title: mESC_Mettl3KO_m6A-CT_scCUT&Tag

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing and gene counting were made using the Cell Ranger software cellranger-arc-2.0.2 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10, hg39
Supplementary files format and content: Tab-separated values files and matrix files
Library strategy: scCUT&Tag

extract_protocol:
E14 mESCs were harvested, counted and centrifuged for 5 min at 350×g at room temperature. Aliquots of cells (~1,000,000 cells) were resuspended in 250 μL Nuclei Extraction Buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.020 % Triton X-100) and incubated on ice for 1 min. The mixture was diluted with 1 mL Nuclei Dilution Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 0.1 U/μL RiboLock RNase inhibitor) and centrifuged for 5 min at 600×g at 4 °C. Nuclei were washed once with 1 mL Nuclei Dilution Buffer and counted. E14 EPSC and MEF nuclei were similarly isolated with the following conditions: 1 min on ice 0.015 % Triton X-100 for EPSC; 5 min on ice in 0.2 % NP-40 for MEF. Aliquots of nuclei (150,000 nuclei in total per assay) were incubated with 1 μg primary antibody (rabbit IgG or rabbit anti-m6A: MilliporeSigma, #ABE572) in 50 μL Antibody Binding Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) for 2 hours on rotator at 4 °C. Afterwards, nuclei were incubated with 1 μg secondary antibody (guinea pig anti-rabbit: Novus Biologicals, #NBP1-72763) in 50 uL Antibody Binding Buffer for 30 min on the nutator at room temperature. Nuclei were then centrifuged for 5 min at 600×g at room temperature and two rounds of washes with 200 μL Wash Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 0.1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) were performed. The nuclei were resuspended in 50 μL high-salt Binding Buffer (20 mM HEPES pH7.5, 300 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) with 2.5 μL pAG-Tn5 (EpiCypher, #15-1117) and incubated for 1 hour on the nutator at room temperature. The nuclei were then washed twice with high-salt Wash Buffer (20 mM HEPES pH7.5, 300 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 0.1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) and resuspended in 50 μL Reverse Transcription & Tagmentation Buffer (1X SuperScript IV buffer along with, 1 M Betaine, 5 mM DTT, 2 μM Random Hexmer Primers, 0.5 mM each dNTP mix, 1X Roche Complete Protease Inhibitor Cocktail, 1 U/μL RiboLock RNase inhibitor, 4 U/μL SuperScript IV) for 2 hours at 37 °C. When reaction was done, the nuclei were resuspended with 50 μL cold Antibody Binding Buffer and placed on ice. All steps after nuclei extraction were performed in 0.2-ml tubes to minimize nuclei loss.  The nuclei were counted and diluted to a concentration based on the targeted nuclei number. Seven microliters of 10x Genomics ATAC Buffer B was added to 8 μl of nuclei, pipetting several times to mix. Most of the remaining steps were performed according to the Chromium Next GEM Single Cell Multiome protocol Rev E (10x Genomics), skipping the Step 1 ‘Transposition’ and continuing from Step 2.0 ‘Generation & barcoding’. A qPCR cycle check was performed after pre-amplification step using 1:100 amplicon. Indexed CT libraries were prepared with the following PCR program: 45 sec at 98 °C, followed by 23 cycles (ESC IgG-CT) or 15 cycles (ESC and mixed cell-lines m6A-CT) of 15 sec at 98 °C and 10 sec at 60 °C, with a final extension at 1 min at 72 °C. For cDNA libraries, cycle numbers used in cDNA amplification and sample index PCR were 7 cycles and 12 cycles, respectively. The final cDNA libraries and CT libraries were sequenced using an Illumina NovaSeq 6000, PE150 and PE50 system, respectively.
Library was performed according to the manufacter’s instructions (single cell Multiome Rev E protocol, 10x Genomics).


title: TB-only_Diagnosis_South_Africa_SUN129

status: Public on Sep 05 2023

type: SRA

city: London

first: Jackie

last: Cliff

address: Keppel Street

department: Immunology and Infection

country: United Kingdom

institute: London School of Hygiene & Tropical Medicine

library_source: transcriptomic

data_processing:
Alignment STAR version 2.5.1b
Quantification HT-seq count version 0.6.1
Normalisation DESeq2 version 1.12.4
Genome_build: Human_g1k_v37
Supplementary_files_format_and_content: read count for each ensembl gene id, un-normalised, un-filtered

molecule: total RNA

organism: Homo sapiens

source_name: Whole Blood

extract_protocol:
Whole blood stored in PAXgene Blood RNA collection tubes and extracted using the PAXgene blood miRNA kit (Qiagen)
Bioscientific NEXTflex-Rapid-Directional mRNA-seq sample preparation (Bioscientific)

tag: patient id

value: TB-only

tag: timepoint

value: Diagnosis


title: TB-IH_Diagnosis_Indonesia_UNPAD23

value: TB-IH

value: Indonesia


title: Ctrl, MeDIP, 1hr, 0X, rep1

city: UNIVERSITY PARK

first: Lu

last: Bai

state: PENNSYLVANIA

address: 406 SOUTH FREAR, UNIVERSITY PARK

department: BMB

country: USA

institute: Penn State University

library_source: genomic

data_processing:
Paired-end reads of 50 bp or 150 bp were processed with fastp (version 0.23.2) to remove low quality reads. Processed reads were aligned to S. cerevisiae genome (sacCer3) with BWA (version 0.7.17). Aligned bam files were deduplicated with Picard MarkDuplicates (version 3.0.0). bigwig files were generated by deeptools bamCoverage (version 3.5.1) for visualization purposes. To detect differentially methylated NDRs, aligned reads on each NDR were counted by Subread featureCounts (version 2.0.3) using a list of NDR coordinates generated from a previous study, with the following parameters “-p -O”. Fold change and P-value were generated by DEseq2 (1.38.0) without outliers filtering. log2 (Fold Change) > 0.7 and the adjusted P-value < 0.05 were used as the cutoff for identifying differentially methylated regions.
Assembly: sacCer3
Supplementary files format and content: table generated by DEseq2
Supplementary files format and content: bigwig

molecule: genomic DNA

organism: Saccharomyces cerevisiae

growth_protocol: Yeast strains were grown in 50 mL synthetic media with/without methionine at 30°C to OD660 = 0.3.

extract_protocol:
Standard MeDIP protocol for preparation of methylated genomic DNA
MeDIP-Seq libraries were prepared with NEBNext Ultra II DNA Library Prep Kit (NEB, E7645L). Next generation sequencing was performed on NextSeq 2000 (Illumina).

treatment_protocol: β-estradiol (Sigma, 50-28-2) was added to a final concentration of 2 nM for 1/2 hrs, allowing the LacI-NLS-M.CviPI fusion protein to express. For MATa and MATalpha viewpoint, glucose (D) or galactose(G) was added according to the experimental conditions.

tag: strain

tag: chip antibody

tag: condition

value: 1hr, 2nM Beta-estradiol

tag: genotype

value: MATa, his3-11,15, leu2-3,112, trp1-1, ura3-1, can1-100, ade2-1, ADH1::2xlexO-GAL1corepr-LacI-SV40NLS-M.CviPI:ADH1pr-LexA-ERHBD-VP16AD::LEU2


title: WT, mock, biolgical rep 1

city: Pozuelo de Alarcón

first: Stephan

last: Pollmann

state: Madrid

address: Campus de Montegancedo, Autovia M-40 (km 38)

country: Spain

institute: CBGP (UPM-INIA/CSIC)

data_processing:
Novogene Novogene Co., LTD Genomics Service (Cambridge, UK)
Original image data files from the Illumina platform were transformed to sequenced reads by CASAVA base recognition.
Sequence reads were trimmed for the adaptor sequence/low-quality reads were deleted using a Novogene in-house script (quality parameter limit: 0.05)
Trimmed sequence reads were mapped against the TAIR10 Arabidopsis genome using Tophat v2.0.12 (mismatch = 2).
Read count extraction and normalization was performed using HTSeq v0.6.3 (–m union parameter).
Assembly: TAIR10 Arabidopsis genome
Supplementary files format and content: tab-delimited text file includes FPKM values for each sample.

molecule: polyA RNA

organism: Arabidopsis thaliana

source_name: Roots

growth_protocol: Seedlings were grown vertically for seven days on 0.5x MS medium. Then, they were transferred to solidified Plant Nutrition Medium. Root tissue was harvested after 14 days.

extract_protocol:
DNA-free total RNA was extracted by employing the phenol/chloroform method described by Oñate-Sánchez & Vicente-Carbajosa, 2008, BMC Res. Notes.
Sequencing libraries were generated using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations.

treatment_protocol: Each seedling was either mock-infected by associating a 5 mm Ø medium plug extracted from sterile AP plates (control) or from AP plates harbouring a 1‐week‐old S. indica mycelium (co-cultivation).

tag: tissue

value: Roots

tag: ecotype

value: wildtype

tag: treatment

value: Control


title: mESC_WT_m6A-CT_scCUT&Tag

city: Singapore

first: Kiyofumi

last: Hamashima

address: 61 Biopolis Drive

country: Singapore

institute: Institute of Molecular and Cell Biology (IMCB)

description: 10x Genomics

organism: Mus musculus

source_name: C57BL/6 wild-type

tag: cell line

value: C57BL/6 wild-type

tag: cell type

value: Mouse embryonic stem cells

tag: antibody

value: m6A (MilliporeSigma, #ABE572)


title: mESC_Mettl3KO_m6A-CT_scRNA-seq

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
The demultiplexing, barcoded processing and gene counting were made using the Cell Ranger software cellranger-arc-2.0.2 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10, hg39
Supplementary files format and content: Tab-separated values files and matrix files

source_name: C57BL/6 Mettl3-/- (KO)

value: C57BL/6 Mettl3-/- (KO)


title: HiC, -met, rep2

data_processing:
150 bp paired-end reads were aligned to S. cerevisiae genome (sacCer3) and analyzed with HiC-pro.
Assembly: sacCer3
Supplementary files format and content: BED file includes Hi-C bins
Supplementary files format and content: matrix file of Hi-C dataset

growth_protocol: Yeast strains were grown in 200 mL synthetic media without methionine to OD660 = 0.3.

extract_protocol:
Standard Hi-C protocol was used for the preparation of Hi-C samples
Hi-C libraries were prepared with NEBNext Ultra II DNA Library Prep Kit. Next generation sequencing was performed on NextSeq 2000.

value: MATa,  his3-11,15, leu2-3,112, trp1-1, ura3-1, can1-100, ade2-1, tor1-1, fpr::NAT, bar1, HIS3::REV1pr-tetR-mCherry-REV1pr-LacI-GFP, ADE2::REV1pr-LacI-REV1pr-tetR, Chr15::497047_LacO, Chr15::834487_TetO

value: methionine depletion


title: TB-DM_Month 2_Indonesia_UNPAD25

value: TB-DM


title: TB-DM_Week 2_South_Africa_SUN51


title: 39-10-3

city: Shenyang City, Liaoning Province

first: XinDi

last: Gao

state: Liaoning

address: 89, North WenHua Road

country: China

institute: Northeastern university

data_processing:
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to whole genome using Bowtie 2 suite (version 4.1.2)
Assembly: Cryptococcus neoformans var. grubii H99 .
Supplementary files format and content: tab-delimited text files include reads values for each Sample.

organism: Cryptococcus neoformans

source_name: Cryptococcus neoformans var.grubii H99

growth_protocol: WT and set302∆ strains were inoculated into 50 ml of YPD broth and cultured overnight at 30°C in a shaking incubator. Cells were subcultured in 100 ml fresh YPD broth at OD600=0.2 until the OD600 reached 1 at 30°C or 39°C, respectively. Three independent cultures for each strain were prepared. Every culture was washed three times with PBS and placed in a liquid nitrogen tank for RNA-seq.

extract_protocol:
Total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies), and the concentration, quality and integrity of total RNA were determined using a NanoDrop spectrophotometer (Thermo Scientific). Three micrograms of RNA were used as input material for the preparations of RNA sample.
Sequencing libraries were constructed using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA).

value: Cryptococcus neoformans var.grubii H99

value: grown at logarithmic phase in 100ml YPD broth at 39°C


title: 39-10-2


title: TB-DM_Month 2_Indonesia_UNPAD19


title: Met4, -met, ChIP, rep2

data_processing:
150 bp paired-end reads were aligned to S. cerevisiae genome (sacCer3). Peak calling was performed with MACS2 (version 2.2.7.1).
Assembly: sacCer3
Supplementary files format and content: bigwig

growth_protocol: Yeast strains were grown in 50 mL synthetic media without methionine at 30°C to OD660 = 0.3.

