title: experimental B: Liver specific Cpt2 KO maternal genotype WT fetal mouse liver, Rep_4

city: Baltimore

first: Michael

middle: J.

last: Wolfgang

state: MD

address: 855 N Wolfe Street

department: Institute for Basic Biomedical Sciences

institute: The Johns Hopkins University School of Medicine

description:
CF_2968-3
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt

data_processing:
Single-end 75nt sequencing on Illumina NextSeq 500.
Base calling with RTA version 2.4.11.
Data filtering to strip adapters with bcl2fastq 2.17.1.14.
Read alignment to Mus musculus sequence GRCm38.p6 Apr-'18, with CLCGenomicsServer 10.0.1 with: Mismatch cost =2, Insertion cost = 3, Deletion cost = 3, Length fraction = 0.8, Similarity fraction = 0.8.
FPKM calculation with CLCGenomicsServer 10.0.1 as above.
FPKM values log2 converted (FPKM = 0 ignored) and quantile normalized with Partek Genomics Suite v7.0.
Genome_build: Mouse NCBI build GRCm38.p6 (RefSeq)
Supplementary_files_format_and_content: MWol032218_LogQuant-Signals.txt: Single tab-delimited text file of quantile normalized log2 FPKM values for all samples.

source_name: E17.5 Fetal Liver

growth_protocol: E17.5 fetal livers were collected from timed pregnant mice that were fasted for 24hrs.

extract_protocol:
Trizol followed by QIAGEN column clean up.
RNA libraries were prepared for sequencing using standard Illumina TruSeq Stranded mRNA protocols for 1x75bp.

treatment_protocol: None, samples were snap-frozen.

tag: strain/background

tag: maternal genotype

value: Cpt2f/f; Albumin-Cre

tag: nutrition

value: 24hr fasting

tag: fetal genotype

value: Fetal liver

tag: fetal developmental stage

tag: fetal sex

value: Female


title: experimental B: Liver specific Cpt2 KO maternal genotype WT fetal mouse liver, Rep_3

status: Public on Sep 20 2019

type: SRA

country: USA

description:
CF_2966-3
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt

library_source: transcriptomic

molecule: total RNA

organism: Mus musculus

tag: tissue

value: Male


title: experimental B: Liver specific Cpt2 KO maternal genotype WT fetal mouse liver, Rep_2

description:
CF_4537-2
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt


title: Tree_19_Tolf_T0

city: Gainesville

first: Leandro

middle: Octavio

last: Balzano

state: FL

address: 1275 Center Drive

department: Pathology, Immunology, and Laboratory Medicine

institute: University of Florida

data_processing:
Alignment of reads  to the Clementina genome with TopHat2 (Kim et al. 2013)
Count of gene expression with HTSEQ-count (Anders et al. 2015)
Genes with less than 10 reads collectively for all samples were discarded
Gene expression values were normalized using the TMM method (Robinson and Oshlack, 2010)
Batch effects were removed witrh ARSyNseq (Tarazona et al. 2011).
Genome_build: ncbi_gca_000493195_1_citrus_clementina_v1_0
Supplementary_files_format_and_content: csv, gene expression counts

organism: Citrus sinensis

source_name: RNA-seq

growth_protocol: ‘Valencia’ sweet orange trees (Citrus sinensis [L.] Osbeck), ranging in age from twenty to thirty years old. Standard grove care and maintenance was performed in each grove throughout the field trials, which included irrigation, fertilization and pest management.

extract_protocol:
Fifty leaves were collected randomly from the canopy of each tree. Following collection, the midrib and petiole of each leaf was excised and immediately submerged in RNAlater. Prior to RNA extraction, the tissue samples were freeze (-80°C), dried and homogenized.  25 mg of each powdered leaf was used. Total RNA was extracted with Isolate II RNA Plant Kit (Bioline, Taunton, MA). RNA integration number (RIN) was measured using BioAnalyzer (Agilent Technologies) RNA 6000 Nano Kit.
TruSeq Stranded total RNA with Ribo-Zero Plan Kit (Illumina)

treatment_protocol: Three group of infected trees each were established: control plants, plants treated with benzbromarone and plants treated with tolfenamic acid. RNA was obtained two months before treatment (T0) and 2 weeks after treatment (T2).

tag: cultivar

value: Valencia

tag: infected with

value: Liberibacter asiaticus

value: leaf midrib and petiole

tag: tree number

tag: treatment

value: Tolfenamic acid

tag: treatment time


title: Tree_20_Tolf_T0


title: Tree_06_Benz_T0

value: Benzbromarone


title: control B: WT maternal genotype WT fetal mouse liver, Rep_4

description:
FF_4166-3
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt


title: control B: WT maternal genotype WT fetal mouse liver, Rep_2

description:
FF_4134-5
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt


title: experimental: Ppar-alpha KO maternal genotype Ppar-alpha KO fetal mouse liver, Rep_4

description:
NN_4796-2
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt

value: Ppar-alpha Knockout


title: experimental B: Liver specific Cpt2 KO maternal genotype WT fetal mouse liver, Rep_1

description:
CF_2969-1
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt


title: control B: WT maternal genotype WT fetal mouse liver, Rep_1

description:
FF_2849-5
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt


title: experimental: Ppar-alpha KO maternal genotype Ppar-alpha KO fetal mouse liver, Rep_2

