title: Kidney, old, female, SGLT2

city: BAR HARBOR

first: CGDS

last: Data

state: ME

address: 159 OLD BAR HARBOR RD

institute: MDI Biological Laboratory

description: Library name: Old Female SGLT2

data_processing:
Data processing was done using nf-core/scrnaseq version 2.6.0 with aligner set to cellranger.
Processed data was analyzed with Seurat v5.0.2
Doublets were removed with DoubletFinder v2.0.4
Assembly: Ensembl Version 111 (Nfu_20140520.111)
Supplementary files format and content: Tab-seperated values files and matrix files (cellranger filtered)
Supplementary files format and content: Cell-by-cell metadata extracted from Combined Seurat Object (All samples share same file)

organism: Nothobranchius furzeri

source_name: Kidney

growth_protocol: All the work reported here was performed in wild-type African turquoise killifish (Nothobranchius furzeri) of the strain GRZ (origin: Gona-Re-Zhou National Park). All procedures were approved by the MDIBL Institutional Animal Care and Use Committee (IACUC, #24-02) and complies with the MDI Biological Laboratory Institutional Assurance # D16-00341. Our husbandry protocol was adapted from Sandhi et al, manuscript in preparation. Fish were maintained at 27°C in an Aquaneering® recirculating system under a 12h light/dark cycle. Water parameters were continuously monitored (pH 7.0–7.4, conductivity 2800 µS). Adult fish were housed in age-matched groups, with adult males in 3 L tanks adjacent to 2-4 females in 6 L tanks and mated once weekly. Based on tank and group size, plastic plants (Aquaneat®) or igloos (Plexx®, Catalog #13220B) were added for environmental enrichment. Embryos were collected, disinfected in 550ppm PVP Iodine (Ovadine®, Syndel, USA), and incubated at 28°C in Ringer’s Solution. At the black-iris stage (12-14 days post fertilization (dpf)), embryos were transferred to coconut plates with Whatman® Filter paper and stored at 27°C for three weeks. Golden eyed embryos were hatched using a salt solution (1g Instant Ocean® in 1L RO water, pH=6, 4°C). Hatchlings were reared at controlled densities and transitioned to system water at 7 days post-hatch (dph). Fish were fed Artemia and Killifeast® (Brine Shrimp Direct, USA) according to age-specific schedules. Under these conditions, the median lifespan was 18 weeks for both sexes. Group housing males and females led to a relative decrease in male lifespan in our facility (female median lifespan: 20 weeks, male median lifespan: 16 weeks, data now shown). "Young" and "old" time points were defined as 6–8 weeks, where sexual maturity was reached, and 18 weeks, respectively. Experimental fish for the SGLT2 inhibitor trial were housed separately from the circulating system. Males and females were group-housed in a 6 L tank with 1 male and 2-3 females. Large cohorts hatched on the same day were split in a 1:1 ratio to a control and interventional group at the age of 4 weeks.

extract_protocol:
We established and verified our own single nuclei RNA isolation protocol for killifish kidneys. Our protocol was established by combining two existing protocols, one for Killifish brain (Teefy et al., 2022) and a kidney-specific protocol for mice (Leiz et al., 2021). For the experiment the following numbers of fish were used: young female n=9, young males n=6, old females n=6, old males n=5. Pooling of multiple kidneys was necessary to achieve an average tissue weight of 20 mg. Young time points were defined as 6 weeks old animals; old time point was defined by the median lifespan of the cohort (in this case: 20 weeks of age). All old fish were transcardially perfused with ice-cold PBS before kidney collection (Mariën et al., 2022). Kidneys were pooled into RNA-later solution and stored at 4°C for 24h. Then all remaining liquid was removed, and tissue was moved to -80°C until isolation. Nuclei isolation: On day of experimentation tissue was removed from the cryotube, rehydrated with ice-cold PBS on ice. PBS was removed and tissue was resuspended in Nuclei Lysis Buffer (NLB: 10mM Tris-HCl (pH 7.4), 10mM, NaCl, 3mM MgCl2, 0.01875% v/v NP-40, 0.2U/µl RNase Inhibitor). Tissue in buffer was transferred into a 2ml dounce homogenizer on ice and homogenized with pestle A for 30 sec. The homogenate was passed through 70µM strainer into a 15 ml tube, previously precoated with 5% BSA overnight. The filter was washed with another 1ml of NLB. The dissociated tissue was moved back into the douncer and homogenized using pestle B for 1 minute. The homogenate was then strained through a 40µm strainer into a 15 tube precoated with 5% BSA. The homogenate was diluted with Nuclei wash buffer (NWB: 2% BSA in PBS, 0.2U/µl RNase Inhibitor) up to 5 ml and gently inverted. The suspension was centrifuged at 500g at 4°C for 10 minutes. Nuclei purification: Nuclei were purified with an additional wash step. The supernatant was discarded by gently tipping over the tube and the pellet was resuspended in 1 ml NWB using a wide bore P1000. The volume was filled up to 5 ml with NWB and the tube centrifuged 500g at 4°C for 5 minutes. Debris removal: The supernatant was carefully discarded by gently tipping over the tube. The pellet was resuspended in 1 ml NWB with a wide bore P1000. 300µl debris removal solution (Miltenyi Biotec®, USA) was added and slowly mixed. The solution was overlayed with 1ml NWB and centrifuged at 3000g for 10 minutes at 4°C. The top two phases were gently removed. The remaining phase was diluted to 8ml and centrifuged at 1000g for 10 minutes at 4°C.  The supernatant was removed and the pellet resuspended in 1 ml NWB. The solution was filtered through a 20µm filter into a 1.5ml low-bind DNA tube. Samples were processed which have shown an RNA Integrity number of >7 as measured on a Agilent Bioanalyzer using a Bioanalyzer High Sensitivity RNA Analysis Kit (Agilent®, USA). Nuclei were counted using Acridine Orange/Propidium Iodide Assay on a CellDrop®( DeNovix®, USA) automated cell counter set to “Nuclei AO/PI”.
A Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 (10x genomics) was used for library construction following the user’s manual. Nuclei were concentrated to 1000nuclei/µl for a targeted cell recovery of 7000.

treatment_protocol: Experimental diet consisted of custom-made food. 200ml of reverse osmosis water was brought to a boil in a microwave. 3 teaspoons (tsp) of Repashy® Grub Pie Insectivore Gel Premix (Repashy® Speciality Pet Products, USA) and ¾ tsp of Repashy® Redrum Carotenoid Gel Premix (Repashy® Speciality Pet Products, USA; for additional food coloring) were added, immediately stirred and let to cool to ~45°C. The liquid premix was poured onto aluminum-covered trays as a thin layer and let to set at room temperature. For drugged food, the SGLT2 inhibitor Dapagliflozin was added for a final drug concentration of 2µM. DMSO was added to Control food; final concentration of DMSO in both foods was 0.1% v/v. After gel solidified into thin sheets, these were dehydrated at 45°C for 12 hours in a food dehydrator (SeptreeTM, USA). Once completely dried, sheets were cut into small flakes using a SlapTMChop (USA). Fish were fed 3x times daily with a spoon, with approximately 60mg food/fish/day, resulting in a SGLT2i dosage of approximately 0.04mg/kg fish/d (equivalent to 3mg Dapagliflozin daily for a 70kg person). Diet was supplemented with Artemia once daily. Water changes were performed daily with fresh system water and the remaining food was cleaned out. Dosages of equivalent to 10mg Dapagliflozin/d/70kg led to a decrease in lifespans compared to control diet, therefore a lower concentration was chosen for downstream experiments.

value: Kidney

value: WT

value: Old


title: HepG2, siDUS2, SR, treated, rep#3

status: Public on Jul 30 2025

type: SRA

city: Chicago

first: Cheng-Wei

last: Ju

state: Illinois

address: 929 East 57th Street, GCIS, Room E313

country: USA

institute: The University of Chicago

library_source: transcriptomic

data_processing:
The sequencing data were all trimmed with cutadapt tool (v4.8) to remove adapters and low-quality reads. PCR duplicates were removed with BBMap tool (v.38.73), 5-mer random barcodes at reads ends were trimmed and low-quality or short reads (less than 20 nt) were removed using cutadapt tool.
For tRNAs, remaining reads were aligned to tRNA sequence obtained from GtRNAdb (https://gtrnadb.ucsc.edu/) using hisat-3n (v.2.2.1-3n-0.0.3). Mitochondria and chloroplast tRNA sequences are obtained from published papers.
Meanwhile, we have also aligned remaining reads to hg38, mm10, or TAIR10 genome using hisat3n to investigate D sites in other RNA species.
The generated table from hisat-3n were parsed and analyzed by inhouse scripts. Internal mutation ratio at each D candidate site suggested by hisat-3n, was further confirmed by direct IGV visualization (v.2.8.0).
Assembly: mm10 (GRCm38.102), hg38 (GRCh38.107), TAIR10
Supplementary files format and content: For samples with cellular RNA, the mutation ratio at D sites in treated, input samples are shown. For samples with RNA probes, mutations at D sites are shown.
Library strategy: Chemical Reduction Assisted Cytosine Incorporation sequencing (CRACI)

molecule: total RNA

organism: Homo sapiens

growth_protocol: Human HepG2 cell lines and mouse embryonic stem cells (mESC) used in this study were all purchased from ATCC (the American Type Culture Collection). HepG2 cell lines were maintained in DMEM (GIBCO, 11995), supplemented with 10% FBS and 1% 100X Pen/Strep (GIBCO). mESC cells were grown under a typical feeder-free culture condition: cells were cultured in DMEM (GIBCO, 11995), supplemented with 15% FBS, 1% Pen/Strep (GIBCO), 1X Glutamax (GIBCO), 1X non-essential amino acids (GIBCO), 1X 2-Mercaptoethanol (GIBCO), and 1000 U/ml leukemia inhibitory factor (Millipore, ESG1107), together with two inhibitors: 3 mM CHIR99021 (STEMCELL Technologies, dissolved in DMSO) and 1 mM PD0325901 (STEMCELL Technologies, dissolved in DMSO). mESC cells were distinctively cultured on gelatinized culture plates (0.2% Gelatin). The culture of mESC cells were passaged every 2 days. All cells were cultured at 37 Co under 5.0% CO2.