extract_protocol:
Standard ChIP protocol was used for the preparation of ChIP samples
ChIP and input libraries were prepared with NEBNext Ultra II DNA Library Prep Kit. Next generation sequencing was performed on NextSeq 2000.

value: anti-TAP(Thermo Fisher, CAB1001)


title: KSC1

city: Birmingham

first: Susan

middle: L

last: Bellis

state: ALABAMA

address: MCLM 350, 1918 University Boulevard

institute: University of Alabama at Birmingham

data_processing:
Basecalls were performed using Illumina's bcl2fastq v2.18.12.
Raw sequence FASTQ reads were aligned with STAR version 2.7.10a using parameters --outReadsUnmapped Fastx --outSAMtype BAM SortedByCoordinate --outSAMattributes All
Transcript abundances were calculated from the STAR alignment BAM files using HTSeq-count version 2.0.2 using parameters -m union -r pos -t exon -i gene_id -a 10 -s no -f bam
Assembly: Gencode GRCm39 Release M32
Supplementary files format and content: tab-delimited Excel file containing Ensembl ID#s for each gene in each sample

source_name: pancreas

growth_protocol: Cells were dissociated from the pancreas of GEMMs and placed into organoid cultures in Matrigel. Organoid lines were propagated according to standard organoid culture methods.

extract_protocol:
RNA was extracted using the Rneasy kit from Qiagen according to the vendor's protocol.
RNA libraries for RNA-seq were prepared using NEB Ultra II Directional Kits following manufacturer's protocols.

treatment_protocol: Organoids were generated from 4 genotypes: SC = ST6GAL1 transgene expression in pancreas; KC = KRASG12D expression in pancreas; KSC = ST6GAL1 transgene plus KRASG12D expression in pancreas; WT = wild-type pancreas

value: KSC organoid line 1

value: organoids generated from the pancreas


title: 39-10-1


title: Met4, -met, ChIP, rep1


title: mESC_WT_m6A-CT_scRNA-seq

description: 10x Genomics


title: TB-only_Week 2_Romania_ROM17

value: TB-only

value: Romania


title: KC1

value: KC organoid line 1


title: WT, infected, biolgical rep 2

value: Roots

value: wildtype

value: S. indica infected


title: 30-10-3

value: grown at logarithmic phase in 100ml YPD broth at 30°C


title: TB-preDM_Diagnosis_South_Africa_SUN140

value: TB-preDM

value: Diagnosis


title: YBP2, 4C, rep2

data_processing:
150 bp paired-end reads were merged and aligned to S. cerevisiae genome (sacCer3) and analyzed with pipe4C.Bigwig file was generated by pipe4C, peak calling was performed with PeakC.
Assembly: sacCer3
Supplementary files format and content: bigwig

extract_protocol:
Standard 4C protocol was used for the preparation of 4C samples
4C-seq libraries were prepared with NEBNext Ultra II DNA Library Prep Kit.


title: WT, mock, biolgical rep 2

value: Control


title: YBP2, 4C, rep1

value: methionine depletion


title: StingKO_AngII2000

city: Houston

first: Yanming

last: Li

state: TX

address: One Baylor Plaza

department: Michael E. DeBakey Department of Surgery

institute: Baylor College of Medicine

description: 10X Genomics

data_processing:
FASTQ sequence data were processed to obtain a unique molecular identifiers (UMIs) count in each cell by using “Cell Ranger.” A mouse reference genome (mm10) was used for gene alignment.
Assembly: mm10
Supplementary files format and content: H5 file, matrix files

source_name: ascending aorta

extract_protocol:
For each sample, the ascending aortas of 3 mice were pooled together in ice-cold Hanks’ balanced salt solution (HBSS, without Ca2+ and Mg2+) (Gibco, Waltham, MA) with 10% fetal bovine serum (FBS). Aortas were cut into small pieces with microsurgical scissors and then placed into an enzyme cocktail prepared with 3 mg/ml collagenase type II (LS004176, Worthington Biochemical Corp., Lakewood, NJ), 0.15 mg/ml collagenase type XI (H3506, Sigma Corp., Kanagawa, Japan), 0.1875 mg/ml lyophilized elastase (LS002292, Worthington), 0.24 mg/ml hyaluronidase type I (H3506, Sigma), and 2.38 mg/ml 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, H4034, Sigma) in HBSS (with Ca2+ and Mg2+) (14025092, Thermo Fisher Scientific, Waltham, MA). Tissues were digested in a 37 °C water bath for 1 to 2 hours, and dissociation was examined under a microscope. Cell suspensions were collected by using a 40-µm cell strainer (CLS431750-50EA, Corning, Inc., Corning, NY), centrifuged at 300 x g for 10 minutes, and resuspended in HBSS (without Ca2+ and Mg2+) (14175095, Thermo Fisher) with 5% FBS, followed by incubation on ice for 30 minutes. Cells were then stained by using a live and dead cell kit (L3224, Thermo Fisher) and were submitted for flow cytometry (BD Biosciences) to collect live singlet cells. The living cell rate was determined with a microscope by using trypan blue (T8154, Sigma Corp.) staining.
Single-cell suspensions were separately dispensed onto the Chromium Controller (10x Genomics, Pleasanton, CA); we targeted 10,000 cells per sample. Our scRNA-seq library was constructed by using the Chromium Single-Cell 3′ v3 Reagent Kit (10X Genomics).

value: ascending aorta

value: C57BL/6J-Sting1 gt/gt

value: AngII infusion at week 9-10, for 7 days


title: TB-DM_Week 2_Indonesia_UNPAD25

value: TB-DM

value: Indonesia


title: TB-only_Month 2_Indonesia_UNPAD16


title: HiC, -met, rep1

value: MATa,  his3-11,15, leu2-3,112, trp1-1, ura3-1, can1-100, ade2-1, tor1-1, fpr::NAT, bar1, HIS3::REV1pr-tetR-mCherry-REV1pr-LacI-GFP, ADE2::REV1pr-LacI-REV1pr-tetR, Chr15::497047_LacO, Chr15::834487_TetO


title: 39-h-3

value: Cryptococcus neoformans var.grubii H99

value: wild type

value: grown at logarithmic phase in 100ml YPD broth at 39°C


title: 30-10-1


title: WT_Saline

description: 10X Genomics

value: Wild type (C57BL/6J)

value: Saline infusion at week 9-10, for 7 days


title: MATalpha, 4C, rep1


title: TB-IH_Diagnosis_South_Africa_SUN127

value: TB-IH


title: TB-only_Month 6_Indonesia_UNPAD30

value: TB-only


title: 39-h-2


title: IMH/D.GFP

description: 10X Genomics

value: Myh11-CreERT2+ mT/mG

value: GFP+ cells

tag: aorta status

value: IMH/D

value: Tamaxifen at week 6, AngII infusion at week 9


title: MATa, 4C, rep2

value: methionine depletion


title: TB-only_Month 2_Romania_ROM16

value: Romania


title: nonIMH/D.RFP

description: 10X Genomics

value: ascending aorta

value: RFP+ cells

value: nonIMH/D


title: 39-h-1


title: MATa, 4C, rep1


title: TB-DM_Month 2_Indonesia_UNPAD31

value: TB-DM

value: Indonesia


title: TB-DM_Diagnosis_South_Africa_SUN112

value: Diagnosis


title: 30-h-3

value: Cryptococcus neoformans var.grubii H99

value: wild type

value: grown at logarithmic phase in 100ml YPD broth at 30°C


title: 30-10-2


title: nonIMH/D.GFP

description: 10X Genomics


title: TB-only_Diagnosis_Indonesia_UNPAD14


title: HEK293_WT_RBM33_rep1

city: Chicago

first: Allen

last: Zhu

state: Illinois

address: 929 E 57th St, Room E331

institute: University of Chicago

description:
HEK293_WT_RBM33_rep1
hek293.siCtrl.RBM33.merge.GRCh38.T2C.cluster.bed

data_processing:
Adapters of all raw PAR-CLIP data sets were removed by Cutadapt v1.18. Reads longer than 17 were first mapped to the human rRNAs and small RNAs with Bowtie2 v2.4.5. Then unmapped reads were aligned to the human genome (hg38) and the GENCODE v36 gene annotation using STAR v 2.7.9a with parameters according to the ENCODE RNA-seq pipeline, except that the option (--outFilterMismatchNoverReadLmax 0.06) was modified in order to capture more potential T to C mutations. Only reads with proper pair were remained. Reads with mate unmapped and not in primary alignment were discarded. MarkDuplicates was used to remove PCR duplicates. To get aggregated T to C mutation counts, alignment files of two replicates were merged. T positions with coverage >= 10 were selected and they were concatenated as clusters if they were next to each other within 20 nt. Clusters with total mutation counts >= 3 were considered as potential binding regions. De novo motif enrichment analysis was performed in the binding regions using HOMER v4.9. Differentially ALKBH5 binding regions before and after RBM33 knock out were defined according to the following steps: 1) T to C mutation rates of each selected T positions in the clusters were calculated and summed up as the mutation levels of the clusters. 2) Total mutation counts >= 3 in the clusters of the control group, the RBM33 knock out group or both were required. 3) clusters with the mutation level difference >= 20% between the two groups were considered as differentially binding regions.
Assembly: hg38
Supplementary files format and content: ALKBH5 and RBM33 binding sites in BED format

growth_protocol: Human HEK293T cell line was purchased from ATCC. HEK293T cell line, HEK293T stably expressing 3XFlag-ALKBH5/3XFlag-RBM33 were grown in DMEM (Sigma, Cat# D5796) media supplemented with 10% FBS (Gibco) and 1% 100× Pen/Strep (Gibco) and cells were cultured at 37°C under 5.0% CO2.

extract_protocol:
Add 4SU to final concentration of 200 uM when cells confluency reaches 70-80%. The cells were cultured at 37℃ for 14 h.Then the cells were UV crosslinked with 0.15J/cm2. After that, the cells were rinsed with 1× PBS (Gibco), and lysed by the PAR-CLIP-seq lysis buffer (50 mM Tris-HCl, pH7.5+ 150 mM NaCl+ 2 mM EDTA+ 0.5% NP-40+ 1 mM DTT+ 1X protease inhibitor. Then the ALKBH5 and RBM33 bound RNA fragments were enriched by anti-Flag antibody. After the RNAase T1 digestion, and end repair, the enriched RNA fragments were extracted by phenol-choloroform.
All the libraries were constructed by the NEBNext Multiplex Small RNA library library Prep Set for Illumina Set1 (Cat# E7300). The resulting library was purified by AMPure XP beads (Beckman Coulter) following the manufacturer’s directions and subjected to next-generation sequencing.

treatment_protocol: To generate a RBM33 knock out 293T cells, 293T cells were infected with Lenti-CRISPR-V2-RBM33 gRNA lentivirus. Then the cells were diluted and single colony was picked up after puromycin selection. After validation by TA-clone and DNA sequencing, RBM33 knockout HEK293T cells were infected with Pmscv-blasticindin-3XFlag-ALKBH5 retrovirus and cells were selected by both blasticidin and puromycin to get the 293T_RBM33 K0_Flag-ALKBH5 stable cell line. To obtain the 293T cells stably expressing 3XFlag-ALKBH5 and 3XFlag-RBM33, 293T cells were infected with pMSCV-balsticidin-3XFlag-ALKBH5 and pMSCV-puromycin-3XFlag-RBM33 retrovius, and the infected cells were selected by blasticidin and puromycin respectively.


