12h-2

city: Kunming

first: Shimin

last: Gong

address: Wujiaying

institute: Life Sciences, Yunnan University

data_processing:
Raw reads were trimmed for adaptor sequence and the low-quality reads were excluded using Trim galore (Version:1.3.0)
Then, the reads were mapped to human and Neurospora crassa(spike-in) using STAR (Version: 2.7.10b)
Assembly: hg38
Supplementary files format and content: raw counts for each Sample

source_name: colon

extract_protocol:
The HCT116-AID2 cell lines were induced by 1 µg/ml of doxycycline for 24h, and wereadded or not added (as the control) to 10 µM 5-Ph-IAA (MCE, HY-134653) for several hours. Then transferring cultures to fresh medium supplemented with 10μg/mLactinomycin D (HY-17559, MCE). Samples were harvested at time points of 0, 4, 8 and 12h followingactinomycin Dtreatment. Three replicates were collected at each time point.To determine decay rate of RNA, 500ng HCT116-AID2 cellRNA and 50ngNeurospora RNA were input RT reaction. For tested genes, random RT primers were employed in RT reaction.
the VAHTS Universal V8 RNA-seq Library Prep Kit (Vazyme, NRM605)

value: colon

value: UPF1 -AID

value: Actinomycin-D


title: Kira8-2


title: Nobody 22-41 overexpressed in HEK293T 3

value: NBDY_22-41 overexpressed


title: D0_C06


title: HCT116-UPF1,12h-1


title: Kira8-1


title: D0_C05


title: HCT116-UPF1,8h-3


title: None3


title: D0_C04


title: HCT116-UPF1,8h-2


title: Nobody 22-41 overexpressed in HEK293T 1


title: None2


title: HCT116-UPF1,8h-1


title: None1


title: HCT116-UPF1,12h-3


title: Nobody 22-41 overexpressed in HEK293T 2


title: D0_C03


title: D0_C08


title: wt3_nkp46, Bulk RNA seq

city: Saint Louis

first: Alina

last: Ulezko Antonova

state: MO

address: 660 S. Euclid Ave

department: Path/Immuno

institute: WUSM

data_processing:
Base calls and demultiplexing were performed with Illumina’s bcl2fastq software and a custom python demultiplexing program with a maximum of one mismatch in the indexing read. RNA-seq reads were then aligned to the Ensembl release 76 primary assembly with STAR version 2.7.9a. Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 2.0.3. Isoform expression of known Ensembl transcripts were quantified with Salmon version 1.5.2. Sequencing performance was assessed for the total number of aligned reads, total number of uniquely aligned reads, and features detected. The ribosomal fraction, known junction saturation, and read distribution over known gene models were quantified with RSeQC version 4.0. Aligned gene counts were processed using the DESeq2 package with R (version 4.1). Genes with fewer than 10 counts among all of the samples were excluded.
Assembly: mm10
Supplementary files format and content: Bulk_all.gene_counts.tsv is a tab-delimited file that contains the gene counts from all samples.

source_name: Small intestine lamina propria

growth_protocol: Cell isolation protocol: Small intestines were flushed to remove luminal contents, and Peyer’s patches and mesenteric fat were removed. Intestines were opened lengthwise, gently agitated for 20 min in Hanks’ balanced salt solution (HBSS) containing 1M HEPES (Corning), 10% bovine calf serum (BCS) (HyClone) and 0.5M EDTA (Corning) and vortexed to remove epithelial cells. Small intestines were subjected to a second round of gentle agitation and vortexing in fresh HBSS/BCS/EDTA buffer. The tissue was then rinsed with HBSS before digestion with collagenase IV (Sigma-Aldrich) in complete RPMI 1640 for 40 min at 37 °C under agitation. Digested tissues were filtered through 100-µm strainer and subjected to density gradient centrifugation using 40 and 70% Percoll (GE Healthcare) solutions.