description:
NN_4476-2
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt


title: experimental: Ppar-alpha KO maternal genotype Ppar-alpha Het fetal mouse liver, Rep_3

description:
NH_113-2
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt

value: Ppar-alpha Heterozygote


title: control: Ppar-alpha Het maternal genotype, Ppar-alpha Het fetal mouse liver, Rep_4

description:
HH_585-1
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt


title: experimental: Ppar-alpha KO maternal genotype Ppar-alpha Het fetal mouse liver, Rep_4

description:
NH_114-2
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt


title: experimental: Ppar-alpha KO maternal genotype Ppar-alpha KO fetal mouse liver, Rep_3

description:
NN_4540-5
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt


title: experimental: Ppar-alpha KO maternal genotype Ppar-alpha Het fetal mouse liver, Rep_2

description:
NH_179-3
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt


title: control: Ppar-alpha Het maternal genotype, Ppar-alpha Het fetal mouse liver, Rep_3

description:
HH_306-5
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt


title: control: Ppar-alpha Het maternal genotype, Ppar-alpha Het fetal mouse liver, Rep_1

description:
HH_304-3
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt


title: control: Ppar-alpha Het maternal genotype, Ppar-alpha Het fetal mouse liver, Rep_2

description:
HH_305-4
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt


title: experimental: Ppar-alpha KO maternal genotype Ppar-alpha Het fetal mouse liver, Rep_1

description:
NH_179-1
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt


title: Tree_01_Tolf_T0


title: MTECs_ALI3

city: Valbonne - Sophia-Antipolis

first: Kevin

last: Lebrigand

address: 660 route des lucioles

country: France

institute: IPMC/CNRS

description: Sample was prepared 22 days after air-liquid interface. MTECs were cultivated as described in Revinsky et al. (Nature Communications, 2018). Cells were incubated with 0.10% protease type XIV from Streptomyces griseus in HBSS for 4 hours at 4°C degrees. Cells were  detached from the Transwells by pipetting and then transferred to a microtube. 50 units of DNase I per 250µl, were directly added and cells were further incubated at room temperature for 10 min. Cells were washed in 500 µL HBSS 10% Fetal Bovine Serum then in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a Scienceware® Flowmi™ Cell Strainer (40µm porosity), then centrifuged (150g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed and 5000 cells were used for single-cell sequencing.

data_processing:
The Cell Ranger Single Cell Software Suite 2.0 was used to perform sample demultiplexing, barcode processing, and single cell 3’ gene counting (https://support.10xgenomics.com/single-cell/software/pipelines/latest/what-is-cell-ranger).
Cell Ranger command used to produce output directory for each sample was executed using transcriptome based on human genome release hg19, mouse genome mm10 and pig genome Sscrofa11.2
Supplementary_files_format_and_content: Cellranger raw gene / barcode hdf5 matrix file.

source_name: Mouse tracheal epithelial cells differentiated for 3 days

extract_protocol:
Samples were run on the Chromium machine from 10x Genomics following the  User Guide for v2 Chemistry (https://support.10xgenomics.com/single-cell/sample-prep/doc/user-guide-chromium-single-cell-3-reagent-kit-v2-chemistry).
Single cell RNA-Seq libraries were made following the User Guide for v2 Chemistry (https://support.10xgenomics.com/single-cell/sample-prep/doc/user-guide-chromium-single-cell-3-reagent-kit-v2-chemistry). Library was sequenced as follows: 26bp Read 1 (16bp Chromium barcode and 10bp UMI), 57bp Read 2 (transcript), and 8bp i7 sample barcode for each scRNA-Seq library.

tag: cell type

value: airway epithelial cells  (MTECs)

tag: differentiation day

tag: cell origin

value: Primary culture

tag: donor


title: HEK sgNT CEM87 RNA

city: Bethesda

first: Javed

last: Khan

address: 37 Convent Dr.

department: Genetics Branch

institute: NCI, NIH

data_processing:
RNA-seq reads were mapped to UCSC reference for hg19 using STAR
RNA-seq read abundance for each gene counted (TPM method) using RSEM
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: RNA-seq processed files are quantification matrices of TPM (transcripts per million) at the gene level.

molecule: polyA RNA

organism: Homo sapiens

source_name: 293 HEK

growth_protocol: Cell lines were grown in high-glucose DMEM supplemented with 10% FBS serum, 10 mM HEPES, NEAA, Pen/Strep, and 55 μM 2-Mercaptoethanol.

extract_protocol:
RNA extraction was performed with a RNeasy Plus Mini Kit (Qiagen, 74134).
Standard Illumina barcodes were introduced during library preparation.

treatment_protocol: (dCas9 & ms2-FKBPx2)-infected cells were grown to 70% confluence, transfected with sgRNA using a PEI protocol. Media was changed 16 hours later, and cells were treated with 50 nM of CEM87 for 48 hours.

tag: cell line

value: HEK 293

value: 50nM CEM87


title: HEK sgMYOD cntrl RNA

value: 0nM CEM87


title: HEK sgNT cntrl RNA


title: HEK sgMYOD CEM87 input

library_source: genomic

data_processing:
ChIP enriched DNA reads were mapped to reference genome using BWA version 0.7.10
The ChIP-seq peaks were called by MACS2 version 2.1.0.
Enrichment of known and de novo motifs were found using HOMER version 4.8.2
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: Peak files are tab-delimited text files for each sample.