title: WT_AngII2000

description: 10X Genomics

value: Wild type (C57BL/6J)

value: AngII infusion at week 9-10, for 7 days


title: MATalpha, 4C, rep2


title: 30-h-2


title: TB-DM_Month 6_Indonesia_UNPAD8


title: HEK293_RBM33KO_ALKBH5_rep2

description:
HEK293_RBM33KO_ALKBH5_rep2
hek293.RBM33KO.ALKBH5.merge.GRCh38.T2C.cluster.bed

value: RBM33 KO


title: 30-h-1


title: IMH/D.RFP

description: 10X Genomics

value: Myh11-CreERT2+ mT/mG


title: Control.GFP

description: 10X Genomics

value: GFP+ cells

value: Normal

value: Tamaxifen at week 6, Saline infusion at week 9


title: HEK293_RBM33KO_ALKBH5_rep1

description:
HEK293_RBM33KO_ALKBH5_rep1
hek293.RBM33KO.ALKBH5.merge.GRCh38.T2C.cluster.bed


title: TB-only_Month 2_South_Africa_SUN132

value: TB-only


title: TB-DM_Month 2_South_Africa_SUN126


title: HEK293_WT_ALKBH5_rep2

description:
HEK293_WT_ALKBH5_rep2
hek293.siCtrl.ALKBH5.merge.GRCh38.T2C.cluster.bed


title: HEK293_WT_ALKBH5_rep1

description:
HEK293_WT_ALKBH5_rep1
hek293.siCtrl.ALKBH5.merge.GRCh38.T2C.cluster.bed


title: TB-DM_Diagnosis_South_Africa_SUN59


title: JEG-3 cells, sgNLRP7,rep1

city: Cambridge

first: Bowen

last: Gu

state: MA

address: 7 Divinity Ave

department: Stem Cell and Regenerative Biology Department

institute: Harvard University

data_processing:
The quality of the sequence reads were accessed by FastQC and the fastq files were quantified with Salmon (version 1.5.2)
Differentially expressed genes were determined by estimating fold change and dispersion using DESeq2
Assembly: GRCh38
Supplementary files format and content: tab-delimited text file of TPM for each sample

growth_protocol: JEG-3 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) with high glucose, 10% fetal bovine serum, and 1x Antibiotic-Antimycotic

extract_protocol:
Cells grown at 60-90% confluency were harvested, and total RNA were extracted by RNeasy Mini Kit.
RNA-seq library was prepared by NEBNext Ultra RNA Library Prep Kit for Illumina

value: NLRP7 knockout


title: JEG-3 cells, sgNLRP7,rep2


title: JEG-3 cells, WT,rep6

value: WT


title: TB-DM_Diagnosis_Indonesia_UNPAD29


title: JEG-3 cells, WT,rep5


title: TB-DM_Week 2_South_Africa_SUN59


title: EHT_RadMito_line1_rep2 [RM2]

city: Stanford

first: Joseph

middle: C.

last: Wu

state: California

address: 240 Pasteur Dr

department: Stanford Cardiovascular Institute

institute: Stanford university

description: EHT_RadMito_line1_rep2 [RM2] [RM2]

data_processing:
Low-quality and adapter sequences have been trimmed from the raw 150 bp paired-end reads by using Trim Galore function within FastQC toolkits Version 0.6 with Phred scoring less than 20.
The trimmed paired-end reads were mapped to the human reference genome GRCh38 to calculate the gene counts by using STAR version 2.7.10a with the parameters: --runThreadN 12 --quantMode GeneCounts --outFilterMultimapNmax 1 --limitBAMsortRAM 90258540544.
The RNA expression count matrix was merged from the read count tables by using Shell command.
Assembly: GRCh38
Supplementary files format and content: csv file, contain RNA expression count matrix

source_name: Engineered heart tissue

growth_protocol: In this study, EHTs were generated from two healthy iPSC lines (#1 and #2) using an established protocol, comprising of 70% cardiomyocytes (CMs), 15% endothelial cells (ECs) and 15% fibroblasts (FBs).

extract_protocol:
Cells were lysised in Tirzol and isolated using Direct-zol™ RNA Purification Kit from Zymo Research.
RNA library is formed by polyA capture (or rRNA removal) and reverse transcription of cDNA.

treatment_protocol:
exposed to 137Cs γ-rays continuously at a dose-rate of 9.3 mGy/hour for 5 days in the presence of 5 uM MitoTempo
After 10 days, the EHTs were exposed to 137Cs γ-rays continuously at a dose-rate of 9.3 mGy/hour for 5 days, with or without the presence of 5 uM MitoTempo.

value: Engineered heart tissue

value: iPSC SCVI15

value: Mixed cardiomyocytes, fibroblasts, endothelial cells

value: Wild type

value: Radiation+MitoTempo


title: EHT_RadMito_line2_rep1 [RM5]

description: EHT_RadMito_line2_rep1 [RM5] [RM5]

value: iPSC SCVI480


title: TB-DM_Diagnosis_South_Africa_SUN155

value: TB-DM

value: Diagnosis


title: TB-DM_Month 6_Indonesia_UNPAD82

value: Indonesia


title: EHT_RadMito_line1_rep1 [RM1]

description: EHT_RadMito_line1_rep1 [RM1] [RM1]


title: JEG-3 cells, WT,rep4


title: JEG-3 cells, sgELF3,rep3

value: ELF3 knockout


title: EHT_Mito_line2_rep2 [CM6]

description: EHT_Mito_line2_rep2 [CM6] [CM6]

treatment_protocol:
treated with 5 uM MitoTempo for 5 days
After 10 days, the EHTs were exposed to 137Cs γ-rays continuously at a dose-rate of 9.3 mGy/hour for 5 days, with or without the presence of 5 uM MitoTempo.

value: MitoTempo


title: HEK293_WT_RBM33_rep2

description:
HEK293_WT_RBM33_rep2
hek293.siCtrl.RBM33.merge.GRCh38.T2C.cluster.bed

value: wild type


title: JEG-3 cells, sgNLRP7,rep3


title: TB-DM_Week 2_Romania_ROM7

value: Romania


title: TB-DM_Week 2_Indonesia_UNPAD12


title: JEG-3 cells, sgELF3,rep1


title: JEG-3 cells, sgELF3,rep2


title: EHT_Mito_line2_rep1 [CM5]

description: EHT_Mito_line2_rep1 [CM5] [CM5]


title: JEG-3 cells, WT,rep3


title: TB-only_Month 6_Indonesia_UNPAD16


title: JEG-3 cells, WT,rep2


title: EHT_Mito_line1_rep2 [CM2]

description: EHT_Mito_line1_rep2 [CM2] [CM2]


title: EHT_Mito_line1_rep1 [CM1]

description: EHT_Mito_line1_rep1 [CM1] [CM1]


title: EHT_RadMito_line2_rep2 [RM6]

description: EHT_RadMito_line2_rep2 [RM6] [RM6]


title: JEG-3 cells, WT,rep1


title: TB-only_Diagnosis_Indonesia_UNPAD16


title: EHT_Radiation_line2_rep2 [RD6]

description: EHT_Radiation_line2_rep2 [RD6] [RD6]

treatment_protocol:
exposed to 137Cs γ-rays continuously at a dose-rate of 9.3 mGy/hour for 5 days
After 10 days, the EHTs were exposed to 137Cs γ-rays continuously at a dose-rate of 9.3 mGy/hour for 5 days, with or without the presence of 5 uM MitoTempo.

value: Radiation


title: TB-IH_Month 6_South_Africa_SUN121

value: TB-IH


title: Control.RFP

description: 10X Genomics

value: ascending aorta

value: Myh11-CreERT2+ mT/mG

value: RFP+ cells

value: Normal

value: Tamaxifen at week 6, Saline infusion at week 9


title: EHT_Radiation_line2_rep1 [RD5]

description: EHT_Radiation_line2_rep1 [RD5] [RD5]

value: Engineered heart tissue

value: Mixed cardiomyocytes, fibroblasts, endothelial cells

value: Wild type


title: EHT_Radiation_line1_rep2 [RD2]

description: EHT_Radiation_line1_rep2 [RD2] [RD2]

value: iPSC SCVI15


title: orange-derived microRNAs

city: Pamplona

first: Fermín

middle: Ignacio

last: Milagro Yoldi

state: Navarra

department: Department of Nutrition, Food Science and Physiology/Center for Nutrition Research

institute: University of Navarra

data_processing:
Adapter were removed from data using skewer.
Read with a size ranging from 15 to 30 bases were aligned to the reference genome with microRNA annotation from miRBase (maximum 2 mismatches in the seed area) using ShortStack.
Mapped tags were counted using htseq-count.
Reads were normalized with DESeq2.
Assembly: Prunus Persica (NCBIv2 and annotation NCBIv2.52 restricted to microRNAs)
Supplementary files format and content: text files that include raw counts for each sample
Supplementary files format and content: text file that includes normalized counts for each Sample

organism: Citrus sinensis

source_name: edible portion

extract_protocol:
0.1 of fruits (apple, olive, orange, pear, tomato), 0.1 of vegetables (spinach) and 0.5 g of nuts (almond and nut) were ground in QIAzol Lysis Reagent for 1 min using Ultra-Turrax T25 Basic (Basic IKA- WerkeTotal RNA). Total RNA was isolated with miRNeasy Serum/Plasma Kit (Qiagen) according to manufacturer protocol.
Libraries were prepared using the NEBNext® Small RNA Library Prep Set for Illumina kit

value: edible portion


title: almond-derived microRNAs

organism: Prunus dulcis


title: EHT_Radiation_line1_rep1 [RD1]

description: EHT_Radiation_line1_rep1 [RD1] [RD1]


title: apple-derived microRNAs

organism: Malus domestica


title: EHT_Control_line2_rep2 [CD6]

description: EHT_Control_line2_rep2 [CD6] [CD6]

treatment_protocol:
No treatment
After 10 days, the EHTs were exposed to 137Cs γ-rays continuously at a dose-rate of 9.3 mGy/hour for 5 days, with or without the presence of 5 uM MitoTempo.

value: iPSC SCVI480

value: Control


title: EHT_Control_line2_rep1 [CD5]

description: EHT_Control_line2_rep1 [CD5] [CD5]


title: EHT_Control_line1_rep2 [CD2]

description: EHT_Control_line1_rep2 [CD2] [CD2]


title: scRNA-seq_D_9_Aged_3

first: Weiguo

last: Cui

state: IL

address: 303 E Chicago Ave

department: Pathology

institute: Northwestern University

data_processing:
Reads were aligned to mouse reference genome mm10.
Assembly: mm10
Supplementary files format and content: Matrix files were generated using Cell Ranger (10x Genomics);

source_name: Lung

extract_protocol: 10x Genomics; each sample generated one gene expression library (GEX).

value: Lung

value: C57BL/6 mice

value: 9 days p.i.

tag: infection

value: PR8 Influenza

value: Aged


title: scRNA-seq_D_3_Young_3

value: 3 days p.i.

value: Young


title: EHT_Control_line1_rep1 [CD1]

description: EHT_Control_line1_rep1 [CD1] [CD1]


title: scRNA-seq_D_3_Aged_3


title: scRNA-seq_Day_9_Aged_2

data_processing:
Reads were aligned to mouse reference genome mm10
Demultiplexing and alignment were performed with Cell Ranger (v6.0, 10x Genomics) and the mm10 2020-A reference transcriptome (10x Genomics).
Assembly: mm10

extract_protocol: 10x Genomics

value: Day 9

value: Flu PR8 infection


title: Control 21d rep 3

city: Research Triangle Park

first: Brian

last: Chorley

state: NC

address: US EPA, 109 TW Alexander Dr

institute: US EPA

data_processing:
The generated reads were checked for quality and trimmed of adapter sequence (minimum read length =15, adapter trim stringency = 0.9) using Partek Flow (version 8.0.19.1027) software program (Partek, St. Louis, MO). Bases trimmed of adapter sequence from the 3’ prime end (TGGAATTCTCGGGTGCCAAGG) and according to Quality Score from both 5’ and 3’ ends if the Phred quality score was less than 20..
Contaminant reads (rRNA, tRNA, and mtDNA) were filtered out using Bowtie 2 v2.2.5.
Samples were aligned with Bowtie version 1.0.0 to the mm10 reference index using up to a 1 base pair mismatch and seed length of 10.
Aligned sequences were then annotated to miRBase mature miRNA v21 with the Partek E/M (expectation maximum) algorithm with 90% minimum  read overlap with feature. Features (miRNAs) were excluded if the geometric mean across all samples was less than or equal to 10 this eliminated 338 miRNAs of the 655 miRNAs aligned.
Normalization and differentially expressed miRNAs (DEmiRS) were then determined by DESeq2. Significance testing was corrected for multiple tests and was determined by a FDR value of p ≤0.05.
Assembly: mm10
Supplementary files format and content: tab-delimited file contains filtered count values for all samples and mapped featues before DESeq2 analysis.

source_name: liver

extract_protocol:
Total RNA was extracted from frozen liver tissue (n=4-5/treatment) using a ceramic bead-based homogenization combining an initial RNAzol RT® (MRC, Cincinnati, OH) extraction with subsequent purification using the RNeasy MinElute kit (Qiagen, Germantown, MD).  Clean-up used the modifications for RNA binding indicated in the MinElute kit protocol supplement to retain the small RNA fractions.  Total RNA from whole blood (n= 4-5/treatment) was isolated using the mirVANA RNA isolation kit (ThermoFisher Scientific, Waltham, MA) and from in vitro samples (n=2/treatment) using the miRCURY RNA isolation kit (Exiqon Inc., Woburn, MA).  RNA was quantified by Nanodrop spectrophotometer (NanoDrop Technologies, Wilmington, DE) and/or Qubit fluorometric assay (ThermoFisher Scientific).  All RNA exhibited high purity with A260/280 ratios >2.0.
To create sequencing libraries, the TruSeq Small RNA Library Prep Kit was utilized following manufacturer’s instructions. Quality and size range of prepped libraries were check using Agilent Bioanalyzer before loading on flow cell.