extract_protocol:
Total RNA was extracted using RNeasy Micro Kit (Qiagen) per manufacturer’s instructions. Samples were prepared according to library kit manufacturer’s protocol (SMARTer Low Input RNA Kit, Clontech Takara Bio), indexed, pooled, and sequenced on an Illumina NovaSeq 6000.
Library preparation was performed with 10 ng total RNA with a Bioanalyzer RNA integrity number score greater than 8. Double standard complementary DNA (cDNA) was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara-Clontech) according to the manufacturer’s protocol. cDNA was fragmented using a Covaris E220 sonicator using the following settings: peak incident power 18, duty factor 20%, and cycles per burst 50 for 120 s. cDNA was blunt ended, had an A base added to the 3′ ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12 to 15 cycles using primers, incorporating unique dual index tags. Fragments were sequenced on an an Illumina NovaSeq 6000

value: Small intestine lamina propria


title: HCT116-UPF1,4h-3


title: Spinal organoid at day 35 of differentiation (hiPS cell line 4)

city: Stanford

first: Sergiu

last: Pasca

state: California

address: 318 Campus Drive

department: Psychiatry

institute: Stanford University

description: 10x genomics

data_processing:
Gene expression levels were quantified for each putative cell barcode using the 10x Genomics CellRanger analysis software suite (version 7.1.0).
CellRanger count command with –include-introns=TRUE to include reads mapping to intronic regions.
Raw count matrices outputted from CellRanger were further processed using the R (version 4.1.2) package Seurat (version 4.3.0) (Stuart et al., 2019). Low quality cells with less than 2,000 unique genes detected and with mitochondrial counts accounting for greater than 15% of the total counts were identified and removed. Gene normalization (sctransform), PCA anaylsis (top 30 PCs), clustering (FindNeighbors/FindClusters, dataset dimension = 30, resolution=1)) were performed using Seurat with default paramaters.
Assembly: Human (GRCh38) 2020-A
Supplementary files format and content: tsv files with feature and cell barcode information corresponding to row and column indices of gene count data for each sample
Supplementary files format and content: matrix exchange format for sparse matrices of gene count data for each sample

source_name: in vitro

extract_protocol:
Four or five randomly selected organoids or assembloids were pooled to obtain single cell suspension and then incubated in 30 U/mL papain enzyme solution (Worthington Biochemical, LS003126) and 0.4% DNase (12,500 U/mL, Worthington Biochemical, LS2007) at 37°C for 45 min. After enzymatic dissociation, organoids were washed with a solution including protease inhibitor and gently triturated to achieve a single cell suspension. Cells were resuspended in 0.04% BSA/PBS (Millipore-Sigma, B6917-25MG) and filtered through a 70 μm Flowmi Cell Strainer (Bel-Art, H13680-0070).
Dissociated cells were then resuspended in PBS containing 0.04% BSA and loaded onto a Chromium Single cell 3’ chip. scRNA-seq libraries were prepared with the Chromium Single cell 3’ GEM, Library & Gel Bead Kit v3 (10x Genomics).

value: in vitro

value: Spinal organoid


title: Spinal organoid at day 35 of differentiation (hiPS cell line 1)

description: 10x genomics


title: HCT116-UPF1,4h-2


title: D0_C07

department: Bioinformatics

value: embryonic stem cell


title: D0_C01


title: wt3_ilc1, Bulk RNA seq


title: Floor plate-assembled spinal organoid at day 11 of differentiaiton (hiPS cell line 3)

description: 10x genomics


title: D0_B12


title: HCT116-UPF1,4h-1


title: wt3_dn, Bulk RNA seq


title: D0_B11


title: HCT116-UPF1,0h-3


title: wt3_ccr6, Bulk RNA seq


title: D0_B10


title: Floor plate-assembled spinal organoid at day 11 of differentiaiton (hiPS cell line 1, replicate 2)

description: 10x genomics


title: D0_C02


title: Spinal organoid at day 35 of differentiation (hiPS cell line 5)

description: 10x genomics


title: wt2_ilc1, Bulk RNA seq


title: wt2_nkp46, Bulk RNA seq


title: Spinal organoid at day 11 of differentiaiton (hiPS cell line 3)

description: 10x genomics


title: HCT116-UPF1,0h-1


title: D0_B08


title: D0_B07


title: wt2_dn, Bulk RNA seq


title: HCT116-Control,re,12h-2

value: Contro


title: Floor plate-assembled spinal organoid at day 11 of differentiaiton (hiPS cell line 1, replicate 1)

description: 10x genomics


title: HCT116-Control,re,12h-3


title: Spinal organoid at day 11 of differentiaiton (hiPS cell line 2)