extract_protocol:
Generally, with harvesting cells as the first step, the media was aspirated, and the cells were washed once with proper volume of ice cold DPBS buffer for each plate. Then total RNA was isolated from cells with TRIzol reagent (Invitrogen) and then extracted following manufacturer’s protocol by isopropanol precipitation. Small RNA fraction was purified from total RNA with RNA Clean & Concentrator kit (Zymo Research) following manufacturer’s protocol. mRNA was extracted by two rounds of polyA+ purification with Dynabeads mRNA DIRECT kit (Ambion).
CRACI for D site detection Wild-type HepG2, mESC, and arabidopsis thaliana seedling cells (two replicates for each sample, one 10-cm plate per replicate); HepG2 siControl or DUS knockdown cells were prepared as described (two replicates for each sample, one 10-cm plate per replicate). After extracting the polyA+ RNA or small RNA from mammalian cells or plant cells, around 100 ng RNA was fragmented using RNA Fragmentation Reagents (catalog no. AM8740, Invitrogen) and heated at 70 °C for 14 min, followed by purification with Oligomer Clean and Concentrator (Zymo Research). 3′-End repair and 5′-phosphorylation were conducted with T4 polynucleotide kinase (PNK) (catalog no. EK0032, Thermo Fisher Scientific). RNA was mixed with 3 µL 10× T4 PNK reaction buffer (catalog no. B0201S, NEB) and 3 µl T4 PNK, diluted to a final volume of 30 µl, and incubated at 37 °C for 45 min; then 1.5 µl T4 PNK and 1.5 µl 10 mM ATP were added for another incubation at 37 °C for 45 min, followed by Oligomer Clean and Concentrator (Zymo Research) purification eluting with 10 µl RNase-free water.  To perform 3′-adapter ligation, 10 µl 3′-repaired and 5′-phosphorylated RNA fragments were incubated with 1.0 µl 20 µM RNA 3′ SR Adapter (5′App-NNNNNATCACG AGATCGGAAGAGCACACGTCT-3SpC3, with ATCACG as the inline barcode) at 70 °C for 2 mins and placed immediately on ice. Then, 2.5 µl 10× T4 RNA Ligase Reaction Buffer (catalog no. M0373L, NEB), 7.5 µl PEG8000 (50%), 1 µl SUPERase•In RNase Inhibitor and 2 µl T4 RNA Ligase 2 truncated KQ (catalog no. M0373L, NEB) were added to the RNA–adapter mixture48,49. The reaction was incubated at 25 °C for 2 h followed by 16 °C for 10 h. The reaction was further diluted to 47 µl with nuclease-free water, and the excessive adapters were removed with 2 µl 5′-deadenylase (catalog no. M0331S, NEB) at 30 °C for 45 min followed by adding 1 µl RecJf (catalog no. M0264L, NEB) for ssDNA digestion at 37 °C for 45 min. The 3′-end-ligated RNA was extracted by RNA Clean and Concentrator (Zymo Research) and eluted with 10 µl RNase-free water. The purified RNA was incubated with 1.2 µl 10 µM 5′ SR Adapter (5′-GUUCAGAGUUCUACAGUCCGACGAUC -3′) at 70 °C for 2 mins and placed immediately on ice. Then 2.5 µl 10× T4 RNA ligase reaction buffer, 1.0 µl 25 mM ATP, 10 µl PEG8000 (50%) and 1 µl T4 RNA Ligase 1 (high concentration, catalog no. M0437M, NEB) were added to the RNA–adapter mixture. The reaction was mixed well and incubated at 25 °C for 8 h, followed by RNA Clean and Concentrator (Zymo Research) purification, eluting with 12 µl RNase-free water.  A 2 µl aliquot of the purified RNA was saved for ‘Input1’ library construction, 2 µl aliquot of the purified RNA was saved for ‘Input2’ library construction, and the rest 6 µl was subjected to CRACI optimized reduction treatment, as the ‘Treated’ sample. The 6 µl RNA was mixed with 40 µl freshly prepared KBH4 (1 M KBH4, prepared by dissolving 54 mg KBH4 in 1000 µl RNase-free water) and Incubated the reaction mixture at 25 °C for 3 h, followed by purification with RNA Clean and Concentrator (Zymo Research) and elute with 10 µl H2O. ‘Input1’, ‘Input2’ and purified ‘Treated’ samples were diluted to 10 µl and mixed with 1.0 µl 2.0 µM SR RT primer (5′-AGACGTGTGCTCTTCCGATCT -3′) at 65 °C for 2 min and moved immediately onto ice. Then for ‘Input2’ and ‘Treated’ samples, a stock with 2 µl 10x AMV RT Buffer (New England BioLabs, cat. no. B0277AVIAL), 2 µl 1 mM dNTP Solution Mix (dilute from 10 mM dNTP Solution Mix, New England BioLabs, catalog no. N0447L), 2 µl 10mM dGTP (dilute from 100 mM dNTP Solution Mix, New England BioLabs, catalog no. N0442S), 1 µl RNaseOUT Recombinant Ribonuclease Inhibitor (catalog no. 10777019, Thermo Scientific) and 2 µl HIV RT (Worthington Biochemical, catalog no. LS05003) was added. For ‘Input1’ sample, a stock with 2 µl RNase-free water, 2 µl 10x AMV RT Buffer, 2 µl 10 mM dNTP Solution Mix (New England BioLabs, catalog no. N0447L), 1 µl RNaseOUT Recombinant Ribonuclease Inhibitor and 2 µl HIV RT was added.  The reaction mixture was heated at 70 °C for 5 min and then cDNA was purified using DNA Clean and Concentrator (Zymo Research). The eluted cDNAs (20 µl) were stored at –80 °C; 4 µl was used for each 15-cycle PCR amplification reaction, which was performed with the SR Primer for Illumina (NEB) and indexed primers (from NEBNext Multiplex Oligos for Illumina). All libraries were purified on a 3.5% low melting point agarose gel and sequenced on Illumina NovaSeqX with single-end 100 bp read length.

tag: genotype

value: siDUS2 Knockdown

tag: cell line

tag: fraction

value: small RNA (<200 nt)


title: HepG2, siDUS3, SR, input1, rep#1

value: siDUS3 Knockdown


title: mmYS-ipe, Young Sed, Imd Post-Ex, Male2

city: Ann Arbor

first: Andrew

last: Ludlow

state: Education

address: 830 N University Ave

institute: University of Michigan

data_processing:
MinKNOW v22.03.4 (parameter: q-score > 8)
Sequence reads were mapped to GRCm39/mm39 using minimap2 v2.14 (parameters: default) and subsequently converted to SAM files using SAMtools v1.13.
Read count extraction was performed using TALON (parameters: coverage=0.75, identity=0.8)
Assembly: GENCODE Release M29 (GRCm39/mm39)
Supplementary files format and content: tab-delimited text file includes raw counts for each sample

molecule: polyA RNA

organism: Mus musculus

source_name: Skeletal Muscle (gastrocnemius)

extract_protocol:
Total RNA from powdered tissues were isolated using the RNeasy Plus Universal Mini Kit (Qiagen, Cat# 73404). 1.2 μg of total RNA was used to isolate poly(A)-containing RNA with the NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England BioLabs, Cat# E7490S).
1 ng of poly(A)-mRNA isolates was used synthesize barcoded PCR-cDNA libraries (Oxford Nanopore Technologies®, Cat# SQK-PCB109).

tag: tissue

value: Skeletal Muscle (gastrocnemius)

tag: time

value: immediately post-exercise

value: hTERT

tag: Sex

value: male


title: Kidney, old, female, control

description: Library name: Old Female Control

library_source: transcriptomic single cell

tag: age

tag: treatment

value: Control

value: Female


title: Kidney, young, female, control

description: Library name: Young Female Control

value: Young


title: mmYS-pre, Young Sed, Pre-Ex, Male5

value: pre-exercise


title: HepG2, siDUS2, SR, treated, rep#2


title: mmYS-pre, Young Sed, Pre-Ex, Female4

value: female


title: mmYS-pre, Young Sed, Pre-Ex, Female3


title: Kidney, old, male, SGLT2

description: Library name: Old Male SGLT2

value: Male


title: Kidney, old, male, control

description: Library name: Old Male Control


title: HepG2, siDUS2, SR, treated, rep#1


title: Kidney, young, male, control

description: Library name: Young Male Control


title: mmYS-pre, Young Sed, Pre-Ex, Male2


title: Pig hrLuc_2, heart, 14 weeks post MI

city: Heidelberg

first: Patrick

last: Most

address: INF 410

country: Germany

institute: University Hospital Heidelberg

description: Library name: Sample 39B

data_processing:
Analysis of fastq files was performed in R (version 4.1.2)
Quality control: fastqcr (version 0.1.2) and FastQC (version 0.11.8)
Mapping against pig genome: Rsubread (version 2.7.1, annotation release 106)
For pathway analyses, pig annotations were mapped to corresponding human annotations using org.Ss.eg.db (version 3.13.0) and org.Hs.eg.db (version 3.13.0). Genome wide annotation for Human. R package version 3.8.2. 2019. Annotations that could not be mapped with these packages (14.9 %) were blasted with blastn from ncbi-blast+ (version 2.9.0-2) with rBLAST (version 0.99.2) and the best hit for mapping was identified as subject hit with sequence identity above 80 % and the lowest mean e-value below 0.01 and assigned to the queried annotation.
Gene expression: DESeq2 (version 1.33.1)
Weighted gene correlation network analysis: (WGCNA, version 1.70-3)
Additional analyses: pcaExplorer (version 2.19.0), PCAtools (version 2.5.0), clusterProfiler (version 4.1.0) and ReactomePA (version 1.37.0) based on variance stabilizing transformation (VST)-normalized feature counts
Assembly: Sscrofa11.1_GCF_000003025.6, PMID: 30783653
Supplementary files format and content: csv file contains VST normalized feature counts

organism: Sus scrofa

source_name: myocardium

extract_protocol:
Total RNA was harvested using AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany).
Sequencing libraries were prepared with TruSeq RiboZero Kits (Illumena, Santa Clara, CA)

value: myocardium

value: hrLuc


title: HepG2, siDUS2, SR, input2, rep#1


title: preosteoclast from femur, LSD1cKO mouse with no ligature 1UL

city: Minneapolis

first: Kim

last: Mansky

state: MN

address: 515 Delaware St SE

institute: University of Minnesota

description: Library name: KO FEM 1 UL

data_processing:
Sequencing reads were mapped to the mouse reference genome (GRCm39 v109) via Hisat2 (v2.1.0).
Quality control on raw sequence data for each sample was performed with FastQC
Gene quantification was done via Feature Counts for raw read counts
Genes differentially expressed between WT and conditional KO and skeletal site were determined using edgeR software package with an Absolute fold-change cutoff set at >2 and FDR < 0.05
Assembly: mouse (GRCm39 v109)
Supplementary files format and content: Mansky044rawcounts.txt

source_name: bone marrow

growth_protocol: Bone marrow was isolated from mandible or femur of ligated and unligated mice

extract_protocol:
RNA was isolated using RNA Plus Mini Kit (Qiagen)
Libraries were constructed by UMN Genomic Center

treatment_protocol: Bone marrow was treated with M-CSF and RANKL for 2 days to stimulate osteoclast differentiation

value: bone marrow

tag: cell type

value: preosteoclasts


title: HepG2, siDUS2, SR, input1, rep#2

value: siDUS2 Knockdown


title: Arsenic treated male brain

city: Greenwood

first: Anurag

last: Chaturvedi

state: SC

address: 114 Gregor Mendel Cir

department: Center for Human Genetics

institute: Clemson University

description: Library name: Male treatment

data_processing:
The mkfastq pipeline in Cell Ranger v3.1 (10X Genomics) was used to convert BCL files into demultiplexed FASTQ files, and the mkref pipeline indexed the D. melanogaster reference genome (release 6, GCA_000001215.4 from NCBI Genbank). Alignment was performed using the count pipeline in Cell Ranger v3.1 with the expected cell count set to 5,000. Raw expression counts were imported into Seurat v3 in R (60), normalized using the scTransform pipeline with regularized negative binomial regression, and integrated using the SCT method. The 1,500 most variable features were selected for downstream analysis
Assembly: D. melanogaster reference genome (release 6, GCA_000001215.4 from NCBI Genbank)
Supplementary files format and content: Matrix, barcodes and features files

organism: Drosophila melanogaster

source_name: Brain

extract_protocol:
We anesthetized CSB flies on ice and dissected 10 brains per sex after six days of exposure to control medium or medium supplemented with 250 µM NaAsO2 in cold Dulbecco's Phosphate-Buffered Saline (DPBS). The brains were flash frozen in a dry ice-ethanol bath, and stored at -80°C.
We prepared the samples for snRNA-seq as described in the 10X Genomics Gene Expression (GEX) protocol (CGOOO338).