treatment_protocol: Archived samples generated from a previously conducted study were used for this evaluation and the animal methods are detailed elsewhere (PMID: 24183702).  Briefly, 6–7-week-old female B6C3F1 mice were housed five per cage in polycarbonate cages in a specific pathogen free (SPF) and Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited facility. Mice were exposed by oral gavage to non-carcinogenic (0, 1 or 2 mg/kg bodyweight per day) or carcinogenic (4 or 8 mg/kg bodyweight per day) doses of furan in corn oil for 21 days. Four hours after the final dosing mice were anesthetized by CO2 inhalation prior to euthanasia by exsanguination. Livers were removed and samples were flash frozen and stored at or below -70°C until used for sequencing.

value: liver

tag: Sex

value: female

tag: age

tag: administration

value: oral gavage

tag: time

tag: chemical

value: furan

tag: dose

value: control


title: MB231-FM34a-rep2

city: West Lafayette

first: Ikjot Singh

last: Sohal

state: IN

address: 4332 Foal Dr

department: Biological Sciences

institute: Purdue University

data_processing:
Reads were trimmed and aligned using Two-Pass alignment mode using STAR aligner (version 2.7.9.a)
Alignments were quantified using SubRead FeatureCounts (version 1.6.1) using the GTF file from Ensembl Release 104 to derive the read counts at Genomic features like Genes, using the unstranded approach.
Alignments were processed using Ngsutils (version 0.5.9) with the Human Annotations from Ensembl Release 104 to check read alignments to the transcriptome.
DESeq2 (v1.36.0) was used to normalize the read count and determine differentially expressed genes.
Assembly: h38
Supplementary files format and content: tab-delimited file includes normalized read counts for each sample
Supplementary files format and content: tab-delimited file includes log2FoldChange, p-value and other key statistics derived from comparison of two samples using DESeq2.

source_name: MB-231 cell line

growth_protocol: MB-231 cells were maintained in folate-free RPMI supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified incubator with 5% CO2 at 37 °C

extract_protocol:
RNA was extracted from the cells after 48 hours of transfection using mirVana™ RNA Isolation Kit (Thermo Fisher, AM1560) including removal of genomic DNA using Dnase I digestion (79254, Qiagen).
RNA sequencing libraries were prepared using poly A enrichment method using NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® to remove ribosomal RNA.

treatment_protocol: MB-231 cells (2 × 105) were seeded into individual wells of a 6 well plate. The next day, cells were transfected with the various miRNA duplexes (50 nM) using Lipofectamine RNAiMAX (Life Technologies).

value: MB-231 cell line

value: Triple negative breast cancer

value: WT

value: Transfected with FM-miR-34a


title: scRNA-seq_Day_9_Aged_1

value: Lung

tag: strain

value: C57BL/6 mice

tag: timepoint


title: MB231-FM34a-rep3

tag: genotype


title: ANT 3

type: RNA

city: Guangzhou

first: Neng

last: Jiang

address: 651 Dongfeng East Road, Guangzhou 510060, P. R. China

institute: Sun Yat-sen University Cancer Center

description: Agilent Whole Genome Oligo Microarrays (one-color): Prepare the RNA Sample; Total RNA Clean-up and RNA QC; Prepare labeling reaction; Purify the labeled/amplified RNA and labeled cRNA QC; Hybridization; Microarray Wash; Scanning; Extract data using Agilent Feature Extraction Software;

hyb_protocol: After stopping the labeling procedure, Agilent Gene Expression Hybridization Kit (Agilent p/n 5188-5242) was used for mRNAs hybridization according to the manufacturer’s instructions. Hybridize at 65°C for 17 hours.

scan_protocol: The hybridized chips were scanned with an Agilent Microarray Scanner (Agilent p/n G2565BA).

data_processing: Agilent Feature Extraction Software is used to read the raw intensity of the image. Add the images (.tif) to be extracted to the FE Project; Set FE Project Properties; Check the Extraction Set Configuration; Save the FE Project (.fep) by selecting File > Save As and browse for desired location; Select Project > Start Extracting and export data to txt.

source_name: primary lung cancer

label_protocol: MRNAs were labeled with the Quick Amp Labeling Kit, One-Color (Agilent p/n 5190-0442), according to the manufacturer’s guidelines.

extract_protocol: Total RNA was extracted from fresh tissue samples by TRIzol Reagent (Invitrogen life technologies) according to the recommended protocols.

tag: tissue

value: adjacent nontumor tissues

tag: status after 5 years

value: Alive

tag: histology

value: NSCLC

value: 61 years

value: Female

tag: Stage

value: IB


title: Control 21d rep 2

value: liver


title: Control 21d rep 4

value: female


title: ANT 2

value: Dead

value: 70 years

value: Male

value: IIIA


title: scRNA-seq_Day_9_Young_2

value: Day 9

value: Flu PR8 infection

tag: condition

value: Young


title: MB231-FM34a-rep1

tag: cell line

value: MB-231 cell line

tag: cell type

value: Triple negative breast cancer

value: WT

tag: treatment

value: Transfected with FM-miR-34a


title: Tumor 2

value: primary lung cancer

value: NSCLC

tag: Sex


title: Tumor 1

value: Alive

tag: age

value: 55 years

value: IIB


title: MB231-PM34a-rep3

value: Transfected with PM-miR-34a


title: scRNA-seq_Day_9_Young_1

value: Lung

value: C57BL/6 mice

tag: infection


title: 8 mg furan 21d rep 5

value: liver

value: female

tag: administration

value: oral gavage

tag: time

value: furan


title: scRNA-seq_D_9_Young_3

value: 9 days p.i.

value: PR8 Influenza

value: Young


title: scRNA-seq_Day_3_Aged_2

value: Day 3

value: Flu PR8 infection

value: Aged


title: MB231-PM34a-rep1

value: MB-231 cell line

value: Triple negative breast cancer

value: WT


title: MB231-PM34a-rep2


title: Control 21d rep 5

tag: chemical

tag: dose

value: control


title: scRNA-seq_Day_3_Aged_1

tag: strain

tag: timepoint


title: 8 mg furan 21d rep 4

tag: tissue


title: Tumor 3

value: primary lung cancer

tag: status after 5 years

value: Alive

value: NSCLC

value: 61 years

value: Female

value: IB


title: MB231-NC-rep3

tag: genotype

value: Transfected with siNC2


title: scRNA-seq_Day_3_Young_2

value: Lung

value: C57BL/6 mice


title: 8 mg furan 21d rep 2

value: liver

value: female

value: oral gavage

value: furan


title: 8 mg furan 21d rep 3


title: ANT 1

value: adjacent nontumor tissues

tag: histology

value: 55 years

value: Male

tag: Stage

value: IIB


title: scRNA-seq_Day_3_Young_1

value: Day 3

value: Flu PR8 infection

tag: condition

value: Young


title: 8 mg furan 21d rep 1


title: 4 mg furan 21d rep 5


title: MB231-Un-rep2

tag: cell line

value: MB-231 cell line

tag: cell type

value: Triple negative breast cancer

value: WT

value: Untreated


title: MB231-NC-rep2

tag: treatment

value: Transfected with siNC2


title: 4 mg furan 21d rep 3

tag: Sex

tag: age


title: MB231-Un-rep1


title: Lung_Day_9_Aged_2

data_processing:
Reads were aligned to mouse reference genome GRCm39
Demultiplexing was performed with bcl2fastq. Alignment was performed with Salmon (v1.4.0) and the GRCm39 reference transcriptome (Ensembl).
Assembly: mm10
Supplementary files format and content: salmon output

extract_protocol:
RNAEasy (Qiagen)
Illumina Nextera

value: Lung

value: C57BL/6 mice

value: Day 9

tag: infection

value: Aged


title: MB231-Un-rep3


title: Control 21d rep 1

value: liver

value: female

tag: administration

value: oral gavage

tag: time

value: furan

value: control


title: Lung_Day_9_Aged_3


title: 4 mg furan 21d rep 2

tag: chemical

tag: dose


title: Lung_Day_9_Aged_1

tag: strain

tag: timepoint


title: 4 mg furan 21d rep 4

tag: tissue


title: Lung_Day_9_Young_1


title: Lung_Day_9_Young_2


title: 4 mg furan 21d rep 1


title: HEK_m6A-CT_scRNA-seq

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing and gene counting were made using the Cell Ranger software cellranger-arc-2.0.2 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10, hg39
Supplementary files format and content: Tab-separated values files and matrix files

extract_protocol:
E14 mESCs were harvested, counted and centrifuged for 5 min at 350×g at room temperature. Aliquots of cells (~1,000,000 cells) were resuspended in 250 μL Nuclei Extraction Buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.020 % Triton X-100) and incubated on ice for 1 min. The mixture was diluted with 1 mL Nuclei Dilution Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 0.1 U/μL RiboLock RNase inhibitor) and centrifuged for 5 min at 600×g at 4 °C. Nuclei were washed once with 1 mL Nuclei Dilution Buffer and counted. E14 EPSC and MEF nuclei were similarly isolated with the following conditions: 1 min on ice 0.015 % Triton X-100 for EPSC; 5 min on ice in 0.2 % NP-40 for MEF. Aliquots of nuclei (150,000 nuclei in total per assay) were incubated with 1 μg primary antibody (rabbit IgG or rabbit anti-m6A: MilliporeSigma, #ABE572) in 50 μL Antibody Binding Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) for 2 hours on rotator at 4 °C. Afterwards, nuclei were incubated with 1 μg secondary antibody (guinea pig anti-rabbit: Novus Biologicals, #NBP1-72763) in 50 uL Antibody Binding Buffer for 30 min on the nutator at room temperature. Nuclei were then centrifuged for 5 min at 600×g at room temperature and two rounds of washes with 200 μL Wash Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 0.1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) were performed. The nuclei were resuspended in 50 μL high-salt Binding Buffer (20 mM HEPES pH7.5, 300 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) with 2.5 μL pAG-Tn5 (EpiCypher, #15-1117) and incubated for 1 hour on the nutator at room temperature. The nuclei were then washed twice with high-salt Wash Buffer (20 mM HEPES pH7.5, 300 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 0.1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) and resuspended in 50 μL Reverse Transcription & Tagmentation Buffer (1X SuperScript IV buffer along with, 1 M Betaine, 5 mM DTT, 2 μM Random Hexmer Primers, 0.5 mM each dNTP mix, 1X Roche Complete Protease Inhibitor Cocktail, 1 U/μL RiboLock RNase inhibitor, 4 U/μL SuperScript IV) for 2 hours at 37 °C. When reaction was done, the nuclei were resuspended with 50 μL cold Antibody Binding Buffer and placed on ice. All steps after nuclei extraction were performed in 0.2-ml tubes to minimize nuclei loss.  The nuclei were counted and diluted to a concentration based on the targeted nuclei number. Seven microliters of 10x Genomics ATAC Buffer B was added to 8 μl of nuclei, pipetting several times to mix. Most of the remaining steps were performed according to the Chromium Next GEM Single Cell Multiome protocol Rev E (10x Genomics), skipping the Step 1 ‘Transposition’ and continuing from Step 2.0 ‘Generation & barcoding’. A qPCR cycle check was performed after pre-amplification step using 1:100 amplicon. Indexed CT libraries were prepared with the following PCR program: 45 sec at 98 °C, followed by 23 cycles (ESC IgG-CT) or 15 cycles (ESC and mixed cell-lines m6A-CT) of 15 sec at 98 °C and 10 sec at 60 °C, with a final extension at 1 min at 72 °C. For cDNA libraries, cycle numbers used in cDNA amplification and sample index PCR were 7 cycles and 12 cycles, respectively. The final cDNA libraries and CT libraries were sequenced using an Illumina NovaSeq 6000, PE150 and PE50 system, respectively.
Library was performed according to the manufacter’s instructions (single cell Multiome Rev E protocol, 10x Genomics).

value: Human embryonic kidney

tag: antibody


title: Lung_Day_3_Aged_3

value: Day 3

value: Flu PR8 infection


title: 2 mg furan 21d rep 5


title: Lung_Day_3_Aged_2


title: AML Patient 20, End of Induction

city: Atlanta

first: Hope

last: Mumme

address: 1760 Haygood Dr NE

institute: Emory University

description: 10x Genomics

data_processing:
Raw scRNA-seq data was demultiplexed, aligned to human reference genome, and processed for single cell gene counting using the Cell Ranger Software from 10x Genomics (v6.1.1).
Assembly: hg38
Supplementary files format and content: Tab separated values files and matrix files

source_name: bone marrow

extract_protocol:
Frozen bone marrow samples were obtained from Aflac Cancer and Blood disorder center from Children’s Healthcare of Atlanta Pediatric Biorepository.The frozen BM were thawed viably using standardized protocol. Briefly the cells were thawed quickly (37C for ~2 minutes till a small ice crystal remains), and warm complete medium (RPMI1640, 5% heat inactivated FBS, 1x Penicillin. Streptomycin) was added gradually. The cells were pelleted (380g, RT, 5min), and washed one more time with complete medium, which was followed by one more wash with PBS containing BSA (0.04 to 1.0%). Finally, the cells were resuspended in PBS with BSA to get an optimal cell concentration of 700-1200 cells/µl. The single cell suspensions were then proceeded using 10x Genomics single cell assay protocols.
The libraries were prepared using the Single Cell 3’V3, Chromium Next GEM single cell 5’V1 reagent kits. Sequencing was performed using the massively parallel sequencing on the Novaseq 6000 platform.