description: 10x genomics


title: wt2_ccr6, Bulk RNA seq


title: wt1_nkp46, Bulk RNA seq


title: Floor plate-assembled spinal organoid at day 11 of differentiaiton (hiPS cell line 2)

description: 10x genomics


title: D0_B09


title: D0_B05


title: Spinal organoid at day 11 of differentiaiton (hiPS cell line 1, replicate 2)

description: 10x genomics


title: Spinal organoid at day 11 of differentiaiton (hiPS cell line 1, replicate 1)

description: 10x genomics


title: HCT116-Control,re,8h-3


title: HCT116-UPF1,0h-2


title: HCT116-Control,re,8h-2


title: Floor plate organoid at day 8 of differentiaiton (hiPS cell line 3)

description: 10x genomics

value: Floor plate organoid


title: wt1_ilc1, Bulk RNA seq


title: D0_B03


title: wt1_dn, Bulk RNA seq


title: Floor plate organoid at day 8 of differentiaiton (hiPS cell line 2)

description: 10x genomics


title: HCT116-Control,re,8h-1


title: HCT116-Control,re,12h-1


title: wt1_ccr6, Bulk RNA seq


title: D0_A12


title: D0_B06


title: ko3_nkp4, Bulk RNA seq

value: DKO


title: ko3_ilc1, Bulk RNA seq


title: D0_B04


title: HCT116-Control,re,4h-1


title: ko3_dn, Bulk RNA seq


title: D0_A11


title: HCT116-Control,re,0h-3


title: ko3_ccr6, Bulk RNA seq


title: IPEC1 cell - VerA_1uM_24hr - Rep5

type: RNA

city: Toulouse

first: Laura

last: Soler Vasco

state: Haute Garonne

address: 180 Chemin de Tournefeuille

country: France

institute: INRAE

hyb_protocol: 600 ng of Cy3-labelled cRNA (specific activity >6 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 10x Agilent fragmentation buffer and 25x Agilent blocking agent following the manufacturer's instructions (Agilent Technologies, Santa Clara, CA). On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 custom GE microarray (8X60K, Design 037880 enclosed in Agilent SureHyb-enabled hybridization chambers for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed sequentially in Wash buffer 1 (Agilent Technologies, 1 min) and Wash buffer 2 (Agilent Technologies, 37°C, 1 min).

scan_protocol: Slides were scanned immediately after washing on a Agilent G2505C Microarray Scanner with Agilent Scan Control A.8.5.1 software

data_processing: The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent Technologies, Santa Clara, CA) using default parameters (protocol GE1_1010_Sep10 and Grid: 037880_D_F_20120213). All subsequent data analyses were done under R (www.r-project.org) using packages of Bioconductor (www.bioconductor.org). Raw data (median of pixels intensity) were imported into R using the read.maimages function from the limma package with the following weight function (assigning a weight of 1 or 0 to each spot): myfunw<-function(x) {okType<-x$ControlType==0; okFoundGreen<-x$gIsFound==1; okPos=x$gIsPosAndSignif==1; okWellAbove<- x$gIsWellAboveBG==1; as.numeric(okType & okFoundGreen & okPos & okWellAbove);} We selected the spots with a minimal weight of 1 for 26 out of 32 microarrays or with a minimal weight of 2 per group from at least one experimental group. At this step, 43416 spots out of 61625 were selected. Data were then stored in an ExpressionSet object and normalized by the quantile method using the normalize.quantiles function from the preprocessCore R library. Replicated probes on the array (identical ProbeName) were resolved by taking the median normalized signal of each set of replicated probes. The resulting matrix has 43418 rows each corresponding to a unique ProbeName (provided as data Matrix).

organism: Sus scrofa

source_name: IPEC1 cell

label_protocol: For each sample, Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng of total RNA using the One-Color Quick Amp Labeling kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions, followed by Agencourt RNAClean XP (Agencourt Bioscience Corporation, Beverly, Massachusetts) purification. Dye incorporation and cRNA yield were checked using Dropsense™ 96 UV/VIS droplet reader (Trinean, Belgium).