value: Brain

value: Canton S (B)

value: 250 µM of  NaAsO2 for 6 days


title: preosteoclast from femur, mouse with no ligature 2UL

description: Library name: WT FEM 2 UL


title: Arsenic treated female brain

description: Library name: Female treatment


title: preosteoclast from femur, mouse with no ligature 1UL

description: Library name: WT FEM 1 UL


title: HepG2, siDUS1, SR, treated, rep#2

value: siDUS1 Knockdown

value: small RNA (<200 nt)


title: No Arsenic treated male brain

description: Library name: Male control

value: No Arsenic


title: preosteoclast from LSD1KO mandible, ligated mouse 2L

description: Library name: KO MAN 2L


title: HepG2, siDUS1, SR, treated, rep#1


title: HepG2, siDUS2, SR, input2, rep#2


title: preosteoclast from LSD1KO mandible, ligated mouse 1L

description: Library name: KO MAN 1L


title: HepG2, siDUS1, SR, input2, rep#2


title: No Arsenic treated female brain

description: Library name: Female control


title: preosteoclast from mandible, ligated mouse 3L

description: Library name: WT FEM 3L


title: HepG2, siDUS1, SR, input2, rep#1


title: zmzfp69 (B73) mutant,leaf,4°C,48h 3

city: Harbin

first: Sinan

last: Li

state: Heilongjaing

address: No.368 Xuefu Road

country: China

institute: Maize Research Institute, Heilongjiang Academy of Agricultural Sciences

description:
Library name: Zmzfp69_48 3
4°C,0h

data_processing:
After library quality control, different libraries were pooled based on the effective concentration and targeted data amount, then subjected to Illumina sequencing.  Raw data (raw reads) of fastq format were firstly processed through fastp software.  Q20, Q30 and GC content the clean data were calculated.  All the downstream analyses were based on the clean data with high quality. Reference genome and gene model annotation files were downloaded from genome website directly(https://phytozome-next.jgi.doe.gov/info/Zmays_RefGen_V4).  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.  The mapped reads of each sample were assembled by String Tie (v1.3.3b) in a reference-based approach. Feature Counts 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 for two conditions/groups was performed using the DESeq2 R package (1.20.0).  DESeq2 provides statistical programs for determining differential expression in digital gene expression data using models based on negative binomial distribution.  The resulting P-value is adjusted using the Benjamini and Hochberg’s methods to control the error discovery rate.  The corrected P-value ≤ 0.05 & |log2(fold change)| ≥1 was set as the threshold of significant differential expression. methods to control the error discovery rate.
The corrected P-value ≤ 0.05 & |log2(fold change)| ≥1 was set as the threshold of significant differential expression. Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the cluster Profiler R package, in which gene length bias was corrected.  GO terms with corrected P-value less than0.05 were considered significantly enriched by differential expressed genes.  We used cluster Profiler R packagetotest the statistical enrichment of differential expression genes in KEGG pathways.
Assembly: Zea mays
Supplementary files format and content: gene_fpkm.txt

organism: Zea mays

source_name: leaf

growth_protocol: In this study, the maize inbred line materials were sown in seed culture boxes and then transferred to a light incubator for cultivation.  The environmental parameters set inside the incubator are as follows: a constant temperature of 25℃, and the photoperiod follows an alternating pattern of 16 hours of light and 8 hours of darkness.  When the seedlings grew to the three-leaf (V2) stage , the plants in the treatment group were subjected to low-temperature stress treatment: they were placed in a light incubator at 4℃ for continuous stress for 48 hours, and then taken out and restored to growth for 24 hours under conditions of 25℃ and 12 hours of light /12 hours of darkness.  The plants in the control group were maintained to grow normally in a light incubator at 25℃ all the time.  This experiment set up three biological replicates, each of which included 50 seedling samples.

extract_protocol:
RNA integrity was assessed using the Bioanalyzer 2100 system(Agilent Technologies, CA, USA) was extracted from the leaves.
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.  Then the second strandcDNA was synthesized using dUTP, instead of dTTP.  The directional library was ready after end repair, A-tailing, adapter ligation, sizeselection, USER enzyme digestion, amplification, and purification.  Thelibrary was checked with Qubit and real-time PCR for quantificationandbioanalyzer for size distribution detection.

treatment_protocol: The leaves at the three-leaf stage of the mutant (zmzfp69) and control (B73) maize inbred lines were treated at low temperature for 0h, 24h and 48h as the experimental materials.

value: leaf

value: ZmZFP69(B73) knock down

value: ZmZFP69 knock down


title: zmzfp69 (B73) mutant,leaf,4°C,48h 2

description:
Library name: Zmzfp69_48 2
4°C,0h


title: HepG2, siDUS2, SR, input1, rep#1


title: preosteoclast from femur, LSD1cKO mouse with no ligature 2UL

description: Library name: KO FEM 2 UL


title: preosteoclast from mandible, ligated mouse 1L

description: Library name: WT  MAN 1L


title: HepG2, siDUS1, SR, input1, rep#2


title: HepG2, siDUS1, SR, input1, rep#1


title: zmzfp69 (B73) mutant,leaf,4°C,48h 1

description:
Library name: Zmzfp69_48 1
4°C,48h


title: preosteoclast from LSD1KO femur, ligated mouse 2L

description: Library name: KO FEM 2L


title: HepG2, siC, SR, treated, rep#2

value: siControl


title: zmzfp69 (B73) mutant,leaf,4°C,24h 2

description:
Library name: Zmzfp69_24 2
4°C,0h


title: preosteoclast from femur, ligated mouse 2L

description: Library name: WT FEM 2L


title: zmzfp69 (B73) mutant,leaf,4°C,24h 3

description:
Library name: Zmzfp69_24 3
4°C,0h


title: preosteoclast from LSD1KO femur, ligated mouse 1L

description: Library name: KO FEM 1L


title: zmzfp69 (B73) mutant,leaf,4°C,24h 1

description:
Library name: Zmzfp69_24 1
4°C,24h


title: zmzfp69 (B73) mutant,leaf,4°C,0h 3

description:
Library name: Zmzfp69_0 3
4°C,0h


title: preosteoclast from femur, ligated mouse 1L

description: Library name: WT Fem 1L


title: zmzfp69 (B73) mutant,leaf,4°C,0h 2

description:
Library name: Zmzfp69_0 2
4°C,0h


title: HepG2, siC, SR, input2, rep#2


title: KO biol rep 4 DFMO

city: Mainz

first: Matthias

last: Klein

address: Langenbeckstrasse 1

department: Inst. for Immunology

institute: University Medical Center Mainz

description: Library name: S29

data_processing:
Sequence reads were trimmed for adapter sequences and further processed using the software CLC Genomics workbench (v24.0.1) with CLC's default settings for RNA-Seq analysis. Reads were aligned to GRCm39 genome.
Assembly: GRCm39
Supplementary files format and content: tab-delimited text files including raw counts, TPM and RPKM. Expression values in manuscript=TPM

source_name: spleen

growth_protocol: 2x10e5 cells were stimulated in culture medium (Iscove’s medium supplemented with 10%(v/v) FCS, 1‰(v/v) penicillin-streptomycin, 2mM L-glutamine ,1mM sodium pyruvate and 50 µM β-mercaptoethanol) in the presence of 250 ng/ml mrIL-2, aCD3/28 T-cell expander beads (Gibco; 11452D; ratio 2:1 (beads:cell)), 1 mM DFMO (Difluoromethylornithine) (HY-B0744B; MedChemExpress) for 72 hours at 37°C (5% CO2) in 96-well plates (flat bottom).

extract_protocol:
RNA preparation was conducted using the RNeasy Micro Kit (Qiagen). RNA quantity was determined with a Qubit 2.0 fluorometer (Invitrogen), and RNA quality  was assessed on a Bioanalyzer 2100 (Agilent) using an RNA Pico Chip (Agilent) to determine the RNA integrity number (RIN value). Samples with a RIN value  of >8 were used for library preparation. Samples were then submitted to Novogene (Cambridge, UK) for high-throughput RNA sequencing.
Samples with a RIN value of >8 were used for library preparation and sequencing (approx. 30mio reads/sample; PE150) at Novogene (Cambridge, UK).

treatment_protocol: 72 hours treatment with 1mM DFMO or left untreated

value: spleen

value: Regulatory T cells

value: KO

value: DFMO


title: HepG2, siC, SR, input2, rep#1


title: HepG2, siC, SR, input1, rep#2


title: zmzfp69 (B73) mutant,leaf,4°C,0h 1

description:
Library name: Zmzfp69_0 1
4°C,0h


title: CK(B73),leaf,4°C,48h 3

description:
Library name: CK_48 3
control

value: WT


title: HepG2, WT, SR, treated, rep#2


title: KO biol rep 3 DFMO

description: Library name: S28


title: KO biol rep 1 DFMO

description: Library name: S26


title: mmYS-pre, Young Sed, Pre-Ex, Male1

value: Skeletal Muscle (gastrocnemius)

value: pre-exercise

value: hTERT

value: male


title: CK(B73),leaf,4°C,48h 2

description:
Library name: CK_48 2
control


title: HepG2, WT, SR, treated, rep#1


title: CK(B73),leaf,4°C,24h 3

description:
Library name: CK_24 3
control


title: CK(B73),leaf,4°C,48h 1

description:
Library name: CK_48 1
control


title: HepG2, siC, SR, treated, rep#1


title: KO biol rep 5 untreated

description: Library name: S25

value: untreated


title: KO biol rep 5 DFMO

description: Library name: S30


title: KO biol rep 4 untreated

description: Library name: S24


title: CK(B73),leaf,4°C,24h 2

description:
Library name: CK_24 2
control


title: CK(B73),leaf,4°C,24h 1

description:
Library name: CK_24 1
control


title: KO biol rep 3 untreated

description: Library name: S23


title: HepG2, WT, SR, input2, rep#1


title: HepG2, WT, SR, input1, rep#2


title: KO biol rep 2 untreated

description: Library name: S22


title: HepG2, WT, SR, input1, rep#1


title: KO biol rep 2 DFMO

description: Library name: S27


title: CK(B73),leaf,4°C,0h 2

description:
Library name: CK_0 2
control


title: HepG2, siC, SR, input1, rep#1


title: HepG2, WT, SR, input2, rep#2


title: CK(B73),leaf,4°C,0h 3

description:
Library name: CK_0 3
control


title: KO biol rep 1 untreated

description: Library name: S21


title: MDA-MB-231 cells, shMSN ,sample 2

city: Jongno-gu

first: chenghyun

last: LEE

state: seoul

address: 71, Daehak-ro

country: South Korea

institute: seoul national university

data_processing:
Sequence reads were trimmed for adaptor sequence/low-quality sequence using fastp
Read count extraction were performed using featureCounts v1.5.0-p3
Normalization were performed using DESeq2 R package v1.20.0(parameter:|log2(FoldChange)| >= 1 & padj <= 0.05)
Assembly: Trimmed sequence reads were mapped to Homo Sapiens(GRCh38/hg38) using hisat2 v.2.0.5
Supplementary files format and content: tab-delimited text file includes raw counts for each sample
Supplementary files format and content: tab-delimited text file includes fpkm values fir each sample

growth_protocol: MDA-MB-231 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% co2 at 37C

extract_protocol:
RNA was harvested using Rneasy mini plus kit (Qiagen). 2ug of total RNA was used for the construction of sequencing libraries.
RNA clustering for RNA-seq were prepared using TruSeq PE Cluster Kit v3-cBot-HS

value: breast cancer cells

value: MSN knockdown


title: WT biol rep 4 DFMO

description: Library name: S9


title: MDA-MB-231 cells, shMSN ,sample 1


title: WT biol rep 3 DFMO

description: Library name: S8


title: WT biol rep 2 DFMO

description: Library name: S7


title: MDA-MB-231 cells, shMSN ,sample 3


title: MDA-MB-231 cells, shControl ,sample 2


title: MDA-MB-231 cells, ADR resistance cell, sample 3

value: ADR resistant


title: CK(B73),leaf,4°C,0h 1

description:
Library name: CK_0 1
control

value: leaf


title: MDA-MB-231 cells, shControl ,sample 1


title: WT biol rep 4 untreated

description: Library name: S4


title: WT biol rep 5 untreated

description: Library name: S5


title: 32TamoCor

city: Oklahoma City

first: Willard

last: Freeman

address: 825 NE 13th St

institute: Oklahoma Medical Research Foundation

description: Library name: 12M-Treated6

data_processing:
Following sequencing, reads were trimmed, aligned, differential expression statistics and correlation analyses were performed in Strand NGS software package (Agilent)
Reads were aligned against the Mm10 build of the mouse genome (2014.11.26). Alignment and filtering criteria included: adapter trimming, fixed 2bp trim from 5’ and 6bp from 3’ ends, a maximum number of one novel splice allowed per read, a minimum of 90% identity with the reference sequence, a maximum of 5% gap, trimming of 3’ end with Q<30.
Alignment was performed directionally with Read 1 aligned in reverse and Read 2 in forward orientation. Reads were filtered based on the mapping status and only those reads that aligned normally (in the appropriate direction) were retained.
Normalization was performed with the DESeq algorithm
Transcripts with an average read count value >20 in at least 100% of the samples in at least one group were considered expressed at a level sufficient for quantitation per tissue and those transcripts below this level were considered not detected/not expressed and excluded, as these low levels of reads are close to background and are highly variable.
For statistical analysis of differential expression, a one-way ANOVA with the factor of TRAP fraction and a Benjamini-Hochberg Multiple Testing Correction followed by Student-Newman Keuls post hoc test were used. For those transcripts meeting this statistical criterion, a fold change >|1.25| cutoff was used to eliminate those genes which were statistically significant but unlikely to be biologically significant and orthogonally confirmable due to their very small magnitude of change.
Assembly: mm10
Supplementary files format and content: Tab-delimited text file contains RPKM values for each sample.