molecule: genomic DNA

extract_protocol:
DNA was extracted from ChIP-seq samples prepared following the Active Motif ChIP-IT High Sensitivity kit protocol.
Standard Illumina barcodes were introduced during library preparation.

tag: antibody

value: none (input)

tag: enrichment target

value: input

tag: target function

value: control


title: HAECs_Pneumacult_ALI28_D389

description: Sample was prepared 28 days after air-liquid interface in Pneumacult medium. HAECs were cultivated as described in Revinsky et al. (Nature Communications, 2018). Cells were incubated with 0.10% protease type XIV from Streptomyces griseus in HBSS for 4 hours at 4°C degrees. Cells were  detached from the Transwells by pipetting and then transferred to a microtube. 50 units of DNase I per 250µl, were directly added and cells were further incubated at room temperature for 10 min. Cells were washed in 500 µL HBSS 10% Fetal Bovine Serum then in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a Scienceware® Flowmi™ Cell Strainer (40µm porosity), then centrifuged (150g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed and 5000 cells were used for single-cell sequencing.

data_processing:
The Cell Ranger Single Cell Software Suite 1.3 was used to perform sample demultiplexing, barcode processing, and single cell 3’ gene counting (https://support.10xgenomics.com/single-cell/software/pipelines/latest/what-is-cell-ranger).
Cell Ranger command used to produce output directory for each sample was executed using --cells=1000 and --transcriptome based on human genome release.GRCh37.p13
Cell Ranger aggregated dataset has been produced with default normalization method based on mapped reads.
BAM: cellranger possorted_genome alignment bam file
Genome_build: hg19
Supplementary_files_format_and_content: HDF5: cellranger filtered gene/barcode matrix.

source_name: Human airway epithellial cells differentiated in Pneumacult medium for 12 days

value: airway epithelial cells  (HAECs)


title: control B: WT maternal genotype WT fetal mouse liver, Rep_3

description:
FF_4166-2
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt

tag: strain/background

tag: maternal genotype

tag: nutrition

value: 24hr fasting

tag: fetal genotype

value: Fetal liver

value: Male


title: experimental: Ppar-alpha KO maternal genotype Ppar-alpha KO fetal mouse liver, Rep_1

description:
NN_4476-1
Total mRNA
processed data file: MWol032218_LogQuant-Signals.txt

tag: tissue

tag: fetal developmental stage

tag: fetal sex


title: HAECs_Pneumacult_ALI26_D395

description: Sample was prepared 31 days after air-liquid interface in Pneumacult medium. HAECs were cultivated as described in Revinsky et al. (Nature Communications, 2018). Cells were incubated with 0.10% protease type XIV from Streptomyces griseus in HBSS for 4 hours at 4°C degrees. Cells were  detached from the Transwells by pipetting and then transferred to a microtube. 50 units of DNase I per 250µl, were directly added and cells were further incubated at room temperature for 10 min. Cells were washed in 500 µL HBSS 10% Fetal Bovine Serum then in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a Scienceware® Flowmi™ Cell Strainer (40µm porosity), then centrifuged (150g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed and 5000 cells were used for single-cell sequencing.

source_name: Human airway epithellial cells differentiated in Pneumacult medium for 7 days


title: HAECs_BEGM_ALI31_D395

description: Sample was prepared 31 days after air-liquid interface in BEGM medium. HAECs were cultivated as described in Revinsky et al. (Nature Communications, 2018). Cells were incubated with 0.10% protease type XIV from Streptomyces griseus in HBSS for 4 hours at 4°C degrees. Cells were  detached from the Transwells by pipetting and then transferred to a microtube. 50 units of DNase I per 250µl, were directly added and cells were further incubated at room temperature for 10 min. Cells were washed in 500 µL HBSS 10% Fetal Bovine Serum then in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a Scienceware® Flowmi™ Cell Strainer (40µm porosity), then centrifuged (150g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed and 1500 cells were used for single-cell sequencing.

source_name: Human airway epithellial cells differentiated in BEGM medium for 2 days


title: HEK sgMYOD CEM87 RNA

tag: treatment


title: WT_sysy202111 rep2

city: Shenzhen

first: Wee Siong Sho

last: Goh

state: Guangdong

address: Shenzhen Bay Laboratory, No. 5, Kelian Road, Goh Lab, A1819, Guangming District