value: bone marrow

tag: sample type

value: end of induction

value: Bone marrow blast and immune cells

tag: who classification


title: 2 mg furan 21d rep 4


title: Lung_Day_3_Aged_1

tag: condition


title: AML Patient 20, Diagnosis

description: 10x Genomics

value: diagnosis


title: AML Patient 19, End of Induction

description: 10x Genomics

value: AML with mutated RUNX1


title: Lung_Day_3_Young_3

value: Lung

value: Young


title: 2 mg furan 21d rep 3


title: MB231-NC-rep1

tag: cell line

value: MB-231 cell line

value: Triple negative breast cancer

tag: genotype

value: WT

value: Transfected with siNC2


title: 2 mg furan 21d rep 2

value: liver

value: female

value: oral gavage


title: AML Patient 19, Diagnosis

description: 10x Genomics

tag: cell type


title: HEK_m6A-CT_scCUT&Tag

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing and gene counting were made using the Cell Ranger software cellranger-arc-2.0.2 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10, hg39
Supplementary files format and content: Tab-separated values files and matrix files
Library strategy: scCUT&Tag

value: Human embryonic kidney

value: m6A (MilliporeSigma, #ABE572)


title: 2 mg furan 21d rep 1

tag: Sex

value: furan


title: 1 mg furan 21d rep 4

tag: age


title: Lung_Day_3_Young_2

value: C57BL/6 mice

value: Day 3

tag: infection

value: Flu PR8 infection


title: 1 mg furan 21d rep 3

tag: administration


title: AML Patient 18, Diagnosis

description: 10x Genomics

value: bone marrow

value: diagnosis

value: Bone marrow blast and immune cells

value: APL (AML with t(15;17)(q22;q12)) PML/RARA


title: AML Patient 18, End of Induction

description: 10x Genomics

value: end of induction


title: 1 mg furan 21d rep 2

tag: time


title: Lung_Day_3_Young_1

value: Lung

value: Young


title: Lung_Day_0_Aged_2

value: Day 0

value: Aged


title: AML Patient 17, Diagnosis

description: 10x Genomics

value: Acute megakaryoblastic leukemia


title: Lung_Day_0_Young_2


title: 1 mg furan 21d rep 1

value: liver

value: female

value: oral gavage

tag: chemical


title: AML Patient 16, End of Induction

description: 10x Genomics

value: AML with myelodysplasia-related changes


title: Lung_Day_0_Aged_1

value: C57BL/6 mice


title: AML Patient 17, End of Induction

description: 10x Genomics


title: C3H10T1/2 MSCs, shMLL1, day 7

city: Incheon

first: Jangyhun

last: Choi

state: Incheon

address: 5N521, 100 Inha-ro, Michuhol-gu

department: Department of biological sciences

country: South Korea

institute: Inha University

data_processing:
Raw reads were trimmed to remove adaptor sequence and low-quality reads by Trimmomatic version 0.39 with the following parameters; ILLUMINACLIP:adapters/TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36.
The resulting reads were mapped to the mouse genome assembly GRCm38 (mm10) using HISAT2 version 2.2.1 under the default parameters.
Reads with mapping quality score (MAPQ) > 30 were selected and converted into BAM files using SAMtools version 1.12 with -q 30 option. PCR duplicates were then removed and the resulting BAM files were sorted by name using rmdup tool and sort tool in SAMtools, respectively.
Sorted reads were assigned to gene and counted using htseq-count version 0.11.1 as follows parameters; -f bam --strand=no --mode=intersection-nonempty -r name.
Assembly: GRCm38/mm10
Supplementary files format and content: tab-delimited text files including raw count for each sample.

growth_protocol: C3H10T1/2 mesenchymal cells were maintained in DMEM (WelGene) supplemented with 10% FBS (WelGene) and 1% penicillin-streptomycin (Hyclone) in humidified atmosphere with 5% CO2 at 37ºC.

extract_protocol:
Total RNA was prepared using an RNeasy mini plus kit (Qiagen) according to the manufacturer’s instructions.
Total RNAs were subjected to library preparation using a TruSeq RNA-seq kit (Illumina) and libraries were quantified by Agilent Technologies 2100 Bioanalyzer (Agilent Technologies).

treatment_protocol: To induce osteogenic differentiation, cells were cultivated in osteogenic medium containing 20 mM β-glycerophosphate (Sigma), 100 μg/mL ascorbic acid 2-phosphate (Sigma), and 0.1 μM dexamethasone (Sigma) for 2 days. The osteogenic medium was replenished every other day for 21 days.

value: Mesenchymal stem cells

value: MLL1 knockdown

tag: time point

value: Day 7

tag: treatment

value: Osteogenic media


title: WT3

data_processing:
Basecalls were performed using Illumina's bcl2fastq v2.18.12.
Raw sequence FASTQ reads were aligned with STAR version 2.7.10a using parameters --outReadsUnmapped Fastx --outSAMtype BAM SortedByCoordinate --outSAMattributes All
Transcript abundances were calculated from the STAR alignment BAM files using HTSeq-count version 2.0.2 using parameters -m union -r pos -t exon -i gene_id -a 10 -s no -f bam
Assembly: Gencode GRCm39 Release M32
Supplementary files format and content: tab-delimited Excel file containing Ensembl ID#s for each gene in each sample

value: WT organoid line 3

value: Organoids from wild-type mice

value: organoids generated from the pancreas


title: Lung_Day_0_Young_1

value: Lung

tag: strain

tag: timepoint

value: Flu PR8 infection


title: AML Patient 16, Diagnosis

description: 10x Genomics

tag: tissue

value: bone marrow

value: diagnosis

value: Bone marrow blast and immune cells


title: Liver_Day_9_Aged_2

data_processing:
Reads were aligned to mouse reference genome GRCm39
Demultiplexing was performed with bcl2fastq. Alignment was performed with Salmon (v1.4.0) and the GRCm39 reference transcriptome (Ensembl).
Assembly: mm10
Supplementary files format and content: Salmon output

source_name: Liver

value: Liver

value: Day 9

value: Aged


title: WT2

value: WT organoid line 2


title: Liver_Day_9_Aged_1

value: C57BL/6 mice


title: C3H10T1/2 MSCs, shNFIB, day 7

value: Mesenchymal stem cells

value: NFIB knockdown


title: AML Patient 15, End of Induction

description: 10x Genomics

tag: sample type

value: end of induction

value: AML, NOS


title: AML Patient 14, End of Induction

description: 10x Genomics

tag: who classification


title: C3H10T1/2 MSCs, shControl, day 7

value: WT

value: Day 7

value: Osteogenic media


title: WT1

value: WT organoid line 1

value: Organoids from wild-type mice

value: organoids generated from the pancreas


title: SC3

value: SC organoid line 3


title: C3H10T1/2 MSCs, shMLL1, day 0

value: MLL1 knockdown

value: Day 0

value: Untreated


title: Liver_Day_9_Young_2

value: Liver

value: Day 9

value: Flu PR8 infection

tag: condition

value: Young


title: AML Patient 14, Diagnosis

description: 10x Genomics

value: bone marrow

value: diagnosis

value: Bone marrow blast and immune cells


title: C3H10T1/2 MSCs, shNFIB, day 0

tag: cell line

value: Mesenchymal stem cells

value: NFIB knockdown


title: Liver_Day_9_Young_1

value: C57BL/6 mice


title: SC1

value: SC organoid line 1

tag: genotype


title: KSC3

value: KSC organoid line 3


title: AML Patient 13, Diagnosis

description: 10x Genomics

tag: cell type


title: C3H10T1/2 MSCs, shControl, day 0

value: WT


title: AML Patient 12, Diagnosis

description: 10x Genomics


title: Liver_Day_3_Aged_3

value: Day 3

value: Aged


title: Liver_Day_3_Aged_2


title: AML Patient 11, Diagnosis

description: 10x Genomics


title: SC2

value: SC organoid line 2

value: organoids generated from the pancreas


title: Liver_Day_3_Aged_1

value: Liver


title: AML Patient 10, Diagnosis

description: 10x Genomics

value: AML with mutated NPM1


title: KSC2

value: KSC organoid line 2


title: Mix_m6A-CT_scRNA-seq

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing and gene counting were made using the Cell Ranger software cellranger-arc-2.0.2 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Mix (E14 and CF1)

extract_protocol:
E14 mESCs were harvested, counted and centrifuged for 5 min at 350×g at room temperature. Aliquots of cells (~1,000,000 cells) were resuspended in 250 μL Nuclei Extraction Buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.020 % Triton X-100) and incubated on ice for 1 min. The mixture was diluted with 1 mL Nuclei Dilution Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 0.1 U/μL RiboLock RNase inhibitor) and centrifuged for 5 min at 600×g at 4 °C. Nuclei were washed once with 1 mL Nuclei Dilution Buffer and counted. E14 EPSC and MEF nuclei were similarly isolated with the following conditions: 1 min on ice 0.015 % Triton X-100 for EPSC; 5 min on ice in 0.2 % NP-40 for MEF. Aliquots of nuclei (150,000 nuclei in total per assay) were incubated with 1 μg primary antibody (rabbit IgG or rabbit anti-m6A: MilliporeSigma, #ABE572) in 50 μL Antibody Binding Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) for 2 hours on rotator at 4 °C. Afterwards, nuclei were incubated with 1 μg secondary antibody (guinea pig anti-rabbit: Novus Biologicals, #NBP1-72763) in 50 uL Antibody Binding Buffer for 30 min on the nutator at room temperature. Nuclei were then centrifuged for 5 min at 600×g at room temperature and two rounds of washes with 200 μL Wash Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 0.1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) were performed. The nuclei were resuspended in 50 μL high-salt Binding Buffer (20 mM HEPES pH7.5, 300 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) with 2.5 μL pAG-Tn5 (EpiCypher, #15-1117) and incubated for 1 hour on the nutator at room temperature. The nuclei were then washed twice with high-salt Wash Buffer (20 mM HEPES pH7.5, 300 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 0.1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) and resuspended in 50 μL Reverse Transcription & Tagmentation Buffer (1X SuperScript IV buffer along with, 1 M Betaine, 5 mM DTT, 2 μM Random Hexmer Primers, 0.5 mM each dNTP mix, 1X Roche Complete Protease Inhibitor Cocktail, 1 U/μL RiboLock RNase inhibitor, 4 U/μL SuperScript IV) for 2 hours at 37 °C. When reaction was done, the nuclei were resuspended with 50 μL cold Antibody Binding Buffer and placed on ice. All steps after nuclei extraction were performed in 0.2-ml tubes to minimize nuclei loss. The nuclei were counted and diluted to a concentration based on the targeted nuclei number. Seven microliters of 10x Genomics ATAC Buffer B was added to 8 μl of nuclei, pipetting several times to mix. Most of the remaining steps were performed according to the Chromium Next GEM Single Cell Multiome protocol Rev E (10x Genomics), skipping the Step 1 ‘Transposition’ and continuing from Step 2.0 ‘Generation & barcoding’. A qPCR cycle check was performed after pre-amplification step using 1:100 amplicon. Indexed CT libraries were prepared with the following PCR program: 45 sec at 98 °C, followed by 23 cycles (ESC IgG-CT) or 15 cycles (ESC and mixed cell-lines m6A-CT) of 15 sec at 98 °C and 10 sec at 60 °C, with a final extension at 1 min at 72 °C. For cDNA libraries, cycle numbers used in cDNA amplification and sample index PCR were 7 cycles and 12 cycles, respectively. The final cDNA libraries and CT libraries were sequenced using an Illumina NovaSeq 6000, PE150 and PE50 system, respectively.
Library was performed according to the manufacturer’s instructions (single cell Multiome Rev E protocol, 10x Genomics).

value: Mix (E14 and CF1)

value: Mix (Mouse embryonic stem cells, Expanded potential stem cells and Mouse embryonic fibroblast)


title: Liver_Day_3_Young_3

value: Flu PR8 infection

value: Young


title: Aged_PDK4IN_03

city: Shanghai

first: Yu

last: Sun

address: 320 Yueyang Rd, Life Sciences Building A #1508

department: Shanghai Institute of Nutrition and Health

institute: Chinese Academy of Sciences

description: PE150 high throughout data

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mouse genome (UCSC GRCm39/mm39) using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata.
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Assembly: GRCm39/mm38
Supplementary files format and content: Tab-delimited text files include FPKM values for each Sample.
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample.

source_name: Pulmonary alveolus

growth_protocol: Mice (C57BL/6J background, males) were allowed to naturally age until 20 months old, then subject to treatment by vehicle or PDK4-IN (an anthraquinone derivative, PDK4 inhibitor). Animals were treated once every 2 weeks, in an intermittent manner for 4 months before physical tests at 24 months of age, then sacrificed for further histological and molecular examinations. Tissues were acquired freshly upon mouse dissection and embedded as snap-frozen chunks.