source_name: Brain Cortex

extract_protocol:
RNA was extracted using Rneasy Mini Kit (Qiagen) according to manufacturer protocol.
RNA libraries for RNA-Seq were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina along with the NEBNext Poly(A) mRNA Magnetic Isolation Module according to manufacturer's protocol.

treatment_protocol: At 45 days of age, mice were treated with tamoxifen (60 mg/kg, intraperitoneally) or corn oil for 5 consecutive days

value: Brain Cortex

value: Slick-H Cre - Rosa Mlkl

value: Tamoxifen

tag: batch

value: 12 months


title: 30TamoCor

description: Library name: 12M-Treated4


title: MDA-MB-231 cells, ADR resistance cell, sample 2


title: 31TamoCor

description: Library name: 12M-Treated5


title: 27TamoCor

description: Library name: 12M-Treated1


title: MDA-MB-231 cells, ADR resistance cell, sample 1


title: 28TamoCor

description: Library name: 12M-Treated2


title: 26CornCor

description: Library name: 12M-Control6

value: Corn oil


title: High 7 Testis R

type: RNA

city: Kawasaki

first: Satoshi

last: Yokota

state: Kanagawa

address: Tonomachi, Kawasaki-ku

department: 1.	Division of Cellular & Molecular Toxicology, Center for Biological Safety & Research

country: Japan

institute: Research, National Institute of Health Sciences

hyb_protocol: The labeled RNA was hybridized using GeneChip® Hybridization Oven 645 (Applied Biosystems); the plate used in the microarray was miRNA 4.0 Array (Applied Biosystems).

scan_protocol: GeneChips were scanned using GeneChip® Scanner 3000 7G system (Applied Biosystems)

data_processing:
The data were analyzed using Array-Pro Analyzer Ver. 4.5 (Media Cybernetics, Silver Spring, MD, USA).
The data were normalized and expressed as fold increase in comparison to the control, using the Expression Console™ Software (Applied Biosystems).

label: biotin

label_protocol: The RNA samples were labeled with biotin using the FlashTag™ Biotin HSR RNA Labeling Kit (Applied Biosystems, Foster City, Union City, USA), according to GeneChip® miRNA Arrays User Manual.

growth_protocol: The left testes (n=4 per group for microarray) of 12-week-old C57BL/6J mice were immediately isolated, frozen in liquid nitrogen, and kept at –80°C

extract_protocol: Total RNA was isolated using the miRNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol, and suspended in pure water. On-column DNase digestion was performed using an RNase-free DNase set (Qiagen). RNA was quantified using a NanoDrop® Lite (Thermo Fisher Scientific, Franklin, MA, USA). RNA integrity was checked on a Bioanalyzer 2100 instrument (Agilent Technologies). All the 12 individual RNA samples displayed RNA integrity number scores of >9.2. The RNA extracted from each sample was used for microarray

treatment_protocol: Pregnant mice (n=4/group) were injected intratracheally with 0.1 mL of saline or 50 or 500 μg/mL of AgNP suspension, once each on E7.5 and E14.5.The pregnant mice of each group received doses of 5 μg/injection (Ag-5, n=4) or 50 μg/injection (Ag-50, n=4).

tag: strain

value: 12 weeks

value: Testis


title: WT biol rep 3 untreated

description: Library name: S3

value: spleen

value: Regulatory T cells

value: WT


title: WT biol rep 2 untreated

description: Library name: S2


title: MDA-MB-231 cells, shControl ,sample 3


title: High 5 Testis R


title: WT biol rep 1 DFMO

description: Library name: S6

value: DFMO


title: Low 12 Testis R


title: Low 8 Testis R


title: 25CornCor

description: Library name: 12M-Control5


title: High 2 Testis R


title: High 3 Testis R


title: MDA-MB-231 cells, parent cell ,sample 2


title: WT biol rep 1 untreated

description: Library name: S1


title: Low 9 Testis R


title: 24CornCor

description: Library name: 12M-Control4


title: 23CornCor

description: Library name: 12M-Control3


title: Macaca_2

city: Sherbrooke

first: Michelle

middle: S

last: Scott

state: QC

address: 3201 Jean-Mignault

country: Canada

institute: Université de Sherbrooke

description:
Library name: Sample 4
small RNAs (<300 nt)

data_processing:
Base callings were converted to fastq using bcl2fastq v2.20.0 with the following parameters for S. pombe, S. cerevisiae and M. musculus: --minimum-trimmed-read-length 13 --mask-short-adapter-reads 13 --no-lane-splitting -p 32. Base callings were converted to fastq using bases2fastq v1.2.0 with the following parameters for G. gallus and M. mulatta: -p 36 –detect-adapters --filter-mask R1:N*-R2:N*.
Trimming of paired-end reads was performed using Trimmomatic v0.36 (parameters: ILLUMINACLIP:<fastaWithAdaptersEtc>:2:12:10:8, TRAILING:30, LEADING:30, MINLEN:20) in order to remove adapters and low-quality reads.
FastQC v0.11.5 was used before and after trimming to assess the quality of the reads.
Alignment of reads to the reference genome was performed using STAR v2.6.1a (parameters: --runMode alignReads, --outSAMunmapped None, --outSAMtype BAM SortedByCoordinates, outFilterScoreMinOverLread 0.3, --outFilterMatchNminOverLread 0.3, --outFilterMultimapNmax 100, --winAnchorMultimapNmax 100, --alignEndsProtrude 5 ConcordantPair). The index needed to align reads was generated using STAR v2.6.1a (parameters: --runMode genomeGenerate, --sjdbOverhang 74)
Gene expression normalized in transcript per million (TPM) was obtained using CoCo v0.2.1p4 (parameters: cc, --countType both, --strand 1 --paired)
Assembly: GRCg7b (G. gallus), Mmul_10 (M. mulatta), GRCm39 (M. musculus), R64-1-1 (S. cerevisiae), ASM294v2 (S. pombe)
Supplementary files format and content: A tab-separated (.tsv) file showing the gene id, gene name and abundance (in TPM) of all annotated genes for each sample in the given species.

organism: Macaca mulatta

source_name: Universal Monkey RNA

growth_protocol: Wild type 10H3 S. cerevisiae cultures were grown in Yeast Extract Peptone Dextrose (YEPD) rich media at 26°C. Wild type yAS99 S. pombe cultures were grown to mid-log phase in 25 mL of YES media at 32°C.

extract_protocol:
Phenol-chloroform RNA extraction was performed for M. musculus, S. cerevisiae and S. pombe, whereas no RNA extraction was needed for the Biochain-purchased RNA for G. gallus and M. mulatta.
TGIRT-Seq libraries were constructed as described previously (Boivin et al., RNA, 2018) using ribodepleted total RNA (S. cerevisiae and M. musculus) or gel-purified and size-selected small RNAs (S. pombe, M. mulatta and G. gallus).

value: Universal Monkey RNA


title: Specific Pathogen Free (SPF) IV+

first: Nathan

last: Schuldt

address: 2231 6th St. SE

department: Pediatrics

description:
Library name: Sample 4
10X Chromium

data_processing:
Primary analysis and de-multiplexing: Base call (.bcl) files for each cycle of sequencing are generated by Illumina Real Time Analysis (RTA) software.  The base call files and run folders are streamed to servers maintained at the Minnesota Supercomputing Institute.  Primary analysis and de-multiplexing are performed using Illumina’s bcl-convert v4.0.3.  The end result of the bcl-convert workflow is de- multiplexed FASTQ files that are released to client accounts for subsequent analysis by the mapping software and aligner of their own choosing. The FASTQ files were analyzed using the Cellranger package from 10X Genomics, generating an HTML summary report and feature-barcode expression matrices for further analysis.
Assembly: Mouse mm10
Supplementary files format and content: Tab-separated values and matrix files

source_name: Maternal Fetal Interface (Placenta and decidua)

extract_protocol:
At 14.5 days gestation dams received a bolus of anti CD45.2-APC antibody and were euthanized three minutes later. The maternal fetal interface (combined placenta and decidua) were harvested and mechanicall separated into single cell solutions in 2% BS 1X PBS solution. Cells were stained for extracellular markers (including CD45.2-BV605 to identify IV- leukocytes) using standard flow cytometry procedures. IV+ and IV- populations were sorted and collected from SPF and NME samples for library preparation using the 10X chromium platform and the Chromium Next GEM Single Cell 5' HT Reagent kit v2 (dual index).
Capture and cDNA generation: Cell suspensions are aliquoted onto a microfluidic platform (e.g. The Chromium X) partitioning thousands of cells into nanoliter-scale Gel Beads-in-emulsion (GEMs), where all generated cDNA share a common 10X Barcode. 10X Barcodes are used to associate individual reads back to the individual partitions. Please visit www.10xgenomics.com for a detailed list of kit contents and methods. Incubation of the GEMs produces barcoded, full-length cDNA from poly-adenylated mRNA and amplified via PCR to generate sufficient mass for library construction. cDNA generated from the cell suspensions are quantified using a fluorometric PicoGreen assay and capillary electrophoresis (e.g. Agilent BioAnalyzer 2100) to assess quality. Library Creation: cDNA generated from the cell suspensions are converted into dual index libraries compatible with Illumina sequencers using 10X Genomics 5’ Gene Expression Library Construction kits (PN-1000190). In summary, the cDNA undergoes an enzymatic fragmentation, end repair, A-tailing, adaptor ligation, and PCR. Final library size distribution is validated using capillary electrophoresis and quantified using fluorimetry (PicoGreen) and via Q-PCR. Cluster generation and sequencing: Pooled libraries are denatured and diluted to the appropriate clustering concentration. The libraries are then loaded onto the NovaSeq paired end flow cell and clustering occurs on board the instrument. Once clustering is complete, the sequencing reaction immediately begins using Illumina’s 2-color SBS chemistry. Upon completion of read 1, 2 separate 8 or 10 base pair index reads are performed. Finally, the clustered library fragments are re-synthesized in the reverse direction thus producing the template for paired end read 2.

treatment_protocol: Preconception, female C57BL/6 mice were cohoused with pet store mice for >4 wks to passively share their diverse microbial communities. Cohousing continued post conception and throughout gestation. Specific pathogen free control females were maintained in conventional SPF housing.

value: CD45.2+ cells

value: Leukocytes

value: Natural microbial exposure

value: SPF IV+


title: 22CornCor

description: Library name: 12M-Control2


title: Specific Pathogen Free (SPF) IV-

description:
Library name: Sample 3
10X Chromium

value: Maternal Fetal Interface (Placenta and decidua)

value: SPF IV-


title: MDA-MB-231 cells, parent cell ,sample 1

value: breast cancer cells


title: 21CornCor

description: Library name: 12M-Control1

value: Brain Cortex

value: Slick-H Cre - Rosa Mlkl


title: WT biol rep 5 DFMO

description: Library name: S10


title: MouseBrain_3

description:
Library name: Sample 7
Ribodepleted total RNA

value: Brain


title: MDA-MB-231 cells, parent cell ,sample 3


title: MOP promoter N-terminal HA-tagged MAB_1638 anti-HA immunoprecipitation

city: Worcester

first: Scarlet

last: Shell

state: Massachusetts

address: 60 Prescott St, WPI

department: Biology and Biotechnology

institute: Worcester Polytechnic Institute

description: Library name: 353_IP

library_source: genomic

data_processing:
Reads were aligned to the GenBank CU458896.1 reference genome and GCA_000069185.1_ASM6918v1 annotation using Bowtie2 v2.2.6 with default parameters, filtering reads for unique alignment using samtools v1.3.
Peaks detection was done using MACS2 v2.1.1.
Assembly: CU458896.1
Supplementary files format and content: csv files containing information on the peaks found in each immunoprecipitation sample

molecule: genomic DNA

organism: Mycobacteroides abscessus

source_name: ATCC19977 derivative

growth_protocol: M. abscessus grown to an OD600 of ~0.4-0.6 in 50 mL of 7H9 supplemented with 0.5% glycerol and 0.05% Tween 80.