department: Institute of Molecular Physiology

country: China

institute: Shenzhen Bay Laboratory

data_processing:
Library strategy: m6ACE-sesq
Basecalls performed using FASTQ Generation v1.0.0.Fastq sequences were first trimmed of 5’ and 3’ adapter sequences and poly(A) tails using Cutadapt. The 8-mer ‘N7B’ (N=A/C/G/T, B=C/G/T) UMI located at the first 8 nucleotides of read 1 was registered and trimmed. Any complementary UMI sequence in read 2 was also trimmed. Reads were mapped to the methylated spike-in using Bowtie2, or to the hg38 assembly transcriptome (Gencode v28 comprehensive gene annotations) using STAR. Aligned pairs that had the same mapping coordinates and UMIs were filtered out as PCR duplicates. Read-start coordinates in hg38-mapped reads that began with an adenosine nucleotide, and had a minimum mean read count of 1 across the triplicate samples were collated. m6A or m6Am sites were identified as read starts that were at least 2-fold enriched in m6ACE libraries than in the corresponding input libraries. This enrichment was calculated using DESeq2 performed on A-only sites across triplicate pairs of m6ACE and corresponding input libraries (FDR<0.1, padj<0.05). Based on read-start patterns observed from m6ACE-seq of methylated spike-ins, we considered identified “sites” that were 1-4 nucleotides upstream of another identified significant Rm6AC site or “sites” found within clustered read-starts to be m6ACE-seq false-positives and filtered them out. To identify m6A or m6Am sites that were differentially methylated between sample conditions, we calculated the RML of each site in each sample: The read-start counts at positions -4 to 0 of each site in the m6ACE library were summed and divided by the read-start counts at positions -51 to 0 of the same site in the input library to give ‘X’. Similarly, the read-start counts at positions -4 to 0 of the spike-in m6A site in the m6ACE library were summed and divided by the read-start counts at positions -21 to 0 of the same spike-in m6A site in the input library to give ‘Y’. X was normalized to Y to give RML. RML values of each site was averaged across triplicates for each sample condition. A site was denoted as differentially methylated if the average RML differs between sample conditions with a log2fold-change (LFC) cutoff of 2.0 (for methylase-KO or demethylase-OE induced RML reduction) or 1.0 (for demethylase-KO induced RML accumulation), as well as a one-tailed T-test p-value cutoff of <0.05.
Genome_build: hg38
Supplementary_files_format_and_content: Bedgraph files of first nucleotide coordinates of forward reads. Text files comparing RML of WT versus gene-KO/KD samples.

source_name: Human embryonic kidney cells

growth_protocol: ATCC HEK293T CRL-3216 cells were cultivated in a sterile 5% CO2 incubator at 37oC, and in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin, and used within passage number 3-20.

extract_protocol:
Total RNA was isolated using Trizol-LS.
Poly(A) RNA was purified using Poly(A)Purist Mag kit (Thermofisher AM1922) according to manufacturer’s instructions. Poly(A) RNA was fragmented using RNA fragmentation buffer (Ambion AM8740) and dephosphorylated at its 3’ end with T4 PNK (NEB M0201) for 30min at 37oC. 5’ phosphorylation was then initiated by adding 1mM ATP and incubating for 30min at 37oC. 3’ ligation was then performed as previously described 2, where 5’-adenylated,3-dideoxyC DNA adapters were ligated with truncated T4 RNA ligase 2 (NEB M0242) in 1X ATP-free T4 RNA ligase buffer [50mM Tris pH 7.5, 60µg/ml BSA, 10mM MgCl2, 10mM DTT, 12.5% PEG8000] for 2hr at 25oC. 3’-ligated methylated RNA spike-in (Table S1) was added to ligated Poly(A) RNA and the mixture was denatured for 5min at 65oC before incubating for 2min on ice. This denatured RNA mixture was incubated overnight at 4oC with anti-m6A antibody (Synaptic Systems 202003) in 1X IP buffer [150mM NaCl, 10mM Tris pH 7.4, 0.1% IGEPAL CA-630 (Sigma I8896)] supplemented with RNasin Plus (Promega N2611). In parallel, Protein-A-dynabeads (Life Technologies 10002D) was blocked overnight at 4oC in 1X IP buffer supplemented with 0.5mg/ml BSA (Sigma A7906). The iced antibody-RNA mixture was crosslinked with 0.15J/cm2 254nm UV radiation six times. 1% of the antibody-RNA mixture was set aside as “input” and the rest (designated as “m6ACE”) was mixed with decanted BSA-blocked Protein-A-dynabeads for 1.5hr at 4oC. Beads bound with crosslinked RNA were then washed with the following cold buffers in this order: Wash buffer 1 [1M NaCl, 50mM HEPES-KOH pH 7.4, 1% Triton X-100, 0.1% Sodium Deoxycholate, 2mM EDTA], Wash buffer 2 [0.5M NaCl, 50mM HEPES-KOH pH 7.4, 1% IGEPAL, 0.1% Sodium Deoxycholate, 2mM EDTA], Wash buffer 3 [1% Sodium Deoxycholate, 25mM LiCl, 10mM Tris pH 8, 1% Triton X-100, 2mM EDTA], TE [10mM Tris pH 8, 1mM EDTA] and finally 10mM Tris pH 8. m6ACE RNA was subjected to 5’ to 3’ exonuclease treatment with XRN-1 (NEB M0338) for 1hr at 37oC. The m6ACE RNA-bead mixture was then washed with Wash buffer 1, Wash buffer 2, Wash buffer 3, TE and 10mM Tris pH 8. Both input and m6ACE RNAs were reverse crosslinked in elution buffer [1%SDS, 200mM NaCl, 25mM Tris pH 8, 2mM EDTA, 1mg/ml Proteinase K (Thermo Scientific EO0491)] for 1.5hr at 50oC. RNAs were ethanol-precipitated and ligated to 5’adapters (Table S1) with T4 RNA ligase (Ambion AM2140) for 16hr at 16oC. Reverse transcription primer (Table S1) was annealed (72oC 2min, ice 2min) and reverse transcription was performed with SuperscriptIII (Invitrogen 18080) for 1hr at 50oC, with the reaction stopped by incubating for 15min at 70oC. The cDNA was used for PCR amplification with Phusion High-fidelity PCR mastermix (NEB M0530) and Truseq PCR primers. Finally, primer-dimer and adapter-dimers were removed with AMpure XP beads before undergoing PE75 sequencing on the Illumina Nextseq platforms.