growth_protocol: The non-tumorigenic and non-transformed intestinal cell line IPEC1 was chosen as a model due to their competence in terms of cytochrome P450 and p53 status and thus well-suited for the study of AF and other pro-toxicants. IPEC1 cells were grown in 100 × 20 mm Petri dishes (Cellstar, Greiner Bio-One, Germany) using DMEM/HAMs F12 medium (Sigma,), supplemented with 5% fetal bovine serum (FBS, Eurobio, Courtaboeuf, France), 1% streptomycin/penicillin (Eurobio), 1% L-glutamine (Eurobio), 1% insulin transferrin-selenium (Sigma), and 5 μg/mL epidermal growth factor (Becton-Dickinson, Le Pont de Claix, France). Cells were consistently maintained under controlled conditions at 39 °C in a humidified environment containing 5% CO2. To mimic real conditions, IPEC1 cells were differentiated into monolayers using transwell devices prior to exposure to toxins as previously described (Al-Ayoubi et al., 2022).  In brief, 2.105 cells were initially seeded in Transwell inserts featuring 0.4 μm polyethylene terephthalate membranes (Coring Falcon, Corning, NY, USA). Once confluence was attained, cells were systematically differentiated into monolayers within the same medium, enriched with 20 μg/mL dexamethasone but devoid of FBS. This differentiation process remained until a stable transepithelial electrical resistance was established, which normally takes 8-10 days.

extract_protocol: For total RNA extraction, cells were washed in phosphate-buffered saline (PBS; Sigma) and lysed in Extract-All reagent (Eurobio; Les Ulis, France). the total RNA extraction was followed according to Chomczynski and Sacchi (1987).  Following extraction, the concentration and quality of the RNA were assessed using a Dropsense 96 UV/VIS droplet reader (Trinean, Belgium) and an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA). The mean (± SD) RNA Integrity Number (RIN) was 9.53 ± 0.36.

treatment_protocol: Monolayers were washed with Phosphate saline buffer exposed apically to 1 µM and 3 µM VerA, 1 µM AFB1, or solvent control (DMSO) for 6 or 24 h in cell culture medium without serum (SVF) nor phenol rouge. These conditions were not cytotoxic as described previously (Al-Ayoubi et al., 2023).

value: VerA

tag: dose


title: IPEC1 cell - VerA_3uM_24hr - Rep4


title: D0_A10


title: HCT116-Control,re,4h-3


title: Floor plate organoid at day 8 of differentiaiton (hiPS cell line 1)

description: 10x genomics


title: HCT116-Control,re,0h-2


title: D0_B02


title: ko2_nkp46, Bulk RNA seq


title: ko2_ilc1, Bulk RNA seq


title: D0_A08


title: D0_A09


title: ko2_dn, Bulk RNA seq


title: HCT116-Control,re,4h-2


title: HCT116-Control,re,0h-1


title: D0_B01


title: HCT116-eRF1,12h-3


title: D0_A07


title: ko2_ccr6, Bulk RNA seq


title: HCT116-eRF1,12h-2


title: D0_A06


title: HCT116-eRF1,12h-1


title: ko1_nkp46, Bulk RNA seq


title: D0_A05


title: HCT116-eRF1,8h-3


title: IPEC1 cell - VerA_1uM_6hr - Rep2


title: IPEC1 cell - AFB1_1uM_6hr - Rep1


title: ko1_dn, Bulk RNA seq


title: IPEC1 cell - DMSO_0_24hr - Rep1


title: IPEC1 cell - AFB1_1uM_24hr - Rep1


title: HCT116-eRF1,8h-2


title: HCT116-eRF1,8h-1


title: ko1_ccr6, Bulk RNA seq


title: IPEC1 cell - DMSO_0_6hr - Rep5


title: IPEC1 cell - VerA_3uM_6hr - Rep1


title: IPEC1 cell - AFB1_1uM_6hr - Rep4


title: D0_A03


title: HCT116-eRF1,4h-3


title: IPEC1 cell - DMSO_0_6hr - Rep4


title: Multiome_KO3, Paired scRNA/ATACseq

data_processing:
The CellRanger Software Suite (v6.0.0) from 10x Genomics was used for sample demultiplexing, barcode processing, and single cell counting.
Assembly: mm10
Supplementary files format and content: For GEX, tab-delimeted files contain the features, barcodes and count matrix files contained in the "filtered_feature_bc_matrix" subfolder of the CellRanger "outs" folder.
Supplementary files format and content: For ATAC, tab-delimeted files contain the fragment files and the frament file indices of the CellRanger "outs" folder.
Library strategy: Multiome scRNA/ATAC-seq

molecule: nuclear RNA

extract_protocol:
Nuclei extraction was performed as described in the 10x Genomics demonstrated protocol (CG000365).
For sample preparation on the 10x Genomics platform, the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle was used. Briefly, barcoded cDNA was amplified for 6 cycles before being purified using SPRIselect beads and the precise concentration of each library was determined.