extract_protocol:
equilibrated to room temperature. Formaldehyde (1% final concentration) was added, followed by a 20-minute shaking-incubation at room temperature. Cross-linking was quenched by adding glycine (final concentration of 0.25 M) for 5 minutes at RT. Fixed cultures were collected by centrifugation at 4500 x g for 10 minutes. Pellets were washed twice with cold PBS and resuspended in 600 µL of ChIP lysis buffer (20 mM HEPES-KOH pH=7.9, 50 mM KCl, 0.5 mM DTT, and 10% glycerol) supplemented with a protease inhibitor cocktail (Roche cOmplete #11836153001). The resuspended cells were then transferred to tubes containing 500-700 mg of 0.1 mM glass beads (Sigma-Aldrich G8772). Bead beating was performed through three cycles, each consisting of 30 seconds of beating followed by a 5-minute pause on ice, at 4260 RPM. Samples were centrifuged at 100 x g for 2 minutes at 4°C, and supernatants were collected.  DNA shearing was achieved using a Covaris M220 instrument with the following parameters: 10% duty cycle, 10 minutes, and 200 cycles per burst. Insoluble materials were removed by centrifugation at 11,000 x g for 10 minutes at 4°C, and the supernatant was collected. Efficiency of shearing was confirmed by electrophoresis on a 2% agarose gel. Anti-HA antibodies (3 µg per sample, Abcam, Ab#9110) were added to 25 µg of sheared DNA from each sample, followed by a 1-hour incubation at 4°C with rotation. Subsequently, 25 µL of pre-washed magnetic beads (NEB-S1425S) were added to the samples, and incubation was continued overnight with rotation at 4°C. The next day, the samples were subjected to magnetic separation to remove liquids. The beads were washed five times with 900 µL of IPP50 buffer (10 mM Tris-HCl buffer pH=8, 150 mM NaCl, and 0.1% NP-40), followed by two washes with TE buffer (50 mM Tris-HCl pH=8, 10 mM EDTA). Beads-antibody-DNA complexes were collected by magnetic separation and then resuspended in 70 µL of elution buffer (50 mM Tris-HCl pH=8, 1% SDS). This suspension was incubated at 65°C for 15 minutes. The elution step was repeated with additional 70 µL, and the eluents were combined. RNA was removed from the eluted DNA using RNase A (NEB T3018-2, 20 mg/mL) at 37°C for 1 hour. Subsequently, reverse crosslinking was carried out by adding 3 µL of proteinase K (20 mg/mL, MAN-740506) and incubating for 2 hours at 37°C, followed by an overnight incubation at 65°C. The next morning, DNA was extracted using phenol:chloroform. Finally, the extracted DNA was resuspended in 500 µL of 10 mM Tris-HCl pH=8.
Single-end Illumina library construction was performed by the Technion Genomics Center, Haifa, Israel.

value: ATCC19977 derivative

value: Log phase growth


title: Natural microbial exposure (NME) IV+

description:
Library name: Sample 2
10X Chromium

value: NME IV+


title: ATAC_PM3

city: Baltimore

first: Bongsoo

last: Park

state: Maryland

address: 251 Bayview Blvd

department: Translational Gerontology Branch

institute: National Institute on Aging

data_processing:
Raw sequencing fragments were trimmed using TrimGalore (0.4.3), cutadapt
Alignment to the mouse reference genome (mm10) was performed using Bismark (v0.23.1)
Alignment to the mouse reference genome (mm10) was performed using BWA
Peak calling using MACS2
Assembly: mm10
Supplementary files format and content: DNA methylation level (cov files)
Supplementary files format and content: Open chromatin narrowPeaks (text files)

source_name: Brown adipose tissue

extract_protocol: Qiagen DNA extraction kit

value: Brown adipose tissue

tag: disease

value: M

tag: replicate


title: Macaca_1

description:
Library name: Sample 3
small RNAs (<300 nt)


title: Control 12 Testis R

value: 12 weeks

value: Testis


title: Control 10 Testis R


title: mESC, SR, treated, rep#2

source_name: mESC

value: WT

value: mESC

value: small RNA (<200 nt)


title: Control 6 Testis R


title: MOP promoter N-terminal HA-tagged MAB_1638 input DNA

description: Library name: 353_Input


title: 29TamoCor

description: Library name: 12M-Treated3

value: Tamoxifen

value: 12 months


title: MouseBrain_2

description:
Library name: Sample 6
Ribodepleted total RNA


title: mESC, SR, treated, rep#1


title: Low 6 Testis R


title: Native promoter C-terminal HA-tagged MAB_1638 input DNA

description: Library name: 352_Input


title: MouseBrain_1

description:
Library name: Sample 5
Ribodepleted total RNA


title: mESC, SR, input2, rep#2


title: ATAC_FA3

value: Filtered air


title: GFP positive EECs

city: Chapel Hill

first: Kendall

last: Lough

state: North Carolina

address: 160 N Medical Drive

institute: University of North Carolina at Chapel Hill

description:
Library name: Sample 1
10X Genomics

data_processing:
De-multiplexing alignment to the mm10-2020-A transcriptome and unique molecular identified (UMI)-collapsing were performed using Cell Ranger (v7.0.0). A gene-barcode matrix was generated with ~5.77e8 reads over 8,912 cells (~65,000 mean reads per cell and ~3,000 mean genes detected per cell).
Assembly: mm10-2020-A
Supplementary files format and content: Tab separated values files and matrix files

source_name: small intestine

extract_protocol:
intestines were dissected out and bisected longitudinally to expose the luminal epithelia and submerged in cold dissociation cocktail (2.25 mg/mL DNAse (Sigma Cat. #: DN25), 10 mg/mL protease (Sigma Cat. #: P5380), and 9 µM Y-27632 (Selleck Chem. Cat. #: S1049) in PBS). Samples were incubated in dissociation cocktail on ice for 30 minutes with trituration every 5 minutes until achieving >90% single cells in the suspension. Cells were then rinsed 3x in cold PBS, passed through a 40 µm cell strainer, pelleted and resuspended in cold DMEM+10% FBS with 2.25% DNAse and 9 µM Y-27632 to preserve viability during cell sorting. Final cell counts were ~2.75e6 cells from E15.5 embryos, ~7.5e6 cells from E16.5 embryos and ~7.5e6 cells from E17.5 embryos, all of which were stained with Sytox and Annexin V (to label dead cells) and pooled prior to sorting. FACS isolation of GFP-POS EECs was performed on a Sony SH800 cell sorter with gating based on  GFP-NEG controls (one Cre-NEG ROSAmTmG littermate from each age), excluding SytoxPOS and Annexin VPOS cells. Sorted ~65,000 GFP-POS cells and viability was determined to be ~89% by LUNA-FL Cell Counter before library preparation.
10,000 single cells were processed through the Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 (Dual Index) with Feature Barcode technology for Cell Surface Protein (10X Genomics, Pleasanton, CA) as per the manufacturer’s instructions. Single-cell cDNA libraries were sequences on the Illumina NextSeq 2000 platform with a NextSeq P3 flow cell.

value: small intestine

value: enteroendocrine


title: Natural microbial exposure (NME) IV-

description:
Library name: Sample 1
10X Chromium

value: Maternal Fetal Interface (Placenta and decidua)

value: CD45.2+ cells

value: Leukocytes

value: Natural microbial exposure

value: NME IV-


title: ATAC_PM1

value: Brown adipose tissue


title: ATAC_FA2

value: M


title: mESC, SR, input1, rep#2


title: D3, scRNAseq

city: Nanjing

first: yang

last: sun

state: Jiangsu

address: Nanjing University Academy of Sciences, No. 163 Xianlin Avenue, Qixia District, Nanjing City, Jiangsu Province

department: Life Sciences

institute: Nanjing University

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v2.1.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: Human with GRCh38 and mouse with mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: colon

extract_protocol:
After the tumor was removed, it was digested into single cell suspension with tumor tissue dissociation kit on ice, and 10x-RNA reagents were added
Library was performed according to the manufacter’s instructions (single cell 3’ v2 protocol, 10x Genomics). Briefly, cell suspension were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.

value: colon

value: Intestinal tissue single cell suspension

value: Epithelial cells, Stromal cells, Immune cells

value: D (SBJD represents the abbreviation of a compound Chinese medicine)


title: Control 4 Testis R

value: 12 weeks

value: Testis


title: mESC, SR, input1, rep#1

value: WT

value: mESC

value: small RNA (<200 nt)


title: Native promoter C-terminal HA-tagged MAB_1638 anti-HA immunoprecipitation

description: Library name: 352_IP

value: ATCC19977 derivative

value: Log phase growth


title: ATAC_FA1

value: Filtered air


title: D2, scRNAseq

description: 10x Genomics


title: WT anti-HA immunoprecipitation

description: Library name: WT_IP


title: WGBS_PM3


title: mab_1638::tn replicate 3

description: Library name: mDB292_3

data_processing:
Reads were aligned to the NC_010397 reference genome using the Burrows-Wheeler Aligner
FeatureCounts was used to align reads to genomic features.
Assembly: NC_010397
Supplementary files format and content: tsv file contains results of FeatureCounts analysis (counts per gene for each replicate sample)

growth_protocol: M. abscessus WT and mab_1638::tn were grown in triplicates (each starting from a different single colony) to an OD600 of 0.5 in 7H9 supplemented with 0.5% glycerol and 0.05% Tween 80.

extract_protocol:
Frozen cultures were thawed on ice and centrifuged at 4,000 rpm for 5 min at 4°C. The pellets were resuspended in 1 ml Trizol (Life Technologies) and placed in tubes containing Lysing Matrix B (MP Bio). Cells were lysed by bead-beating twice for 40 s at 9 m/sec in a FastPrep 5G instrument (MP Bio). RNA was purified using Direct Zol RNA miniprep kit (Zymo) according to manufacturer’s instructions.
rRNA depletion, library preparation and RNA sequencing were performed in the Technion Genomics Core Facility (Haifa, Israel).