tag: genotype/variation

value: Wild type

tag: passage number

value: anti-M6A Synaptic Systems 202111


title: WT_sysy202111 rep2_input


title: WT_sysy202111 rep3


title: WT_abcam151230 rep3

value: anti-m6A Abcam 151230


title: WT_abcam151230 rep3_input


title: WT_sysy202111 rep3_input


title: WT_sysy202111 rep1


title: WT_abcam151230 rep2_input


title: A224_ITLN1_2

type: RNA

city: Houston

first: Chi Lam

last: Au Yeung

state: TX

address: 1515 Holcombe Blvd

institute: UT MD Anderson Cancer Center

description: Gene expression data from A224 cell line treated with recombinant human ITLN1 protein

hyb_protocol: Following fragmentation, 15 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Human Genome U133 Plus 2.0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.

scan_protocol: GeneChips were scanned using the GeneChip Scanner 3000 7G

data_processing: The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method.

label: biotin

source_name: Ovarian cancer A224 cells - ITLN1 treatment

label_protocol: Biotinylated cRNA were prepared using the MessageAmp Premier RNA Amplification Kit (Ambion) according to the manufacturer's instruction from 100ng total RNA.

growth_protocol: Cells were cultured in humidified incubator at 37C and 5% carbon dioxide. Culture medium RPMI1640 supplemented with 10% fetal bovine serum was used as growth medium.

extract_protocol: Total RNAs were isolated using TRI reagent (Molecular Research Center) according to the manufacturer's instructions.

treatment_protocol: A224 cells were cultured in 24-well plate overnight before treatment with PBS (Control) or 500 ng/mL recombinant human ITLN1 protein for 24 hours according to the treatment group.

tag: cell type

value: High grade serous ovarian cancer cell line


title: A224_Control_2

description: Gene expression data from A224 cell line treated with PBS (Control)

source_name: Ovarian cancer A224 cells - control

value: PBS


title: WT_abcam151230 rep2


title: WT_sysy202111 rep1_input


title: A224_Control_1


title: A224_ITLN1_1

value: 500 ng/mL recombinant human ITLN1 protein for 24 h


title: WT_abcam151230 rep1_input


title: WT_abcam151230 rep1


title: CAA6

description: Microdissected omental adipose tissue from patients with high grade serous ovarian cancer

source_name: Cancer-associated adipose tissue

tag: disease state

value: high grade serous ovarian cancer

value: Cancer-associated adipose tissue


title: CAA1


title: CAA4


title: CAA9


title: CAA5


title: CAA2


title: CAA8


title: NA4

description: Microdissected omental adipose tissue from patients with benign gynecologic diseases

source_name: Normal adipose tissue

value: benign gynecologic disease

value: Normal adipose tissue


title: CAA10


title: WT 0% Mettl3-KO 100% input rep3

data_processing:
Library strategy: m6ACE-seq
Basecalls performed using FASTQ Generation v1.0.0.Fastq sequences were first trimmed of 5’ and 3’ adapter sequences and poly(A) tails using Cutadapt. The 8-mer ‘N7B’ (N=A/C/G/T, B=C/G/T) UMI located at the first 8 nucleotides of read 1 was registered and trimmed. Any complementary UMI sequence in read 2 was also trimmed. Reads were mapped to the methylated spike-in using Bowtie2, or to the hg38 assembly transcriptome (Gencode v28 comprehensive gene annotations) using STAR. Aligned pairs that had the same mapping coordinates and UMIs were filtered out as PCR duplicates. Read-start coordinates in hg38-mapped reads that began with an adenosine nucleotide, and had a minimum mean read count of 1 across the triplicate samples were collated. m6A or m6Am sites were identified as read starts that were at least 2-fold enriched in m6ACE libraries than in the corresponding input libraries. This enrichment was calculated using DESeq2 performed on A-only sites across triplicate pairs of m6ACE and corresponding input libraries (FDR<0.1, padj<0.05). Based on read-start patterns observed from m6ACE-seq of methylated spike-ins, we considered identified “sites” that were 1-4 nucleotides upstream of another identified significant Rm6AC site or “sites” found within clustered read-starts to be m6ACE-seq false-positives and filtered them out. To identify m6A or m6Am sites that were differentially methylated between sample conditions, we calculated the RML of each site in each sample: The read-start counts at positions -4 to 0 of each site in the m6ACE library were summed and divided by the read-start counts at positions -51 to 0 of the same site in the input library to give ‘X’. Similarly, the read-start counts at positions -4 to 0 of the spike-in m6A site in the m6ACE library were summed and divided by the read-start counts at positions -21 to 0 of the same spike-in m6A site in the input library to give ‘Y’. X was normalized to Y to give RML. RML values of each site was averaged across triplicates for each sample condition. A site was denoted as differentially methylated if the average RML differs between sample conditions with a log2fold-change (LFC) cutoff of 2.0 (for methylase-KO or demethylase-OE induced RML reduction) or 1.0 (for demethylase-KO induced RML accumulation), as well as a one-tailed T-test p-value cutoff of <0.05.
Genome_build: hg38
Supplementary_files_format_and_content: Bedgraph files of first nucleotide coordinates of forward reads. Text files comparing RML of WT versus gene-KO/KD samples.