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

treatment_protocol: Mouse pulmonary alveolus cells, mainly fibroblasts, were precisely collected by laser capture microdissection (LCM). Animals were allowed to naturally age, or subject to treatment by PDK4-IN (an anthraquinone derivative, PDK4 inhibitor).

value: Pulmonary alveolus

value: Normal mouse primary lung fibroblasts

value: Stromal

value: wild type

tag: group

value: Aged plus PDKIN


title: KC2

extract_protocol:
RNA was extracted using the Rneasy kit from Qiagen according to the vendor's protocol.
RNA libraries for RNA-seq were prepared using NEB Ultra II Directional Kits following manufacturer's protocols.

value: KC organoid line 2


title: Liver_Day_3_Young_2

value: C57BL/6 mice

value: Day 3


title: AML Patient 8, Diagnosis

description: 10x Genomics

value: bone marrow

value: diagnosis

value: Bone marrow blast and immune cells

value: AML with mutated CEBPA


title: Aged_PDK4IN_02

description: PE150 high throughout data


title: Liver_Day_3_Young_1

value: Liver


title: Mix_m6A-CT_scCUT&Tag

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing and gene counting were made using the Cell Ranger software cellranger-arc-2.0.2 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files
Library strategy: scCUT&Tag

value: Mix (E14 and CF1)

tag: antibody

value: m6A (MilliporeSigma, #ABE572)


title: AML Patient 7, Diagnosis

description: 10x Genomics

value: AML with minimal differentiation


title: Liver_Day_0_Aged_2

value: Day 0

tag: infection

value: Flu PR8 infection

value: Aged


title: Aged_03

description: PE150 high throughout data

value: Pulmonary alveolus

value: Normal mouse primary lung fibroblasts

value: Stromal


title: AML Patient 6, Relapse

description: 10x Genomics

value: relapse

value: AML with myelodysplasia-related changes


title: 24h post-infection (hpi) uninduced [UI σ28/σ54 OE 3]

city: Omaha

first: Scot

middle: P.

last: Ouellette

state: NE

address: 985900 Nebraska Medical Center

department: Pathology and Microbiology

institute: University of Nebraska Medical Center

description: PolyA and rRNA depleted for both human and bacteria

data_processing:
original fastq format reads were trimmed by fqtrim tool (https://ccb.jhu.edu/software/fqtrim) to remove adapters, terminal unknown bases (Ns), and low quality 3’ regions (Phred score < 30)
trimmed fastq files were processed by FastQC
fastq files were mapped to Chlamydia trachomatis 434/Bu by CLC Genomics Workbench 12 for RNAseq analyses
read counts were modeled using Generalized Linear Model (GLM), assuming a negative binomial distribution, and reported as transcripts per million (TPM)
Wald test was used for statistical analysis of two-group comparisons
Assembly: http://bacteria.ensembl.org/Chlamydia_trachomatis_434_bu_gca_000068585/Info/Index
Supplementary files format and content: excel file showing fold changes and statistical significance between strains and treatment conditions

organism: Chlamydia trachomatis L2/434/Bu

source_name: Infected McCoy cell line

growth_protocol: McCoy cells were infected with C. trachomatis L2 and induced or not at 4 or 14 hpi with aTc. Samples were collected at 24 or 18 hpi, respectively.

extract_protocol:
Total RNA was collected from C. trachomatis infected cell cultures using Trizol. Total RNA was DNased then further purified to remove eukaryotic mRNA and both prokaryotic and eukaryotic rRNA using Oligotex, MicrobEnrich, MicrobExpress, and Ribozero.
Illumina Ribo-Zero plus Microbiome

treatment_protocol: Indicated above in the the first characteristics column.

value: C. trachomatis L2 434/Bu

tag: induction

value: Uninduced

tag: plasmid

value: pBOMBL-σ28_FLAG + σ54_6xH

tag: knockdown/overexpression

value: Overexpression


title: AML Patient 6, End of Induction

description: 10x Genomics

value: bone marrow

value: end of induction

value: Bone marrow blast and immune cells


title: Liver_Day_0_Aged_1

value: Liver

value: C57BL/6 mice


title: Aged_02

description: PE150 high throughout data

value: wild type


title: Aged_01

description: PE150 high throughout data


title: 24h post-infection (hpi) uninduced [UI σ66 OE 3]


title: AML Patient 6, Diagnosis

description: 10x Genomics

value: diagnosis


title: 24h post-infection (hpi) induced [I σ28/σ54 OE 3]

value: 10 nM aTc @14hpi


title: Liver_Day_0_Young_2

value: Day 0

value: Flu PR8 infection

value: Young


title: Liver_Day_0_Young_1


title: Young_03

description: PE150 high throughout data

value: Pulmonary alveolus

value: Normal mouse primary lung fibroblasts

value: Stromal


title: AML Patient 5, Relapse

description: 10x Genomics

value: relapse

value: AML, NOS


title: 24h post-infection (hpi) induced [I σ54 OE 3]

value: C. trachomatis L2 434/Bu

value: Overexpression


title: KC3

value: KC organoid line 3

value: organoids generated from the pancreas


title: Organoids from proximal part of small intestine of male old mice (105 weeks old)

city: Kiel

first: Seyed Mohammd Mahdi

last: Rasa

state: Schleswig-Holstein

address: Michaelisstrasse 5

department: Institut for Immunology

country: Germany

institute: Universitätsklinikum Schleswig-Holstein

description:
control group and treated with IFNγ.
Single cell RNA sequencing

data_processing:
For single cell RNA sequencing: The fastq files are mapped against mm10.e87 genome following by counting the UMI and removing duplicates for single cells using 'cellranger count' (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/count ) with default parameters.
Genome_build: mm10.e87
Supplementary_files_format_and_content: Organoids_scRNAseq_combined2.rds: The processed data is a seurat object saved in RDS file with assigned meta data in trascriptome matrix of intesine

extract_protocol:
Small intestinal crypts were isolated using the established protocol (Sato and Clevers, 2013) with some modifications. Briefly, mouse small intestine was dissected, washed in cold PBS. The villi-free intestinal pieces (2 cm) were washed with cold PBS and transferred to 5 mM EDTA/PBS, followed by two 30-min incubations at 4°C on a rotator. The tissue was transferred to fresh cold PBS and manually shaken for 30 sec. The crypt solution was filtered using a 70 µm cell strainer and centrifuged at 450g for 5 min at 4°C. Isolated crypts were immediately used or snap frozen in liquid nitrogen and stored at -80°C for further experiments. For RNA isolation, the crypts were immediately resuspended in QIAzol Lysis Reagent (Qiagen) and stored at -80°C.  Crypts were resuspended in ice-cold Matrigel (Corning, Cat. 356231) and allowed to solidify at 37°C, then were supplied with culture medium (750ng/ml recombinant human Rspondin-1 (lab-made), 100ng/ml recombinant murine Noggin (PeproTech, Cat. 250-38), 50ng/ml recombinant murine EGF (PeproTech, Cat. 315-09), 10 μM CHIR99021 (Tocris, Cat. 4423/10), 100ng/ml recombinant murine Wnt-3a (PeproTech, Cat. 315-20), N2 (Thermo Fisher Scientific, Cat. 15410294), B27 (Thermo Fisher Scientific, Cat. 11500446), 1X Penicillin-Streptomycin (Thermo Fisher Scientific, Cat. 15140122) in Advanced DMEM/F12 (ThermoFisherScientific, Cat. 11550446)). Organoids were cultured for at least 2 weeks, passaged, then IFNgamma (2ng/ml) were added at day 3 and 24 hours later being collected for scRNA sequencing.
For single cell RNA sequencing: The 10X Single Cell 3’ v2 reagent kit (10X Genomics) was used for cell partitioning, cDNA amplification and library construction.

treatment_protocol: Organoids devried from old mice were treated with IFNγ 2ng/ml for 24 hours.

value: Epithelial cells

value: Organoids


title: 24h post-infection (hpi) uninduced [UI σ54 OE 3]

value: Uninduced


title: Aged_PDK4IN_01

description: PE150 high throughout data

value: wild type

value: Aged plus PDKIN


title: AML Patient 5, End of Induction

description: 10x Genomics

value: bone marrow

value: end of induction

value: Bone marrow blast and immune cells


title: Young_01

description: PE150 high throughout data

value: Pulmonary alveolus

value: Young


title: Young_02

description: PE150 high throughout data

value: Normal mouse primary lung fibroblasts

value: Stromal


title: 24h post-infection (hpi) induced [I σ28 OE 3]

value: 10 nM aTc @14hpi


title: 14746-7_KSC

data_processing:
Illumina bcl2fastq version 2.18.0.12 was used for basecalling
Raw sequence FASTQ reads were aligned using STAR version 2.7.6a using parameters --outReadsUnmapped Fastx --outSAMtype BAM SortedByCoordinate --outSAMattributes All
Transcript abundances were calculated using HTSeq-count version 0.12.3 using parameters -r pos -t exon -i gene_id -a 10 -s no -f bam
Normalization and differential expression was done by DESeq2 version 1.28.1 following their default parameters in their vignette
Genome_build: Gencode GRCm38 p4 Release M11
Supplementary_files_format_and_content: tab-delimited Excel file containing raw counts for each gene and sample

source_name: Kras+ST6Gal1-expressing pancreas #3

extract_protocol: RNA was harvested from the freshly-excised pancreata of 20 week-old genetically-engineered mouse models.

value: pancreas


title: AML Patient 5, Diagnosis

description: 10x Genomics

value: diagnosis

value: AML, NOS


title: 14758-70_KC

source_name: Kras-expressing pancreas #3


title: AML Patient 4, Diagnosis

description: 10x Genomics


title: 14513_7_KC

source_name: Kras-expressing pancreas #2


title: 24h post-infection (hpi) uninduced [UI σ28 OE 3]

value: C. trachomatis L2 434/Bu

value: Overexpression


title: 24h post-infection (hpi) induced [I σ66 OE 3]


title: AML Patient 3, End of Induction

description: 10x Genomics


title: AML Patient 9, Diagnosis

description: 10x Genomics


title: 24h post-infection (hpi) induced [I σ28/σ54 OE 2]

value: pBOMBL-σ28_FLAG + σ54_6xH


title: 14513_4_KC

source_name: Kras-expressing pancreas #1


title: 24h post-infection (hpi) uninduced [UI σ28/σ54 OE 2]

value: Uninduced


title: 14511_7_ksc

source_name: Kras+ST6Gal1-expressing pancreas #2


title: E14_IgG-CT_scRNA-seq

first: Kiyofumi

last: Hamashima

institute: Institute of Molecular and Cell Biology (IMCB)

description: 10x Genomics

value: Mouse embryonic stem cells


title: AML Patient 2, Diagnosis

description: 10x Genomics

value: AML with myelodysplasia-related changes


title: 24h post-infection (hpi) induced [I σ66 OE 2]


title: 14510_4_WT

source_name: WT control pancreas #3


title: AML Patient 1, Diagnosis

description: 10x Genomics

value: bone marrow


title: 24h post-infection (hpi) uninduced [UI σ66 OE 2]


title: 14509_60_WT

source_name: WT control pancreas #2


title: MEF_100K_Input2

data_processing:
Adapter sequences of paired-end raw reads were trimmed using Trim Galore with parameters -q 30.
Trimmed reads were aligned to the hg19 & mm9 genome assembly using STAR aligner version 2.7.2 with the following parameters: --outFilterMatchNminOverLread 0 --outFilterScoreMinOverLread 0 --outFilterMatchNmin 30  --outFilterMismatchNmax 3 --outFilterMultimapNmax 1.
Peaks were called using Macs2 with parameters -f BAM -B -q 0.01 --nomodel --extsize 100 --keep-dup all and called peaks with log-q-value > 10 were used in downstream analyses.
Assembly: hg19 and mm9
Supplementary files format and content: Bed files were narrowPeak files generated by Macs2.