title: ATAC_PM2


title: mab_1638::tn replicate 2

description: Library name: mDB292_2


title: E550

city: Pasadena

first: Magdalena

last: Zernicka Goetz

state: CALIFORNIA

address: CALIFORNIA INSTITUTE OF TECHNOLOGY

institute: California Institute of Technolog

data_processing:
Cell Ranger software from 10X Genomics was installed for data pre-processing. Raw sequencing data were organized, with the requirement that sequencing reads be demultiplexed into FASTQ format files for each sample. Using cellranger mkfastq to demultiplex raw base call (BCL) files generated by Illumina sequencers into FASTQ files. Reads were mapped to the human hg38 genome and counted with mm10 annotation using cellranger count. The data pre-processing workflow was streamlined and standardized to maintain consistency across samples.
Assembly: GRCm38/mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: embryo

extract_protocol:
For pre-implantation E3.5 and E4.5 embryos, 4-week-old B6SJLF1/J (The Jackson Lab Strain No:100012) females were injected with 10 IU of pregnant mare’s serum gonadotropin (PMSG) and, 48 hours later, with 10 IU of human chorionic gonadotropin (hCG) for superovulation. Females were mated with B6CBAF1/J (The Jackson Lab Strain No:100011) males. Embryos were recovered by flushing the uterine horns with M2 media. For peri-to post-implantation E4.75-E5.5 embryos, 7-week-old B6SJLF1/J (The Jackson Lab Strain No:100012) females were mated with B6CBAF1/J (The Jackson Lab Strain No:100011) males. Embryos were dissected out from the uteri in warmed M2 media. Embryos were kept in M2 media within 2 hours in a 37°C incubator before the dissociation. After collection, embryos were washed twice in DEPC-treated PBS, and were dissociated with Accumax (00-4666-56, Invitrogen) supplemented with 1:100 DNase at room temperature for 30-45 minutes with trituration by mouth pipetting and glass capillaries every 10 minutes. The dissociation reaction was quenched using an equal volume of feeder cell (FC) media (Dulbecco’s modified Eagle’s medium (DMEM) (11995040, Gibco) with 15% FBS, 1% Glutamax, 0.1 mM 2-mercaptoethanol, 1% nonessential amino acids (11140-050, Thermo Fisher Scientific), 1% sodium pyruvate, and 1% penicillin-streptomycin). For E3.5, E4.5 and E5.5, cells were passed through a 20 μm filter and spun down at 0.2 relative centrifugal force(rcf); for 4 minutes at 8C. As for E4.75, E5.0, and E5.25, cells were directly spun down at 0.5 relative centrifugal force(rcf) for 4 minutes at 8C without filtering. Cells were counted on a hemocytometer, and viability greater than 90% was confirmed via Trypan Blue staining.
Cells were resuspended to target volume in 0.04% BSA-PBS, and we performed the Chromium Next GEM Single Cell 3ʹ v3.1 workflow for library generation according to the manufacturer’s protocol (10x Genomics). Libraries were pooled and sequenced to 25,000 reads per cell minimum

value: embryo

value: embryo cells

tag: Stage


title: M3, scRNAseq

description: 10x Genomics

value: M (Model)


title: mab_1638::tn replicate 1

description: Library name: mDB292_1


title: M2, scRNAseq

description: 10x Genomics


title: WGBS_PM2

value: Brown adipose tissue


title: WGBS_PM1

value: M


title: M1, scRNAseq

description: 10x Genomics

value: colon


title: WT replicate 3

description: Library name: ATCC19977_3


title: E500


title: WGBS_FA3


title: E475


title: HA-tagged M. tuberculosis mmaA2 anti-HA immunoprecipitation

description: Library name: 375_IP


title: WT replicate 2

description: Library name: ATCC19977_2


title: D1, scRNAseq

description: 10x Genomics

value: Intestinal tissue single cell suspension


title: NC3, scRNAseq

description: 10x Genomics

value: Epithelial cells, Stromal cells, Immune cells

value: NC (Normal control)


title: WT replicate 1

description: Library name: ATCC19977_1


title: NC2, scRNAseq

description: 10x Genomics


title: E350


title: Pooled_snRNA-seq_Mixed2_4

city: Leuven

first: Llewelyn

last: Roderick

address: Herestraat 49

department: Department of Cardiovascular Sciences

country: Belgium

institute: KU Leuven

description:
Library name: Human sheep heart tissue - Pooled 4
10x Genomics

data_processing:
The sequencing reads of the human dataset were aligned to the GRCh38 reference genome and processed with 10x Genomics Cell Ranger 7.0.0
Species-mixed (human and sheep) data were separately mapped to both the human genome (GRCh38) and the sheep genome (ARS-UI_Ramb_v2.0), whereas 4-sample-multiplexed human data were mapped to the human genome (GRCh38) only.
Assembly: GRCh38, ARS-UI_Ramb_v2.0
Supplementary files format and content: tab-delimited value files and matrix files

molecule: nuclear RNA

organism:
Homo sapiens
Ovis aries

source_name: heart ventricle and atria

extract_protocol:
Nuclei were isolated from heart tissue by mechanical disruption, followed by centrifugation through sucrose and purification from debris by FACS. In brief, frozen heart tissue (~100 mg) was suspended in 5 ml of lysis buffer and thawed on wet ice. When samples were pooled for 10x RNA-Seq (multiple human samples, or sheep and human), samples were either combined after (human real-world dataset) or prior to homogenization (human application dataset and sheep-human species-mixed dataset), where equivalent weights of each tissue sample were pooled. Next, samples were homogenized using a gentleMACS dissociator (Miltenyi) with gentleMACS M tubes and the Protein_01_01 protocol. 5 ml of lysis buffer containing NP-40 was added to the homogenate and incubated on wet ice for 15 min. Then, the suspension was filtered through a 30 μM cell strainer (Miltenyi). Residual nuclei were recovered by washing the M tube and filter with 2 ml of lysis buffer containing NP-40, which was then combined with the remainder of the homogenate. The homogenate was centrifuged in a swinging-bucket centrifuge (Eppendorf, 5920R) at 1000 g for 5 min (4 ℃, ramp speed 4/4). The supernatant was carefully removed and the pellet containing nuclei was resuspended in 2 ml of sucrose buffer, which was then layered on top of 2 ml of sucrose buffer. The sample was then centrifuged at 500 g for 5 min (Eppendorf, 5920R; 4 ℃, ramp speed of 4/4) to recover nuclei. The supernatant was removed and the pellet was resuspended in 500 μl of wash buffer and centrifuged at 500 g for 5 min (Eppendorf, 5920R; 4 ℃, ramp speed of 4/4). The supernatant was carefully removed and stained with 1 ml of staining buffer containing DAPI (1:1000; 7406). DAPI-positive nuclei were separated from cell debris by flow sorting using a SONY sorter instrument (SONY MA900) and collected into 50 μl of collection buffer. Sorted nuclei were then collected by centrifugation at 1000 g for 5 min (Eppendorf, 5920R; 4°C, ramp speed of 4/4), and the concentration was adjusted to 1,000 nuclei/µl targeted for predicted capture 10,000.
Nuclear suspensions were loaded on a 10x Chromium Controller (10x Genomics) and processed with 10x Chromium Single Cell 3’ Reagent Kit v3 (10x Genomics) according to the manufacturer’s protocol. Cell-RT mix was prepared, targeting 10,000 cells per sample. Library quantification and quality assessment were performed using the Bioanalyzer Agilent 2100 with a High Sensitivity DNA chip (Agilent Genomics). Indexed libraries were pooled equimolarly and sequenced on an Illumina NovaSeq 6000 by GenomeScan using paired-end 26x98-bp reads as the sequencing mode. Libraries were sequenced on the NovaSeq 6000 (Illumina) at 30,000 reads per nuclei resolution.

value: heart ventricle and atria


title: E525


title: NC1, scRNAseq

description: 10x Genomics


title: U118-MG sgDDA1_2

status: Public on Jul 29 2025

city: Beijing

first: Xing

last: Chen

address: Capital Medical University

institute: Capital Medical University

description:
Library name: Sample 17
UsgDDA12

data_processing:
Raw sequencing data was preprocessed using fastp v0.19.6
Alignment of the clean sequencing data was conducted using STAR v2.4.0 (RNA-seq)
Abundance measurements of genes in RNA-seq was performed by using HTSeq-Count v0.12.4
Differential gene expression analysis of RNA-seq was conducted using DESeq2.
Assembly: GRCh38/hg38
Supplementary files format and content: xls: gene relative expression values from RNA-seq

source_name: glial cells

growth_protocol: U118-MG, LN229, and T98G cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

extract_protocol:
For RNA-seq assay, total RNAs were extracted from U118-MG, LN229, and T98G cells using Trizol reagent.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: For RNA-seq assay, U118-MG, LN229, and T98G cells that were treated with sgCon and sgDDA1.

value: glial cells

value: Glioma


title: U118-MG sgDDA1_1

description:
Library name: Sample 16
UsgDDA11


title: U118-MG sgCon_2

description:
Library name: Sample 14
UsgCon2

value: sgCon


title: U118-MG sgCon_1

description:
Library name: Sample 13
UsgCon1


title: U118-MG sgCon_3

description:
Library name: Sample 15
UsgCon3


title: T98G sgDDA1_3

description:
Library name: Sample 12
TsgDDA13


title: T98G sgDDA1_2

description:
Library name: Sample11
TsgDDA12


title: T98G sgCon_3

description:
Library name: Sample 9
TsgCon3


title: T98G sgCon_2

description:
Library name: Sample 8
TsgCon2


title: T98G sgDDA1_1

description:
Library name: Sample10
TsgDDA11


title: LN229 sgDDA1_3

description:
Library name: Sample 6
LsgDDA13


title: WGBS_FA2


title: T98G sgCon_1

description:
Library name: Sample 7
TsgCon1


title: LN229 sgDDA1_1

description:
Library name: Sample 4
LsgDDA11


title: LN229 sgCon_3

description:
Library name: Sample 3
LsgCon3


title: WGBS_FA1


title: LN229 sgDDA1_2

description:
Library name: Sample 5
LsgDDA12


title: E450


title: U118-MG sgDDA1_3

description:
Library name: Sample 18
UsgDDA13


title: LN229 sgCon_2

description:
Library name: Sample 2
LsgCon2


title: LN229 sgCon_1

description:
Library name: Sample 1
LsgCon1


title: STAT1 KO Neutrophils, lung, sorted from infected mice, 72h pi. Rep1

city: New York

first: YAHUI

last: GUO

state: NY

address: 407 East 68th street, ZRC1641

department: HOPP

institute: Memorial Sloan-Kettering Cancer Center (MSKCC)

description:
Library name: ATAC_neut_ko_rep1_IGO_14046_N_27_S83
50,000 cells

data_processing:
Raw sequencing reads were trimmed and filtered for quality (Q>15) and adapter content using version 0.4.5 of TrimGalore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore) and running version 1.15 of cutadapt and version 0.11.5 of FastQC.
Version 2.3.4.1 of bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) was employed to align reads to mouse assembly mm10 and alignments were deduplicated using Mark Duplicates in Picard Tools v2.16.0.
Enriched regions were discovered using MACS2 (https://github.com/taoliu/MACS) with a p-value setting of 0.001, filtered for blacklisted regions (http://mitra.stanford.edu/kundaje/akundaje/release/blacklists/ mm10-mouse/mm10.blacklist.bed.gz), and a peak atlas was created using +/- 250 bp around peak summits.
The BEDTools suite (http://bedtools.readthedocs.io) was used to create normalized bigwig files. Version 1.6.1 of featureCounts (http://subread.sourceforge.net) was used to build a raw counts matrix and DESeq2 was employed to calculate differential enrichment for all pairwise contrasts.
Peak-gene associations were created by assigning all intragenic peaks to that gene, while intergenic peaks were assigned using linear genomic distance to transcription start sites (TSS).
Assembly: GRCm38.p6
Supplementary files format and content: Bigwig

source_name: lung

growth_protocol: To prepare single cell lung suspensions for flow cytometry. After perfusing murine lungs, they were placed in a gentle MACS™ C tube and mechanically homogenized in 5 ml RPMI-1640, 10% FBS, and 0.1 mg/ml DNAse using a gentle MACS™ Octo Dissociator (Miltenyi Biotech) without the use of collagenase. The lung cell suspensions were then lysed of RBCs, enumerated, and stained with fluorophore-conjugated antibodies prior to flow cytometric analysis. We used BD Aria for flow cytometric sorting, cell always stays on ice or sorted at 4℃. Neutrophils were identified as CD45+ CD11b+ Ly6Clo Ly6G+ cells

extract_protocol:
ATAC was performed as previously described (Corces et al. Nature Methods 2017) using 50,000 cells per replicate and the Tagment DNA TDE1 Enzyme (Illumina, 20034198)
Sequencing libraries were prepared using the ThruPLEX DNA-Seq Kit (Takarabio, R400676) and sent to the MSKCC Integrated Genomics Operation core facility for sequencing on a NovaSeq 6000

treatment_protocol: cells sorted from infected mice, mice were infected with ECA10 A. fumigatus for 72h

value: lung

value: neutrophil

value: STAT1 KO

value: 72h pi with 3*10^7 CEA10


title: WT Neutrophils, lung, sorted from infected mice, 72h pi. Rep2

description:
Library name: ATAC_neut_wt_rep1_IGO_14046_N_32_S88
50,000 cells

value: wildtype


title: WT Neutrophils, lung, sorted from infected mice, 72h pi. Rep1

description:
Library name: ATAC_neut_wt_rep1_IGO_14046_N_30_S86
50,000 cells


title: STAT1 KO Neutrophils, lung, sorted from infected mice, 72h pi. Rep3

description:
Library name: ATAC_neut_ko_rep3_IGO_14046_N_29_S85
50,000 cells


title: STAT1 KO Neutrophils, lung, sorted from infected mice, 72h pi. Rep2

description:
Library name: ATAC_neut_ko_rep2_IGO_14046_N_28_S84
50,000 cells


title: WT Neutrophils, lung, sorted from infected mice, 72h pi. Rep3

description:
Library name: ATAC_neut_wt_rep2_IGO_14046_N_31_S87
50,000 cells


title: Proximal small intestine, GFP-negative, Replicate 4

first: Bernett

last: Lee

address: 8A Biomedical Grove #03-06 Immunos

country: Singapore

institute: Singapore Immunology Network

description: Library name: SIP_Neg4

data_processing:
The reads were then mapped to the mouse genome build mm10 using STAR based on GENCODE M22 annotations. Gene counts were counted using featureCounts (part of the Subread package) based on GENCODE M22 annotations. The gene counts were then used in edgeR for a differential expression of genes (DEG) analysis with multiple testing correction done using the method of Benjamini and Hochberg.
Assembly: mm10
Supplementary files format and content: Genes counts, TPM and RPKM in long format for all samples as a tab delimited file.