value: WT 0% Mettl3-KO 100%

value: anti-m6A (Synaptic Systems 202003)


title: TKO2

city: Nashville

first: Vito

last: Quaranta

state: TN

address: 2220 Pierce Ave

institute: Vanderbilt University

description: Cells for single cell RNA-seq were harvested 7 months after tumor induction by Adeno-CMV-cre.

data_processing:
Analyzed using 10X genomics analysis pipeline CellRanger for alignment and filtering.
Cells with ≤ 500 detected genes per cell or with ≤ 10% of transcripts corresponding to mitochondria-encoded genes were 137 removed.
Low abundance genes that were detected in less than 10 cells were excluded. Each cell was normalized to a total of 138 10,000 UMI counts, and log2-transformed after the addition of 1.
Top 1000 highly variable genes were selected and clusters of 139 cells were identified by the shared nearest neighbor modularity optimization based on the top 10 PCs using the highly 140 variable genes and visualized by t-SNE in R package Seurat
The k-nearest neighbors (kNN) with k=10 of human cell 141 lines was detected for each mouse cell to predict subtypes of the individual cell based on signature genes of each subtype. If at 142 least 80% nearest human cell line neighbors for a mouse cell belong to one subtype, the mouse cell was assigned to that 143 subtype. Otherwise, the subtype was undetermined (not assigned)
Genome_build: mm10
Supplementary_files_format_and_content: Processed read counts as describe above.

source_name: Small cell lung cancer cells

extract_protocol:
One tumor was dissected from the lung of each mouse, chopped and digested in 1ml of PBS containing 2mg/ml of Collagenase/Dispase (Roche) in a 37°C shaker for 45 minutes. Tumors were then cooled on ice, treated with 2.5μg/ml DNAse (Sigma) for 5 minutes and passed through a 40μm filter. Red blood cells were lysed and the sample was single cell-sorted by FACS.
Single cell capture and library generation was performed using the Chromium Single Cell Controller from 10x Genomics.

tag: strain

value: Mixed genetic background of C57BL/6, 129/SVJ and 129/SvOla

value: Lung

tag: age

value: 8-9 months

tag: genotype

value: Rb-/-; Trp53-/-; Rbl2-/-; Rosa26membrane-Tomato or membrane-GFP


title: WT 25% Mettl3-KO 75% input rep3

tag: cell line

value: WT 25% Mettl3-KO 75%

tag: antibody


title: NA3


title: WT 25% Mettl3-KO 75% rep3


title: WT 50% Mettl3-KO 50% input rep3

value: WT 50% Mettl3-KO 50%


title: TKO1

value: Rb-/-; Trp53-/-; Rbl2-/-; Rosa26membrane-Tomato or membrane-GFPI:IB40H:H


title: CAA7


title: CAA3


title: WT 50% Mettl3-KO 50% rep3


title: WT 75% Mettl3-KO 25% input rep3

value: WT 75% Mettl3-KO 25%


title: ko_48_OPC

city: Charlottesville

first: David

middle: Michael

last: Johanson

state: VA

address: 200 Jeanette Lancaster Way

department: Department of Neuroscience

institute: UVa School of Medicine

description: FACS-Sorted OPC cells from spinal cord of LRP1-/- mice undergoing experimental autoimmune encephalomyelitis

data_processing:
Sequenced Reads were subject to quality control checks using FastQC (Samples 1-29 v0.11.5 and Samples 30-41 v0.11.8)
Reads were filtered and trimmed using Trimmomatic (Samples 1-29 v0.36 and Samples 30-41 v0.12.0)
Reads were aligned to the Gencode mm13 genome using Salmon (Samples 1-29 v0.8.2 and Samples 30-41 v0.12.0)
Genome_build: Gencode mm13
Supplementary_files_format_and_content: Salmon output file with transcript name (ENSEMBL), length, effective length, TPM, and number of reads

source_name: FACS-Sorted OPC spinal cord

growth_protocol: C57BL/6J mice were grown to full adulthood before experimental manipulations.

extract_protocol:
Mice were euthanized and perfused with PBS containing 5U/mL heparin. The brain was harvested and coronal slices were cut using an adult mouse matrix (69080-C, Electron Microscopy Sciences). Coronal slices (cut to include bregma -0.88 to -1.855) were prepared from untreated animals (3mm thick) and cuprizone treated animals (2 mm) to normalize total RNA quantity. The corpus callosum was then micro-dissected, flash-frozen in liquid nitrogen, and RNA was extracted using the Isolate II Kit (Bioline). RNA-seq (Illumina) was performed by the Genomic Sciences Lab or the Genome Analysis and Technology Core at the University of Virginia.
RNA was extracted using the Isolate II Kit (Bioline).
Isolated mRNA was subject to reverse transcriptase to cDNA, 150bp read lengths and paired-end sequencing

treatment_protocol: For EAE, Lrp1fl/flOlig1Cre and Olig1Cre mice were immunized and scored as previously described (Seki et al., 2017).