extract_protocol: For bulk m6A-CT library preparation, E14, EPSC, MEF and HEK293T nuclei were isolated with the following conditions: 1 min on ice in 0.020 % Triton X-100 for E14, 0.015 % Triton X-100 for EPSC; 5 min on ice in 0.2 % NP-40 for MEF, 0.02 % NP-40 for HEK293T. Aliquots of nuclei (5,000–100,000 nuclei) were bound to pre-activated Concanavalin A (Con-A) Conjugated Paramagnetic Beads (EpiCypher, #21-1411) and used in assay. All wash steps were performed on a magnetic separation rack. Undirected cDNA/RNA hybrid tagmentation for INPUT RNA was carried out as described previously. Briefly, nuclei were resuspended in 50 μL Reverse Transcription & Tagmentation Buffer, and incubated for 1 hour at 37 °C. 2.5 μL pAG-Tn5 was then added and vortexed briefly, and incubated for 1 hour at 37 °C.  Antibody directed tagmentation for m6A-CT was carried out as  same as sc-m6A-CT using 1 μg anti-m6A antibody (Synaptic Systems, #202003). When tagmentation was done, 54.5 μL of 0.5 M EDTA, 1 μL of 20 mg/mL Proteinase K and 5.5 μL of 10% SDS were sequentially added to the sample and incubated at 58 °C for 1 hour. An equal amount of Phenol-chloroform-isoamyl alcohol 25:24:1 (PCI), pH 6.7 was added to each sample, and PCI extraction followed by ethanol precipitation were performed. To perform gap-filling, the purified RNA/DNA samples were mixed with 0.32 U/μL Bst 3.0 DNA Polymerase (New England Biolabs) and Q5 High-Fidelilty 2× PCR Master mix (New England Biolabs), and incubated for 5 min at 58 °C, 5 min at 72 °C, and then at 5 min at 80 °C to inactivate Bst 3.0. A qPCR cycle check can be performed at this stage using 1:100 templates. A successful experiment can be identified by comparing cycle threshold values to IgG-CT. A universal i5 and a uniquely barcoded i7 primer, and Q5 HiFi 2× PCR Master mix were used to amplify libraries with the following cycling conditions: 45 sec at 98 °C, followed by 14-21 cycles of 15 sec at 98 °C and 10 sec at 60 °C, with a final extension at 1 min at 72 °C. Post-PCR clean-up was performed by 0.5× right-side followed by 1.2× left-side SPRI beads (Beckman Counter) selection.

value: Mouse embryonic fibroblast

tag: replicate


title: MEF_100K_Input1


title: 24h post-infection (hpi) induced [I σ54 OE 2]


title: 14501_9_SC

source_name: ST6Gal1-expressing pancreas #3


title: HIV113_V1

city: Aarhus

first: Lamin

middle: B

last: Cham

address: Skybe

department: Infectious Disease

country: Denmark

institute: Aarhus University Hospital

description: 10x Genomics

data_processing:
For the preprocessing, individual raw scRNAseq fastq files form each sample were aligned against the human reference genome (GRCh38) through the cell ranger count pipeline (Cell Ranger version 3.1.0, 10x Genomics Technology), which generated the cell-genes count matrices. For data clean up, quality control and analysis, we used R studio and the Seurat package. We performed a clean-up and quality control based on cellular expression of mitochondrial genes (cells with a percentage of mitochondrial genes >25% were discarded). To be considered for further analysis, genes had to be expressed in more than ten cells, cellular barcodes had to be associated to at least 200 genes. We next identified doublets using the DoubletFinder algorithm (version?) and removed these cellular barcodes from the analysis. Each singlet file for each sample was saved and later integrated for further analysis.
Supplementary files format and content: To perform comparison between samples from controls and PWH before and during TLR9 agonist treatment, we integrated the whole expression matrices from different samples using the Harmony algorithm. Using the ‘FindIntegrationAnchors’ Seurat function, we first integrated individual singlet data from HIV baseline and healthy controls as shown in Figure 1 and then integrated data from PWH at baseline and during TLR9 agonist treatment as shown in Figure 2. After log-normalizing and scaling the integrated data, principal component analysis (PCA) was used to reduce the dimensionality of the data set and PCA values were then used for graph-based clustering and for visualization through UMAP. Cells from each integrated file were manually annotated based on their canonical gene expression signature specific to each cell type. Dot plots were used to show the average expression and percentage expression of each gene to specific cell type as shown in the result section.

source_name: PBMC

extract_protocol:
To understand how the transcriptome of pDCs is impacted by HIV-1 infection and exogenous stimulation, we sorted pDCs from people with HIV-1 (PWH) on antiretroviral treatment before and after toll-like receptor 9 (TLR9) agonist treatment, as well as from healthy controls, and performed single-cell (sc)-RNA sequencing.
Briefly after pDCs were isolated, cells and reagents were prepared and loaded into the chip and ran into the Chromium Controller for Gel Bead-In Emulsion (GEM) generation and barcoding. The input number of cells was estimated at 15-20,000 cells per sample. The Chromium Next GEM Single Cell 3’ Gel beads v3.1 kit (10X Genomics, Pleasanton, CA, USA) was used to create GEMs following manufacturer’s instruction. All GEMs generated were used for cDNA synthesis and library preparation using the Chromium Single Cell 3’ Library Kit v3.1 (10X Genomics) following the manufacturer’s instruction. scRNA-seq libraries were then prepared using GemCode Single Cell 3’ Gel bead and library kit (10X Genomics) following the manufacturer’s instruction. cDNA concentration of each sample was measured using a Tapestation 2200 system (Agilent).  Single-cell barcoded cDNA libraries were sequenced on an Illumina Illumina Novaseq6000 system (100-cycle cartridge) with a sequencing depth of at least 50,000 reads per cell.

value: PBMC


title: 14500_7_SC

first: Susan

last: Bellis

institute: University of Alabama at Birmingham

source_name: ST6Gal1-expressing pancreas #2


title: 24h post-infection (hpi) uninduced [UI σ54 OE 2]


title: MEF_100K_m6A-CT2

city: Singapore

address: 61 Biopolis Drive

value: m6A (Synaptic systems, #202003)


title: MEF_100K_m6A-CT1


title: HIV113_V12

description: 10x Genomics


title: E14_m6A-CT_scCUT&Tag

description: 10x Genomics

data_processing:
library strategy: scCUT&Tag
The demultiplexing, barcoded processing and gene counting were made using the Cell Ranger software cellranger-arc-2.0.2 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

value: m6A (MilliporeSigma, #ABE572)


title: E14_IgG-CT_scCUT&Tag

description: 10x Genomics

value: IgG


title: 14500_70_wt

source_name: WT control pancreas #1


title: AML Patient 3, Diagnosis

description: 10x Genomics

value: Bone marrow blast and immune cells


title: EPSC_100K_Input2

value: Expanded potential stem cells


title: E14_m6A-CT_scRNA-seq

description: 10x Genomics


title: 14500_60_ksc

source_name: Kras+ST6Gal1-expressing pancreas #1

value: pancreas


title: 24h post-infection (hpi) uninduced [UI σ28 OE 2]


title: 24h post-infection (hpi) induced [I σ28 OE 2]

value: 10 nM aTc @14hpi


title: HIV112_V1

description: 10x Genomics


title: EPSC_100K_m6A-CT2


title: 24h post-infection (hpi) uninduced [UI σ28/σ54 OE 1]


title: 14500_4_SC

source_name: ST6Gal1-expressing pancreas #1


title: HIV109_V1

description: 10x Genomics


title: 24h post-infection (hpi) induced [I σ28/σ54 OE 1]


title: HIV109_V12

description: 10x Genomics


title: 24h post-infection (hpi) induced [I σ66 OE 1]


title: HIV103_V1

description: 10x Genomics


title: E14_5K_Input2


title: EPSC_100K_m6A-CT1


title: Small_intestinal_crypts_Old (scRNA-seq)

description:
Single cell RNA sequencing
ExpressionMatrix.csv
Index_Cluster_Age.csv

data_processing:
The raw sequencing data was then processed with the ‘count’ command of the Cell Ranger software (v2.1.0, 10X Genomics) with the default options. The required reference was built with the ‘mkref’ command of Cellranger based on the murine genome mm10 as well as the gene annotation from Ensembl (v87) as input. The annotation was filtered with the ‘mkgtf’ command of Cell Ranger to include only protein-coding, lincRNA and antisense gene features (‘--attribute=gene_biotype:protein_coding --attribute=gene_biotype:lincRNA –attribute=gene_biotype:antisense’).
Genome_build: mm10.e87
Supplementary_files_format_and_content: ExpressionMatrix.csv is the expression matrix with genes in row (row names are gene names) and cells in columns (column names are cell index). The information about the cell indexes (index, cell population and sample) is in Index_Cluster_Age.csv. Both files are comma separated text files(csv).  CellIndex-1 refers to Old, CellIndex-2 refers to Young.

source_name: Small intestinal crypts

growth_protocol: 3 Young (2 months old) and 3 old (20 month) male wild-type C57BL6/J mice were group housed and maintained in a specific pathogen-free animal facility

extract_protocol:
Whole intestinal crypts were isolated as previously described (Sato and Clevers, 2013) from the proximal small intestine. After centrifugation, the crypts were re-suspended in 1ml of Single-Cell Isolation Solution (TrypLE supplemented with: 1mg/ml DNaseI, 5mM MgCl2, 80μM Y27) and incubated at 37°C for 20min, vortexing for 5 seconds after first 10min. Reaction was quenched by addition of 29ml ice-cold PBS and cells were centrifuged at 800g for 5minutes at 4°C. Cell pellet was re-suspended in FACS staining medium (FSM - PBS supplemented with 2% Fetal Bovine Serum, 80μM Y27, 3mM EDTA) and treated with CD326 (EpCAM) (G8.8), PE-Cyanine7 coupled rat monoclonal antibody for 30min on ice in the dark. The cells were then centrifuged at 450g for 5min at 4°C and re-suspended in 500μl fresh FSM. An input of 8000 EpCAM positive FACS sorted single cells was added to each 10X Chromium Controller channel with a recovery rate of roughly 3859 cells.
The 10X Single Cell 3’ v2 reagent kit (10X Genomics) was used for cell partitioning, cDNA amplification and library construction.
Single cell RNA sequencing

value: Small intestinal crypts

tag: age

value: Old

tag: gender

value: m


title: Small_intestinal_crypts_Young (scRNA-seq)

tag: strain

value: Young


title: 24h post-infection (hpi) uninduced [UI σ66 OE 1]

value: C. trachomatis L2 434/Bu

value: Uninduced

value: Overexpression


title: HIV103_V12

description: 10x Genomics


title: E14_5K_Input1

value: Mouse embryonic stem cells


title: crypt_m_2m_rep4 (total RNA-seq)

description: transcriptional profiling (RNA sequencing)

data_processing:
Fastq files quality check was performed using FastQC v0.11.5.
The fastq files were mapped to the mm9 genome using TopHat v2.1.0 with the following parameters --bowtie1 --no-coverage-search -a 5.
The number of reads covered by each gene is calculated by HTSeq-Count 0.11.2 with -s no -a 0 -t exon -m intersection-nonempty parameters.
Genome_build: mm9
Supplementary_files_format_and_content: Htseq-count text file

source_name: Small intestinal crypt

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

extract_protocol:
Small intestinal crypts were isolated using the established protocol (Sato and Clevers, 2013) with minor modifications. Briefly, mouse small intestine was dissected, washed in cold PBS and opened longitudinally. Villi were removed by scraping with a coverslip. The villi-free intestinal pieces (2 cm) were washed four times in cold PBS and transferred to 5 mM EDTA/PBS, followed by two 30-min incubations at 4°C on a rotator. The tissue was transferred to fresh cold PBS and manually shaken for 30 sec. The crypt solution was filtered using a 70 µm cell strainer and centrifuged at 120xg for 5 min at 4°C.Isolated crypts were aliquoted, snap freezed in liquid nitrogen and stored at -80°C for further experiments. For RNA isolation, the crypts were immediately resuspended in Qiazol Lysis Reagent (Qiagen) and stored at -80°C.  Total RNA from crypts was isolated using QIAzol Lysis reagent (Qiagen) followed by isopropanol precipitation.
Total ribo-depleted RNA-seq library preparation was performed as described previously (Neri et al., Nature 2017) . In brief, 50 to 500 ng of total RNA were depleted of ribosomal RNA using the Ribo-Zero™ Gold Kit H/M/R Kit (illumina) following manufacturer’s instructions. Ribo-depleted RNA was resuspended in 17 μl of EFP buffer (illumina), heated to 94°C for 8 min, and used as input for first strand synthesis, using the TruSeq™ RNA Library Preparation Kit v2 (illumina) following manufacturer’s instructions.
Total RNA sequencing

tag: tissue

value: Small intestinal crypt


title: HC66

description: 10x Genomics

value: PBMC


title: crypt_m_2m_rep3 (total RNA-seq)


title: E14_5K_m6A-CT2

value: m6A (Synaptic systems, #202003)


title: HIV112_V12

description: 10x Genomics


title: 24h post-infection (hpi) uninduced [UI σ54 OE 1]


title: EPSC_100K_Input1


title: HC65

description: 10x Genomics


title: 24h post-infection (hpi) induced [I σ28 OE 1]


title: crypt_m_2m_rep2 (total RNA-seq)


title: HC63

description: 10x Genomics


title: E14_5K_m6A-CT1


title: crypt_m_2m_rep1 (total RNA-seq)


title: 24h post-infection (hpi) uninduced [UI σ28 OE 1]


title: HC61

description: 10x Genomics


title: E14_25K_Input2


title: 24h post-infection (hpi) induced [I σ28/σ54 KD 3]

value: 10 nM aTc @4hpi

value: Knockdown - dCas12 mediated


title: E14_25K_m6A-CT2


title: crypt_m_26m_rep3 (total RNA-seq)


title: 24h post-infection (hpi) induced [I σ28/σ54 KD 2]


title: 24h post-infection (hpi) induced [I σ54 OE 1]


title: crypt_m_26m_rep2 (total RNA-seq)


title: BLEOPDK4IN_01

description:
PE150 high throughout data
BLEO_vs_BLEOPDK4IN.deseq2.annot

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to human whole genome (UCSC GRCh38/hg38) using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata.
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Assembly: GRCh38/hg38
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample ...
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample.