source_name: Proximal small intestine

growth_protocol: Mouse 8 weeks old

extract_protocol:
Chelation buffer crypt dissociation with TrypLE single cell dissociation. GFP-sorting by FACS in RLTplus + b-mercaptoethanol. Rneasy microkit (qiagen) RNA extraction. A minimum of 30,000 cells for colon and 50,000 cells for small intestine were collected from each GFP fraction.
FASTQ files containing the RNA-Seq reads were checked for quality control issues using FASTQC and MultiQC for which no issues were found.

value: Proximal small intestine

value: Epithelial cells


title: Proximal small intestine, GFP-intermediate, Replicate 2

description: Library name: SIP_Int2


title: Proximal small intestine, GFP-negative, Replicate 3

description: Library name: SIP_Neg3


title: Proximal small intestine, GFP-negative, Replicate 1

description: Library name: SIP_Neg1


title: Proximal small intestine, GFP-intermediate, Replicate 4

description: Library name: SIP_Int4


title: Proximal small intestine, GFP-negative, Replicate 2

description: Library name: SIP_Neg2


title: Proximal small intestine, GFP-intermediate, Replicate 1

description: Library name: SIP_Int1


title: Proximal small intestine, GFP-high, Replicate 3

description: Library name: SIP_Hi3


title: Proximal small intestine, GFP-high, Replicate 1

description: Library name: SIP_Hi1


title: Middle small intestine, GFP-negative, Replicate 3

description: Library name: SIM_Neg3

source_name: Middle small intestine

value: Middle small intestine


title: Proximal small intestine, GFP-high, Replicate 2

description: Library name: SIP_Hi2


title: Middle small intestine, GFP-negative, Replicate 4

description: Library name: SIM_Neg4


title: Middle small intestine, GFP-negative, Replicate 1

description: Library name: SIM_Neg1


title: Middle small intestine, GFP-intermediate, Replicate 3

description: Library name: SIM_Int3


title: Middle small intestine, GFP-negative, Replicate 2

description: Library name: SIM_Neg2


title: Middle small intestine, GFP-high, Replicate 4

description: Library name: SIM_Hi4


title: Middle small intestine, GFP-intermediate, Replicate 1

description: Library name: SIM_Int1


title: Middle small intestine, GFP-intermediate, Replicate 4

description: Library name: SIM_Int4


title: Middle small intestine, GFP-high, Replicate 3

description: Library name: SIM_Hi3


title: Middle small intestine, GFP-intermediate, Replicate 2

description: Library name: SIM_Int2


title: Proximal small intestine, GFP-intermediate, Replicate 3

description: Library name: SIP_Int3


title: Middle small intestine, GFP-high, Replicate 2

description: Library name: SIM_Hi2


title: Middle small intestine, GFP-high, Replicate 1

description: Library name: SIM_Hi1


title: Distal small intestine, GFP-negative, Replicate 3

description: Library name: SID_Neg3

source_name: Distal small intestine

value: Distal small intestine


title: Distal small intestine, GFP-negative, Replicate 4

description: Library name: SID_Neg4


title: Distal small intestine, GFP-negative, Replicate 2

description: Library name: SID_Neg2


title: Distal small intestine, GFP-high, Replicate 1

description: Library name: SID_Hi1


title: Proximal small intestine, GFP-high, Replicate 4

description: Library name: SIP_Hi4


title: Distal small intestine, GFP-intermediate, Replicate 4

description: Library name: SID_Int4


title: Distal small intestine, GFP-intermediate, Replicate 2

description: Library name: SID_Int2


title: Distal small intestine, GFP-intermediate, Replicate 3

description: Library name: SID_Int3


title: Distal small intestine, GFP-intermediate, Replicate 1

description: Library name: SID_Int1


title: Proximal colon, GFP-negative, Replicate 3

description: Library name: COLP_Neg3

source_name: Proximal colon

value: Proximal colon


title: Distal small intestine, GFP-negative, Replicate 1

description: Library name: SID_Neg1


title: Distal small intestine, GFP-high, Replicate 4

description: Library name: SID_Hi4


title: Distal small intestine, GFP-high, Replicate 3

description: Library name: SID_Hi3


title: Proximal colon, GFP-intermediate, Replicate 4

description: Library name: COLP_Int4


title: Proximal colon, GFP-intermediate, Replicate 3

description: Library name: COLP_Int3


title: Distal small intestine, GFP-high, Replicate 2

description: Library name: SID_Hi2


title: Proximal colon, GFP-high, Replicate 4

description: Library name: COLP_Hi4


title: Proximal colon, GFP-negative, Replicate 2

description: Library name: COLP_Neg2


title: Proximal colon, GFP-high, Replicate 3

description: Library name: COLP_Hi3


title: Proximal colon, GFP-negative, Replicate 1

description: Library name: COLP_Neg1


title: Proximal colon, GFP-high, Replicate 2

description: Library name: COLP_Hi2


title: Proximal colon, GFP-high, Replicate 1

description: Library name: COLP_Hi1


title: Distal colon, GFP-negative, Replicate 3

description: Library name: COLD_Neg3

source_name: Distal colon

value: Distal colon


title: Distal colon, GFP-negative, Replicate 2

description: Library name: COLD_Neg2


title: Proximal colon, GFP-intermediate, Replicate 2

description: Library name: COLP_Int2


title: Distal colon, GFP-intermediate, Replicate 4

description: Library name: COLD_Int4


title: Proximal colon, GFP-negative, Replicate 4

description: Library name: COLP_Neg4


title: Distal colon, GFP-high, Replicate 4

description: Library name: COLD_Hi4


title: Distal colon, GFP-intermediate, Replicate 2

description: Library name: COLD_Int2


title: Distal colon, GFP-negative, Replicate 1

description: Library name: COLD_Neg1


title: Distal colon, GFP-high, Replicate 1

description: Library name: COLD_Hi1


title: Distal colon, GFP-high, Replicate 3

description: Library name: COLD_Hi3


title: Distal colon, GFP-high, Replicate 2

description: Library name: COLD_Hi2


title: Small intestine, replicate 2

description: Library name: SI_2_7_2

source_name: Small intestine

value: Small intestine


title: Distal colon, GFP-negative, Replicate 4

description: Library name: COLD_Neg4


title: Small intestine, replicate 3

description: Library name: Si_30_5


title: Colon, GFP-high, replicate 1

description: Library name: CH_2_7_2

source_name: Colon

value: Colon


title: Colon, GFP-negative, replicate 1

description: Library name: CN_2_7_2


title: Colon, GFP-high, replicate 2

description: Library name: CH_27_6_1


title: Colon, GFP-high, replicate 3

description: Library name: CH_30_5


title: Colon, GFP-negative, replicate 3

description: Library name: CN_27_6_2


title: Colon, GFP-negative, replicate 2

description: Library name: CN_27_6_1


title: RNABC8_ExT18088_S42_R1_001.fastq.gz

city: Noda-shi

first: Mikiya

last: Tsunoda

state: Chiba

department: Molecular Preventive Medicine

institute: The University of Tokyo

description: Library name: Sample 11

data_processing:
Sequencing was performed by using Illumina Novaseq 6000 (Illumina, USA). Single-end Fastq files (R1: reverse complement of 5prime region of cDNA) of sample ExT18073 were processed as follows. Adapter trimming of sequencing data was performed by using cutadapt 4.1, and trimmed reads were mapped to reference RNA (build GRCm38 release-101) by using bowtie2-2.4.5 by the following parameters: -p 8 -N 1 -L 16 –very-sensitive-local –seed 656565 –nofw. Then, read counts of each gene in each samples were counted by using awk, sort, and uniq -c commands. Resulted gene-expression count data was summarized by same Gene Symbol and all of the expression table was full-outer joined by Gene Symbol by using dplyr-1.0.7 package.
Assembly: GRCm38 release-101
Supplementary files format and content: Gene-expression matrix file. Rownames are gene symbols, and colnames are sequence file names.

source_name: Tumor

growth_protocol: Lewis lung carcinoma (LLC) cells (5 × 10^5 cells /mouse) were inoculated subcutaneously (s.c.) into the left and right flanks of C57BL/6 mice.