value: C57BL/6J LRP1fl/flPdgfraCre-ERt2

tag: batch date

tag: barcode

value: GAGTGG

value: KO

value: OPC


title: ko_48_Mac

description: FACS-Sorted Macrophage cells from spinal cord of LRP1-/- mice undergoing experimental autoimmune encephalomyelitis

source_name: FACS-Sorted Macrophage spinal cord

value: CCGTCC

value: Macrophage


title: ko_49_Mac

value: GTGAAA


title: ko_49_OPC

value: ATTCCT


title: ko_47_OPC

value: GTTTCG


title: ko_47_Mac

value: AGTTCC


title: WT 75% Mettl3-KO 25% rep3

value: anti-m6A (Synaptic Systems 202003)


title: NA2

value: benign gynecologic disease

value: Normal adipose tissue


title: WT 0% Mettl3-KO 100% rep3

value: WT 0% Mettl3-KO 100%


title: WT 100% Mettl3-KO 0% input rep3

value: WT 100% Mettl3-KO 0%


title: wt_806_OPC

description: FACS-Sorted OPC cells from spinal cord of wild type mice undergoing experimental autoimmune encephalomyelitis

value: ACTGAT

value: WT


title: wt_802_OPC

value: C57BL/6J LRP1fl/flPdgfraCre-ERt2

value: CGTACG


title: NA1


title: WT 25% Mettl3-KO 75% rep2

value: WT 25% Mettl3-KO 75%


title: WT 0% Mettl3-KO 100% rep2


title: wt_806_Mac

description: FACS-Sorted Macrophage cells from spinal cord of wild type mice undergoing experimental autoimmune encephalomyelitis

value: GTCCGC


title: wt_5wk_214

description: Wild type cortex 5 weeks after cuprizone administration

source_name: Whole Cortex

treatment_protocol: For the cuprizone model, adult male mice (at least 10 weeks old) were fed regular chow mixed with 0.3 % cuprizone (Sigma #14690) ad libitum for 5 weeks to induce demyelination as previously described (Sachs et al., 2014).

value: C57BL/6J Lrp1fl/flOlig1Cre

tag: weeks post-demyelination

value: Cortex


title: wt_402_OPC

value: GTGGCC

value: OPC


title: WT 25% Mettl3-KO 75% input rep2


title: WT 75% Mettl3-KO 25% rep2

value: WT 75% Mettl3-KO 25%


title: WT 75% Mettl3-KO 25% input rep2


title: WT 50% Mettl3-KO 50% input rep2

value: WT 50% Mettl3-KO 50%


title: WT 50% Mettl3-KO 50% rep2

value: anti-m6A (Synaptic Systems 202003)