title: HCT116-eRF1,4h-2


title: D0_A02


title: D0_A04


title: M222, CD8 T cells, PD-L1−

city: Heraklion

first: Matthieu

middle: David

last: Lavigne

address: Nikolaou Plastira 100

country: Greece

institute: IMBB-FORTH

description:
Library name: NGS-2356
Column name in raw_counts_All_Samples.txt: M222

data_processing:
Raw sequencing data were assessed for quality using FastQC version 0.12.0 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Adapter sequences and low-quality bases were trimmed with Cutadapt version 1.16 (DOI: https://doi.org/10.14806/ej.17.1.200). Ribosomal RNA sequences were identified and filtered out using RiboDetector version 0.3.1 (https://doi.org/10.1093/nar/gkac112). The resulting single-end reads were aligned to the human reference genome (hg38) using Rsubread version 2.12.3 with default settings. Gene-level counts were obtained using the 'featureCounts' function within the Rsubread package.
Differential expression analysis  (DEA) was conducted by running EdgeR63 version 3.40.2. For the comparison of PD-L1+ vs. PD-L1− CD8 positive sorted cells, DEGs in either group of the comparison were defined by applying the following thresholds: |Log2FC| >1.5 and FDR<0.05, which was considered statistically significant. For the comparison of NR and R, PD-L1+ CD8 T cell samples were divided according to the RECIST 1.1 criteria. DEGs in either group were identified using the thresholds |Log2FC|>0.5 and FDR<0.05, which were considered statistically significant. Heatmaps and boxplots were created in R with an in-house–developed script which is based on the complex heatmap R package.
Assembly: hg38
Supplementary files format and content: tab-delimited text file includes raw counts for each sample

source_name: CD8+ T cells

extract_protocol:
RNA from FACS-sorted CD8 PD-L1 positive or negative cells (21x103 on average).  NSCLC was isolated using Nucleospin RNA XS kit (Macherey-Nagel), according to the manufacturer instructions. The quality and quantity of isolated RNA was measured using  2100 Bioanalyser  (Agilent)  and RNA 6000 Pico Kit reagents (Agilent).
RNA samples with RNA integrity number (RIN)>7 were used for library construction using the 3′ mRNA-Seq Library Prep Kit FWD for Illumina (QuantSeq-LEXOGEN) as per the manufacturer’s instructions. Amplification was controlled for obtaining optimal unbiased libraries across samples by assessing the number of cycles (ranging from 20-24) required by qPCR. DNA High Sensitivity Kit for bioanalyzer was used to assess the quantity and quality of libraries, according to the manufacturer’s instructions (Agilent). Libraries were multiplexed and sequenced on an Illumina Nextseq 500 at the genomics facility of IMBB FORTH according to the manufacturer’s instructions.

tag: patient id

tag: sample id_(p=_plus;_m=minus)

value: CD8+ T cells

value: WT

tag: group id

tag: ici response

value: R


title: Multiome_KO1N, Paired scRNA/ATACseq

tag: cell type


title: ko1_ilc1, Bulk RNA seq

tag: tissue


title: IPEC1 cell - VerA_1uM_6hr - Rep5


title: Multiome_KO2, Paired scRNA/ATACseq


title: M215, CD8 T cells, PD-L1−

description:
Library name: NGS-2354
Column name in raw_counts_All_Samples.txt: M215


title: D0_A01

tag: strain


title: HCT116-eRF1,0h-3

tag: cell line

tag: genotype


title: HCT116-eRF1,0h-2

tag: treatment


title: M212, CD8 T cells, PD-L1−

description:
Library name: NGS-2352
Column name in raw_counts_All_Samples.txt: M212