source_name: Human prostate

growth_protocol: Normal human stromal cells were routinely cultured in stromal complete media supplemented with 10% FBS, until a total number of approximately 1,000,000 was reached in each flask and subject to treatment by the chemotherapeutic agent bleomycin (BLEO) and/or PDK4-IN (an anthraquinone derivative, PDK4 inhibitor).

treatment_protocol: To induce senescence, bleomycin (BLEO) was added to the media at 50 μg/ml, and cells were treated for 12 h before media change. PDK4-IN was used at 5 μM throughout the culture period until cells were collected for lysis.

value: Human prostate

value: Normal human primary stromal cells

value: Stromal

value: wild type

tag: treatment

value: Bleomycin plus PDKIN


title: 24h post-infection (hpi) induced [I σ28/σ54 KD 1]

source_name: Infected McCoy cell line


title: crypt_m_26m_rep1 (total RNA-seq)


title: 24h post-infection (hpi) uninduced [UI σ28/σ54 KD 3]


title: BLEO_03

description:
PE150 high throughout data
CTRL_vs_BLEO.deseq2.annot

value: Bleomycin


title: crypt_m_18m_rep4 (total RNA-seq)

value: m


title: BLEO_02


title: E14_100K_Input2


title: E14_100K_Input1


title: crypt_m_26m_rep4 (total RNA-seq)


title: 24h post-infection (hpi) uninduced [UI σ28/σ54 KD 2]


title: BLEO_01


title: E14_25K_Input1


title: 24h post-infection (hpi) induced [I σ54 KD 3]

value: pL12CRia (σ54)


title: crypt_m_18m_rep3 (total RNA-seq)


title: BLEOPDK4IN_03


title: E14_100K_m6A-CT1


title: crypt_m_18m_rep1 (total RNA-seq)


title: HLI+BMT (tdT cells)

city: Beijing

first: Yuwei

last: Song

address: 38 Xueyuan Road

institute: Peking University

description:
10x Genomics
Mus_HLI[tdT]+HLI BMT[tdT]-barcodes.tsv.gz
Mus_HLI[tdT]+HLI BMT[tdT]-features.tsv.gz
Mus_HLI[tdT]+HLI BMT[tdT]-matrix.mtx.gz

data_processing:
Raw sequencing data were processed using Cell Ranger (version 3.1) to demultiplex raw data and generate FASTQ files, and the resulting data were aligned and counted with Cell Ranger pipelines.
Assembly: mm10-2020-A; GRCh38-2020-A
Supplementary files format and content: Tab-separated values files and matrix files

source_name: skeletal muscle

extract_protocol:
Hindlimb gastrocnemius muscles were removed at 14 days after femoral artery ligation or sham operation (10 mice pre group). A single-cell suspension was prepared from hindlimb muscles by mechanical and enzymatic dissociation with minor modifications. Briefly, hindlimb muscles were dissected and minced into a fragmented muscle suspension. The muscle suspension was digested in Ham’s F10 containing 400 U/ml collagenase II (Worthington) and 11 U/ml dispase (Life Technologies) at 37 ℃ with reciprocal shaking (60 rpm/min) for 1 h. Detached cells were then filtered through a 40 μm cell strainer (BD Biosciences), and red cells were removed by lysis buffer. Subsequently, the remaining cells were washed with prewarmed (37 ℃) RPMI 1640 medium containing 10% FBS by centrifugation at 500 g for 5 min and suspended in PBS. Human muscle tissues were stored in prechilled PBS. A single-cell suspension was prepared from lower limb muscles by mechanical and enzymatic dissociation with minor modifications. Briefly, the muscle was cut into small fragments, and visible fat and connective tissue were removed. Then, the samples were transferred to low-glucose Dulbecco’s modified Eagle’s medium (LG-DMEM) containing 0.1% collagenase D and digested in a 37 °C water bath with agitation (60–70 rpm) for 60 min. Detached cells were then filtered through a 70 μm cell strainer (BD Biosciences), and red cells were removed with lysis buffer. Subsequently, the remaining cells were washed with prewarmed (37 ℃) LG-DMEM containing 10% FBS by centrifugation at 500 g for 5 min and suspended in PBS for further experiments. Cells were then stained with a LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (1:1000) and Hoechst 33342 (Invitrogen, H3570, 1:1000) for 20 mins on ice. After being washed in PBS, cells were re-suspended in PBS. Single live cells (Hoechst+/Dead Cell Stain-) were sorted into PBS with 0.04% BSA using a BD FACS SymphonyS6 (BD Biosciences). In addition, we sorted tdTomato+ cells by the same method.
A Chromium Single Cell 3’ Reagent Kit v3 was used, and Standard 10X Chromium Single Cell 3’ v3 (10X Genomics GemCode Technology) protocols were followed for scRNA-seq. Briefly, single cells with specific 10X barcodes and unique molecular identifiers (UMIs) were generated by partitioning the cells into Gel Bead-in-Emulsions. A cDNA library was generated and sequenced with a Novaseq (S4) 6000 instrument (PE150).

value: muscle mononuclear single cells

value: skeletal muscle

value: Cd34-CreERT2;R26-tdTomato (C57BL/6J)

value: 10-15 weeks old


title: 24h post-infection (hpi) induced [I σ54 KD 2]

value: C. trachomatis L2 434/Bu


title: CTRL_02


title: E14_25K_m6A-CT1

value: Mouse embryonic stem cells


title: BLEOPDK4IN_02


title: crypt_f_2m_rep4 (total RNA-seq)

value: Small intestinal crypt

value: f


title: HLI (tdT cells)


title: 24h post-infection (hpi) induced [I σ54 KD 1]

value: 10 nM aTc @4hpi

value: Knockdown - dCas12 mediated


title: crypt_f_2m_rep3 (total RNA-seq)


title: HEK_100K_Input2

value: Human embryonic kidney


title: E14_100K_m6A-CT2

value: m6A (Synaptic systems, #202003)


title: HLI

description:
10x Genomics
Mus_Sham+HLI-barcodes.tsv.gz
Mus_Sham+HLI-features.tsv.gz
Mus_Sham+HLI-matrix.mtx.gz


title: Sham


title: 24h post-infection (hpi) uninduced [UI σ28/σ54 KD 1]

value: Uninduced


title: crypt_f_2m_rep2 (total RNA-seq)


title: crypt_m_18m_rep2 (total RNA-seq)


title: 24h post-infection (hpi) uninduced [UI σ54 KD 2]


title: CTRL_03

value: Human prostate

value: Normal human primary stromal cells

value: Stromal

value: wild type


title: crypt_f_2m_rep1 (total RNA-seq)


title: HEK_100K_m6A-CT2


title: 24h post-infection (hpi) uninduced [UI σ54 KD 3]

value: pL12CRia (σ54)


title: crypt_f_26m_rep4 (total RNA-seq)


title: Intestinal stem cells, ISX_ATAC_rep3

first: Wei

address: Tsinghua Road, Medical Building

institute: Tsinghua University

data_processing:
For bulk RNA-seq analysis, raw reads were trimmed for base call quality and for adapter sequences using Trim Galore! (v0.6.4_dev) and aligned to the mouse genome (Mus_musculus.GRCm38) using HISAT2 (v2-2.1.0), following quantified using HTSeq (v0.12.4) and differential expression analysis using DESeq2 (v1.24.0).
For ATAC-seq analysis, raw reads were trimmed for base call quality and for adapter sequences using Trim Galore! (v0.6.4_dev) and aligned to the mouse genome (Mus_musculus.GRCm38) using bowtie2 (v2.3.5.1). All mapped reads were shifted by +4 bp for the +strand and -5 bp for the -strand. Peaks were called for each sample using MACS2 (v 2.1.1.20160309) with parameters “-p 0.1 -nomodel -shift 100 -extsize 200 -B -SPMR”, and differential peaks were identified using DiffBind (v3.2.2). ChIPseeker (v1.28.3) was used to annotate accessible genomic regions.
For single cell RNA-seq analysis, the raw data were performed sample demultiplexing, barcode processing and single-cell 3′ gene counting with CellRanger software (v3.1.0) (10x Genomics). Mus_musculus GRCm38 genome with Ensemble transcriptome annotation was used as reference. Most subsequent analysis was performed with the Seurat R Package (v3.1.5). Cells with >1,200 UMIs and <7,000 UMIs were kept for downstream analysis. Cells with high percentage of mitochondrial reads (>15%) were considered as apoptotic and excluded as well. The cells were batch corrected using the Integrate function of Seurat.
Genome_build: Mus_musculus GRCm38
Supplementary_files_format_and_content: For bulk RNA-seq, raw gene counts of every gene and every sample were provided. In addition, differentially expressed gene list (including gene name, fold change, p value) generated using DESeq2 were in supplementary file.
Supplementary_files_format_and_content: For ATAC-seq, bigwig file were provided, scores represent signal intensity.
Supplementary_files_format_and_content: For single cell RNA-seq data, txt files for every sample were provided, the matrix containing gene counts of every gene in every barcoded single cells.

source_name: intestinal stem cells sorted from organoids

growth_protocol: mouse intestinal organoids were cultured in ENR medium: Advanced DMEM/F12 (Thermo Scientific) supplemented with N2 (Thermo Scientific), B27 (Thermo Scientific), HEPES (Thermo Scientific), GlutaMax (Thermo Scientific), Penicillin/Streptomycin (Thermo Scientific), N-acetylcysteine (1.25 mM, Sigma Aldrich), EGF (50 ng/mL, Peprotech), Noggin (100 ng/mL, R&D), and R-spondin1 (500 ng/mL, made in-house).

extract_protocol:
RNA extraction was performed using RNeasy Mini Kit (Qiagen) according to manufacturer’s instruction.
cDNA library for bulk RNA seq was constructed using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB). Sc-RNAseq library construction was prepared following standard 10x genomics protocols. The ATAC-seq libraries were prepared following Omni-ATAC protocol (Corces et al., 2017).

treatment_protocol: oranoids were treated with 0.05% DMSO or 25 μM Isx-9 for indicated period of time, starting at day 2 post passaging

value: Lgr5-EGFP knock-in

value: Isx-9 for 6 hours


title: Intestinal stem cells, ISX_ATAC_rep2

value: intestinal stem cells sorted from organoids


title: crypt_f_26m_rep3 (total RNA-seq)


title: Intestinal stem cells, ISX_ATAC_rep1


title: HEK_100K_Input1


title: 24h post-infection (hpi) induced [I σ28 KD 2]

value: C. trachomatis L2 434/Bu

value: pL12CRia (σ28)


title: PAD

description:
10x Genomics
Homo_PAD-barcodes.tsv.gz
Homo_PAD-features.tsv.gz
Homo_PAD-matrix.mtx.gz

value: muscle mononuclear single cells

value: skeletal muscle

tag: patient

value: A PAD amputation patient

value: 85 years old


title: 24h post-infection (hpi) induced [I σ28 KD 3]

value: 10 nM aTc @4hpi

value: Knockdown - dCas12 mediated


title: CTRL_01