extract_protocol:
Tumors were cut into small fragments and digested for 45 minutes at 37°C with 0.2% collagenase (FUJIFILM Wako) and DNase I (Sigma-Aldrich), except for experiments for single-cell analysis, in which tumors were digested with 0.2% collagenase NB8 (Nordmark Pharma GmbH). The cells were then subjected to two-phase density separation with upper 35% Percoll PLUS (GE Healthcare) (1000g, 10 minutes at room temperature). Leukocytes were recovered, washed and resuspended in preparation medium (PM) [5%FBS, 10mM HEPES (Nacalai), Penicillin-Streptomycin (Nacalai) in RPMI-1640 (Nacalai)]. After RBC lysis with ACK buffer [155mM of NH4Cl (Nacalai), 10mM of KHCO3 (Nacalai), and 0.5mM EDTA pH8.0 (NIPPON GENE)] for 1 minute at room temperature, cells were washed and resuspended in PM, and the cell concentrations were measured using Flow-Count fluorospheres (Beckman Coulter) and a CytoFLEX S flow cytometer (Beckman Coulter).Cells were stained with a mix of Fc Block (anti-mouse CD16/CD32 mAb; clone 2.4G2, BioLegend) and fluorophore-conjugated anti-mouse monoclonal antibodies (Supplementary Table 5). Antibody mixtures were prepared using BD Horizon Brilliant Stain Buffer Plus (BD) diluted 5x with FACS buffer (D-PBS(-) supplemented with 2% FBS, and 0.05% Sodium Azide). Staining was performed for 20 minutes at 4°C. Before sorting, T cells were enriched from tumor suspensions by further staining with BD IMag APC Magnetic Particles-DM (BD Biosciences), then placed on magnetic plate. After washing by MACS buffer (D-PBS(-) supplemented with 10% BSA, and 2 mM EDTA pH8.0), APC and APC-Cy7(CD8, mouse TCRβ)-positive cells were collected as positive fraction. Cell sorting was performed using a FACS Aria II or Aria III (BD Biosciences). Cells were stained with 5µg/mL of propidium iodide (PI, Sigma-Aldrich) immediately before sorting. Nonviable cells were excluded from the analysis based on forward and side scatter profiles and PI staining. For bulk TCR repertoire analysis and bulk RNA-seq analysis, cells were sorted into Lysis buffer [1% w/v LiDS, 100 mM Tris-HCl pH7.5, 500 mM LiCl, 5 mM dithiothreitol, and 10 mM EDTA pH8.0] and stored at −80°C. Cells were sorted into D-PBS(-) with 10% FBS.
Bulk RNA-seq library preparation was performed with on-beads reverse transcription and template-switching. mRNA-trapped oligo-dT-immobilized Dynabeads M270-streptavidin (Thermo Fisher Scientific) were washed as in bulk TCR-seq library preparation, and resuspended in 10 µL of RT mix [1× First Strand buffer (Thermo Fisher Scientific), 1 mM dNTP, 2.5 mM DTT (Thermo Fisher Scientific), 1 M betaine (Sigma-Aldrich), 9 mM MgCl2 (NIPPON GENE), 1 U/µL RNaseIn Plus RNase Inhibitor (Promega, Madison, WI), 10 U/µL Superscript II (Thermo Fisher Scientific), and 1 µM of trP1-TSO], and incubated for 60 min at 42ºC and immediately cooled on ice. Beads were washed once with B&W-T buffer [5 mM Tris-HCl pH 7.5, 1 M NaCl (NACALAI TESQUE), 0.5 mM EDTA, and 0.1% Tween-20 (Sigma-Aldrich)], and once with 10mM Tris-HCl pH 8.0. To amplify the WTA library, cDNA-immobilized beads were resuspended with 25 µL of the first PCR mixture [0.4 μM of primers (illumina-i7-BCXX-trP1, and 5’ BDWTA V2 primer), and 1x KAPA Hifi Hotstart ReadyMix (KAPA Biosystems, Wilmington, MA)], and amplified by PCR with the following conditions: 95°C for 3 min, [98°C for 20 sec, 65°C for 30 sec, 72°C for 5 min] x5, 72°C for 5 min, and hold at 4°C. The first WTA products were purified by an AMPure XP kit (Beckman Coulter, CA) at 0.7:1 ratio of beads to sample and eluted with 20 μL of 10 mM Tris-HCl pH 8.0. 10.5µL of the first PCR products were mixed with 14.5 μL of the second PCR mixture [0.4 μM of primers (5’ BDWTA V2 primer and illumina-i7-primer), and 1x KAPA Hifi Hotstart ReadyMix], and amplified with the same conditions as the first PCR. The second PCR products were purified by an AMPure XP kit at 0.7:1 ratio of beads to sample and eluted with 15 μL of 10 mM Tris-HCl pH 8.0, quantified by a Nanodrop 8000 (Thermo Fisher), and their size distribution was analyzed by a MultiNA system (Shimazu). 100 ng of the WTA library was subjected to fragmentation/end-repair/A-tailing using NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs). Purified adapter-ligated products were subjected to index PCR. The PCR products were purified by double size-selection by using an AMPure XP kit at 0.5:1 ratio and 0.8:1 ratio (final 1.3X) of beads to sample and eluted with 12 μL of dH2O. Amplified products were analyzed by a MultiNA system. Final transcriptome libraries, with lengths of around 300 base pairs, were quantified using the KAPA Library Quantification Kit (KAPA Biosystems). Pooled libraries were sequenced by an Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 140 bp read 2) using NovaSeq 6000 S4 Reagent Kit v1.5.

treatment_protocol: For treatment, anti-LAG-3 (clone C9B7W, BioLegend or Selleck) was injected intraperitoneally at a dose of 200μg per mouse on days 14 and 16 after tumor inoculation.

value: Tumor

value: Lewis lung carcinoma

value: T cells

value: WT


title: RNABC9_ExT18086_S40_R1_001.fastq.gz

description: Library name: Sample 9


title: RNABC8_ExT18083_S37_R1_001.fastq.gz

description: Library name: Sample 10


title: RNABC9_ExT18081_S35_R1_001.fastq.gz

description: Library name: Sample 7


title: RNABC8_ExT18085_S39_R1_001.fastq.gz

description: Library name: Sample 8


title: Distal colon, GFP-intermediate, Replicate 3

description: Library name: COLD_Int3


title: RNABC9_ExT18087_S41_R1_001.fastq.gz

description: Library name: Sample 6


title: RNABC8_ExT18082_S36_R1_001.fastq.gz

description: Library name: Sample 4

value: untreated


title: RNABC9_ExT18082_S36_R1_001.fastq.gz

description: Library name: Sample 5


title: Distal colon, GFP-intermediate, Replicate 1

description: Library name: COLD_Int1


title: RNABC9_ExT18083_S37_R1_001.fastq.gz

description: Library name: Sample 3


title: mESC, SR, input2, rep#1

city: Chicago

first: Cheng-Wei

last: Ju

address: 929 East 57th Street, GCIS, Room E313

institute: The University of Chicago

data_processing:
The sequencing data were all trimmed with cutadapt tool (v4.8) to remove adapters and low-quality reads. PCR duplicates were removed with BBMap tool (v.38.73), 5-mer random barcodes at reads ends were trimmed and low-quality or short reads (less than 20 nt) were removed using cutadapt tool.
For tRNAs, remaining reads were aligned to tRNA sequence obtained from GtRNAdb (https://gtrnadb.ucsc.edu/) using hisat-3n (v.2.2.1-3n-0.0.3). Mitochondria and chloroplast tRNA sequences are obtained from published papers.
Meanwhile, we have also aligned remaining reads to hg38, mm10, or TAIR10 genome using hisat3n to investigate D sites in other RNA species.
The generated table from hisat-3n were parsed and analyzed by inhouse scripts. Internal mutation ratio at each D candidate site suggested by hisat-3n, was further confirmed by direct IGV visualization (v.2.8.0).
Assembly: mm10 (GRCm38.102), hg38 (GRCh38.107), TAIR10
Supplementary files format and content: For samples with cellular RNA, the mutation ratio at D sites in treated, input samples are shown. For samples with RNA probes, mutations at D sites are shown.
Library strategy: Chemical Reduction Assisted Cytosine Incorporation sequencing (CRACI)

extract_protocol:
Generally, with harvesting cells as the first step, the media was aspirated, and the cells were washed once with proper volume of ice cold DPBS buffer for each plate. Then total RNA was isolated from cells with TRIzol reagent (Invitrogen) and then extracted following manufacturer’s protocol by isopropanol precipitation. Small RNA fraction was purified from total RNA with RNA Clean & Concentrator kit (Zymo Research) following manufacturer’s protocol. mRNA was extracted by two rounds of polyA+ purification with Dynabeads mRNA DIRECT kit (Ambion).
CRACI for D site detection Wild-type HepG2, mESC, and arabidopsis thaliana seedling cells (two replicates for each sample, one 10-cm plate per replicate); HepG2 siControl or DUS knockdown cells were prepared as described (two replicates for each sample, one 10-cm plate per replicate). After extracting the polyA+ RNA or small RNA from mammalian cells or plant cells, around 100 ng RNA was fragmented using RNA Fragmentation Reagents (catalog no. AM8740, Invitrogen) and heated at 70 °C for 14 min, followed by purification with Oligomer Clean and Concentrator (Zymo Research). 3′-End repair and 5′-phosphorylation were conducted with T4 polynucleotide kinase (PNK) (catalog no. EK0032, Thermo Fisher Scientific). RNA was mixed with 3 µL 10× T4 PNK reaction buffer (catalog no. B0201S, NEB) and 3 µl T4 PNK, diluted to a final volume of 30 µl, and incubated at 37 °C for 45 min; then 1.5 µl T4 PNK and 1.5 µl 10 mM ATP were added for another incubation at 37 °C for 45 min, followed by Oligomer Clean and Concentrator (Zymo Research) purification eluting with 10 µl RNase-free water.  To perform 3′-adapter ligation, 10 µl 3′-repaired and 5′-phosphorylated RNA fragments were incubated with 1.0 µl 20 µM RNA 3′ SR Adapter (5′App-NNNNNATCACG AGATCGGAAGAGCACACGTCT-3SpC3, with ATCACG as the inline barcode) at 70 °C for 2 mins and placed immediately on ice. Then, 2.5 µl 10× T4 RNA Ligase Reaction Buffer (catalog no. M0373L, NEB), 7.5 µl PEG8000 (50%), 1 µl SUPERase•In RNase Inhibitor and 2 µl T4 RNA Ligase 2 truncated KQ (catalog no. M0373L, NEB) were added to the RNA–adapter mixture48,49. The reaction was incubated at 25 °C for 2 h followed by 16 °C for 10 h. The reaction was further diluted to 47 µl with nuclease-free water, and the excessive adapters were removed with 2 µl 5′-deadenylase (catalog no. M0331S, NEB) at 30 °C for 45 min followed by adding 1 µl RecJf (catalog no. M0264L, NEB) for ssDNA digestion at 37 °C for 45 min. The 3′-end-ligated RNA was extracted by RNA Clean and Concentrator (Zymo Research) and eluted with 10 µl RNase-free water. The purified RNA was incubated with 1.2 µl 10 µM 5′ SR Adapter (5′-GUUCAGAGUUCUACAGUCCGACGAUC -3′) at 70 °C for 2 mins and placed immediately on ice. Then 2.5 µl 10× T4 RNA ligase reaction buffer, 1.0 µl 25 mM ATP, 10 µl PEG8000 (50%) and 1 µl T4 RNA Ligase 1 (high concentration, catalog no. M0437M, NEB) were added to the RNA–adapter mixture. The reaction was mixed well and incubated at 25 °C for 8 h, followed by RNA Clean and Concentrator (Zymo Research) purification, eluting with 12 µl RNase-free water.  A 2 µl aliquot of the purified RNA was saved for ‘Input1’ library construction, 2 µl aliquot of the purified RNA was saved for ‘Input2’ library construction, and the rest 6 µl was subjected to CRACI optimized reduction treatment, as the ‘Treated’ sample. The 6 µl RNA was mixed with 40 µl freshly prepared KBH4 (1 M KBH4, prepared by dissolving 54 mg KBH4 in 1000 µl RNase-free water) and Incubated the reaction mixture at 25 °C for 3 h, followed by purification with RNA Clean and Concentrator (Zymo Research) and elute with 10 µl H2O. ‘Input1’, ‘Input2’ and purified ‘Treated’ samples were diluted to 10 µl and mixed with 1.0 µl 2.0 µM SR RT primer (5′-AGACGTGTGCTCTTCCGATCT -3′) at 65 °C for 2 min and moved immediately onto ice. Then for ‘Input2’ and ‘Treated’ samples, a stock with 2 µl 10x AMV RT Buffer (New England BioLabs, cat. no. B0277AVIAL), 2 µl 1 mM dNTP Solution Mix (dilute from 10 mM dNTP Solution Mix, New England BioLabs, catalog no. N0447L), 2 µl 10mM dGTP (dilute from 100 mM dNTP Solution Mix, New England BioLabs, catalog no. N0442S), 1 µl RNaseOUT Recombinant Ribonuclease Inhibitor (catalog no. 10777019, Thermo Scientific) and 2 µl HIV RT (Worthington Biochemical, catalog no. LS05003) was added. For ‘Input1’ sample, a stock with 2 µl RNase-free water, 2 µl 10x AMV RT Buffer, 2 µl 10 mM dNTP Solution Mix (New England BioLabs, catalog no. N0447L), 1 µl RNaseOUT Recombinant Ribonuclease Inhibitor and 2 µl HIV RT was added.  The reaction mixture was heated at 70 °C for 5 min and then cDNA was purified using DNA Clean and Concentrator (Zymo Research). The eluted cDNAs (20 µl) were stored at –80 °C; 4 µl was used for each 15-cycle PCR amplification reaction, which was performed with the SR Primer for Illumina (NEB) and indexed primers (from NEBNext Multiplex Oligos for Illumina). All libraries were purified on a 3.5% low melting point agarose gel and sequenced on Illumina NovaSeqX with single-end 100 bp read length.

value: mESC

value: small RNA (<200 nt)


title: Small intestine, replicate 1

description: Library name: SI_2_7_1


title: RNABC9_ExT18084_S38_R1_001.fastq.gz

description: Library name: Sample 1


title: RNABC10_ExT18083_S37_R1_001.fastq.gz

description: Library name: Sample 2