title: IPEC1 cell - AFB1_1uM_24hr - Rep4


title: Multiome_WT2N, Paired scRNA/ATACseq


title: IPEC1 cell - VerA_3uM_24hr - Rep1


title: IPEC1 cell - VerA_3uM_6hr - Rep3


title: IPEC1 cell - DMSO_0_24hr - Rep4

value: DMSO


title: HCT116-eRF1,4h-1


title: M209, CD8 T cells, PD-L1−

description:
Library name: NGS-2350
Column name in raw_counts_All_Samples.txt: M209


title: HCT116-eRF1,0h-1


title: M208, CD8 T cells, PD-L1−

description:
Library name: NGS-2348
Column name in raw_counts_All_Samples.txt: M208

value: NR


title: Multiome_WT1, Paired scRNA/ATACseq


title: HCT116-Control,12h-3

value: colon


title: M202, CD8 T cells, PD-L1−

description:
Library name: NGS-2346
Column name in raw_counts_All_Samples.txt: M202


title: IPEC1 cell - VerA_1uM_24hr - Rep1


title: S. coelicolor A3(2) grown in liquid SMM medium, cold-shock, 120 min, total RNA, biol replicate 1

city: Brighton

first: Ruslan

last: Evans

state: East Sussex

address: Lewes Road

department: School of Applied Science

institute: University of Brighton

data_processing:
Reads were quantified against S. coelicolor transcriptome gene annotations from NCBI reference NC_003888.3 using kallisto quant version 0.46.2.
Kallisto quantifications were converted to gene level by tximport version 1.20.0 then analysed for differential expression using DESeq2 version 1.32.0.
Translational Efficiency was evaluated using the deltaTE protocol described in 'deltaTE: Detection of Translationally Regulated Genes by Integrative Analysis of Ribo-seq and RNA-seq Data' Chothani et al. , Current Protocols in Molecular Biology e108 vol 129, 2019'.
Assembly: GCF_000203835.1_ASM20383v1  NC_003888.3
Supplementary files format and content: comma separated files. Table of fold-change and adj pval for each gene for Translational Efficiency, total RNA, and RPF fraction.
Supplementary files format and content: tab-delimited text files. Matrix table of counts for every gene generated by kallisto quant.

organism: Streptomyces coelicolor

growth_protocol: Spores were pre-germinated in 2 x YT medium for 7 hrs at 30°C and 250 rpm then used to inoculate 200ml of SMM medium (Practical Streptomyces Genetics, Kieser et al. 2000, ISBN 9780708406236) in 500 ml spring containing conical flasks at a concentration of approx. 500,000 cfu / ml and incubated for 18 h 20 min at 30°C / 250 rpm.

extract_protocol:
For total RNA extraction (transcriptome analysis), bacterial cultures were treated with RNA protect bacteria reagent ( Qiagen #76506) and incubated for 10 min at room temperature. Cultures were centrifuged and the pellets were stored at -80°C. Pellets from 15 ml of bacterial cultures were resuspended in 200 µl of lysozyme dissolved in TE buffer at a concentration of 0.015 g per ml and incubated at room temperature for 15 min. 600 µl of RNeasy Lysis Buffer (RLT buffer, Qiagen #79216) containing 10 µl / ml β-mercaptoethanol was added and the mixture mixed by vortex mixer then transferred to a 2 ml microfuge tube containing 5 mm stainless steel beads (Qiagen #69989) and placed in a Tissuelyser LT (Qiagen #85600) for efficient mechanical cell disruption / homogenisation for 2 min at 30 rpm. The suspension was then centrifuged at 16,627 RCF for 2 min, the supernatant was recovered and its volume estimated. One volume of acid pH 4.5 phenol:chloroform:isoamyl alcohol (125:24:1, Fisher #AM9720) was added and the mixture mixed by vortex mixer for 30 seconds, then centrifuged at 4°C for 5 min at 16,627 RCF. The upper phase containing the nucleic acids was removed and retained leaving behind the proteins in the interface, and the phenol/chloroform extraction repeated twice more. Then one volume of chloroform was added, the mixture mixed by vortex mixer then centrifuged at 4°C for 2 min at 16,627 RCF, and the upper phase was again transferred to a fresh RNase-free microfuge tube. From this point onward the standard manufacturer’s instructions were followed for the miRNAeasy RNA extraction kit (Qiagen #217004). The total RNA was eluted in 30 µl of nuclease free water and stored at -80°C. Genomic DNA was removed using the Turbo DNA free kit (Invitrogen AM1907). For the Riboseq protocol the protocol followed was based on that described in Jeong et al. Nature Communications 2016. Briefly, the mycelium was ground in lysis buffer and stored at -80°C as described in the treatment protocol. The ground mycelium was defrosted in ice and centrifuged at 20,000 RCF at 4°C. The supernatant was recovered and the RNA concentration was determined by NanoDrop spectrophotometer or Qubit fluorometer. 50 µg of cell lysate was incubated with 400 U of MNase for 2 h at 37°C. The digestion was stopped by adding 2.5 µl of EGTA 0.5M. The monosomes were isolated through Sephacryl S400 Microspin columns (GE cat 27-5140-01) in Recovery buffer (50mM Tris-HCl (pH8); 1% Triton X100; 50mM MgCl2; 25mM EGTA; 250mM NaCl) and the purified ribosomes were stored at -80°C. The ribosome protected RNA was isolated by column purification using the miRNeasy kit (Qiagen #217004) according to manufacturer’s protocol, and RNA between 15 and 45 nt was size selected by electrophoresis in 15% acrylamide gel in TBE. RNA was extracted from the gel in 500 µl of gel extraction buffer (0.25% SDS, 0.3M NaOAc pH 5.5, 1 mM EDTA) and precipitated by adding 16 µl of 5M NaCl, 45 µl 3M NaOAc, 4 µl glycoblue (15 mg / ml, Fisher #10391565) and 1.2 ml absolute ethanol and incubating at -80°C for 1 h. The RNA was centrifuged for 30 min at 20,000 RCF at 4°C. The RNA pellet was washed twice with ice cold 70% ethanol and air dried before resuspension in 9 - 11 µl of RNAse free water. The RNA was subsequently rRNA depleted using NEBNext Core Reagent Set (NEB #E7865) with custom DNA probes designed to cover Streptomyces specific rRNA and tRNA sequences. The RNA was subsequently treated with Polynucleotide Kinase for 1h at 37°C and the RNA was purified from the enzyme and the buffer using miRNeasy mini columns. The RNA was eluted in 30 µl of RNase free water, dehydrated and resuspended in 6 µl of nuclease-free water and used for library construction using the NEB NEBNext Small RNA Library Prep Set for Illumina (cat E7330S) following the manufacturer instructions.
Total RNA were depleted of rRNA by using the NEBNext Core Reagent Set (NEB #E7865) with custom DNA probes designed to cover Streptomyces specific rRNA and tRNA sequences and library constructions were carried out using the NEB directional RNAseq kit (NEB #E7760) and NEB NEBNext Small RNA Library Prep Set for Illumina (NEB #E7330S) following the manufacturer instructions.
directional RNA-seq for total RNA and miRNAseq library prep for the ribosome protected RNAs

treatment_protocol: 15ml of cultures taken immediately prior to the cold-shock as a 'time=0' reference were preserved with RNAProtect (Qiagen #76506) for total RNA extraction. 105ml of culture was taken at the same point for use as a 'time=0' reference in ribosome profiling experiments, vacuum filtered to remove medium and the cells immediately frozen in liquid nitrogen. The frozen cells were ground using a pestle and mortar whilst being kept frozen by adding liquid nitrogen. The pulverized mycelium was dissolved in a lysis buffer (20mM Tris pH 7.6; 5mM MgCl2; 140mM NaCl; 1% Triton X100;chloramphenicol 34 µg / ml) and frozen at -80°C. Cultures (30 ml) were exposed to cold-shock by the addition of 3 volumes of fresh media equilibrated at 4°C to rapidly reduce the temperature to 10°C, whilst control cultures had fresh media at 30°C added. After 120 min 15 ml of cultures was removed for total RNA analysis and preserved as before, and 105 ml of culture was removed for ribosome profiling experiments and processed as before.

tag: medium

value: SMM

value: cold-shock

tag: time (min)

tag: fraction

value: total RNA

tag: biol replicate


title: IPEC1 cell - VerA_1uM_24hr - Rep4


title: S. coelicolor A3(2) grown in liquid SMM medium, cold-shock, 120 min, total RNA, biol replicate 2


title: HCT116-Control,12h-1

value: Actinomycin-D