title: ko_5wk_298

description: LRP1-/- cortex 5 weeks after cuprizone administration

value: KO


title: wt_5wk_213


title: WT 100% Mettl3-KO 0% input rep2


title: WT 100% Mettl3-KO 0% rep3


title: WT 0% Mettl3-KO 100% input rep1


title: ko_5wk_327


title: wt_3wk_210

description: Wild type cortex 3 weeks after cuprizone administration


title: WT 0% Mettl3-KO 100% input rep2


title: WT 100% Mettl3-KO 0% rep2


title: WT 25% Mettl3-KO 75% input rep1


title: ko_5wk_320


title: WT 25% Mettl3-KO 75% rep1


title: wt_3wk_211


title: WT 0% Mettl3-KO 100% rep1


title: wt_3wk_209


title: WT 50% Mettl3-KO 50% input rep1


title: wt_802_Mac

value: ATGTCA

value: Macrophage


title: ko_3wk_326

description: LRP1-/- cortex 3 weeks after cuprizone administration


title: wt_5wk_212


title: ko_3wk_324


title: ko_3wk_323


title: ko_3wk_321


title: wt_5_5wk_644

description: Wild type cortex 5.5 weeks after cuprizone administration


title: wt_5_5wk_643


title: WT 75% Mettl3-KO 25% rep1


title: WT 50% Mettl3-KO 50% rep1


title: Fto OE rep3

value: Fto OE


title: Fto OE input rep3


title: WT 100% Mettl3-KO 0% input rep1

value: WT 100% Mettl3-KO 0%


title: wt_5_5wk_640

value: WT


title: wt_5_5wk_638


title: wt_402_Mac

value: C57BL/6J LRP1fl/flPdgfraCre-ERt2

value: AGTCAA


title: ko_5_5wk_386

description: LRP1-/- cortex 5.5 weeks after cuprizone administration


title: Fto OE input rep2


title: WT 75% Mettl3-KO 25% input rep1


title: wt_5_5wk_641


title: Fto OE input rep1


title: Fto OE rep2


title: ko_5_5wk_383


title: ko_5_5wk_384


title: Pcif1-KO input rep3


title: Fto OE rep1


title: ko_5_5wk_361


title: Pcif1-KO input rep2


title: wt_0wk_854

description: Wild type cortex before cuprizone administration


title: wt_0wk_853


title: Pcif1-KO rep3


title: ko_5_5wk_385


title: WT 100% Mettl3-KO 0% rep1


title: Pcif1-KO rep2


title: wt_0wk_852


title: ko_0wk_466

description: LRP1-/- cortex before cuprizone administration


title: t60_d60CM

city: Suzhou

first: Hang

last: Ruan

state: Jiangsu

address: 199 Ren'ai Road

department: IBMS

institute: Soochow University

data_processing:
Raw sequencing reads were processed in the following four steps: 1) Only read pairs whose read 1 uniquely mapped the pre-defined barcode tag (10nt) and UMI (14nt) were considered as valid. 2) Read pairs were filtered by cutadapt (v1.8.1) with parameters: -m 20 --trim-n --max-n 0.7 –q20. 3) Reads were then aligned to genomes of human, E. coli, mycoplasma, yeast, and adapter sequences by bowtie2 (v2.2.4). Contaminants were filtered by FastQ Screen (v0.5.1.4). Clean reads were then mapped to UCSC human genome (hg38) via STAR (v2.5.2b) and assigned to Ensembl genes41 by featureCounts.
Genome_build: hg38
Supplementary_files_format_and_content: expression matrix file in csv format

source_name: hESC-derived

growth_protocol: Human ESC lines (H1) were maintained on Matrigel-coated plates (BD Biosciences) in Essential 8 Medium (STEM CELL TECHNOLOGIES) and ROCK inhibitor (Selleck). The medium was changed every day. For cardiac differentiation, Human ESC lines (H1) at ∼100% confluence were incubated with differentiation medium comprising RPMI 1640 medium (Gibco) and B27 supplement minus insulin (Invitrogen). On day 0, CHIR99021 (Selleck), a selective glycogen synthase kinase 3β inhibitor, was added to the differentiation medium (3μM final). On day 3, the Wnt antagonist, IWR-1 (Selleck), was added to the differentiation medium (5μM final). On day 5, medium was removed and replaced with differentiation basal medium without any inhibitors. On day 8, the cells were incubated with medium consisting of RPMI 1640 medium and B27 supplement plus insulin (Invitrogen). The medium was changed every 3 days for desired time of culture.

extract_protocol:
Specific day time point of selected six different stages are day 0 (hESC), day 2 (MES), day 5 (CP), day 9 (CM), day 14 (immature CM), day 60(mature CM). Above stage cells were digested into single cell suspension. After digestion, and then filtered through a 70μm cell strainer into 15ml tubes. Single-cell sequencing library was generated by use of iCell8 platform (Takara). In general, isolated cells were stained with a mixture of Hoechst 33342 and Propidium Iodide (R37610, Thermo Scientific) according to the manufacturer’s instruction. After staining, cells were washed by PBS, and counted by MoxiTM Automated Cell Counter. Afterwards, cell suspension (20,000 cells/ml) was submitted to the MultiSample NanoDispenser (MSND, Wafergen Biosystems) for single cell preparation. The dispensed cells were then imaged with the Imaging Station, and single live cells, defined by Hoechst- positive and Propidium Iodide-negative staining, were selected. Selected cells were subjected to reverse transcription and first-step amplification in a Chip Cycler (Bio-Rad), and the resulting cDNA was purified and size-selected with Agencourt AMPure XP beads (A63880, Beckman Coulter)
One ng of purified cDNA was applied to generate a sequencing library by use of Nextera XT DNA sample preparation Kit (FC-131-1024, Illumina). Libraries were sequenced on the NextSeq500 sequencer (Illumina) using the 26nt and 50nt paired-end sequencing protocol.

value: cardiomyocytes

tag: timepoint


title: Pcif1-KO rep1


title: pair15_50mM_Mg

city: Cambridge

first: Conor

middle: James

last: McClune

state: MA

address: 31 Ames St

department: Biology

institute: Massachusetts Institute of Technology

description: sample 15

data_processing:
Reads were mapped to the Echerichia coli MG1655 genome and plasmids with bowtie (default parameters)
RPKM ( Reads Per Kilobase Million) was calculated with custom python scripts
Supplementary_files_format_and_content: csv file containing RPKM for each gene in each sample. First line contains column titles

organism: Escherichia coli str. K-12 substr. MG1655

growth_protocol: Cultures were grown overnight in M9 media containing 2 mM MgSO4 and then diluted 1:25 into fresh M9 containing 2 mM MgSO4 and grown for 2 hours to reach OD600 ~ 0.5.

extract_protocol:
After 30 minutes of growth, cells from each condition were harvested by adding 1.8 mL culture to 200 μM cold stop solution (95% ethanol, 5% acid buffered phenol, 4 °C). The mixture was centrifuged for 30 s at 13,000 rpm on a benchtop centrifuge, and the supernatant was removed with the pellet flash frozen in liquid nitrogen and stored at -80 °C. To extract RNA, Trizol (Invitrogen) was heated to 65 °C, added directly to the pellet, and incubated at 65 °C for 10 minutes with shaking at 2000 rpm (Eppendorf Thermomixer). The mixture was frozen at -80 °C for at least 10 minutes. After thawing, cells were centrifuged at 15,000 rpm, 4 °C for 5 minutes, and the supernatant was removed into 400 μL ethanol. The mixture was applied to a DirectZol spin column (Zymo) and centrifuged for 30 s at 13,000 rpm. The columns were washed with DirectZol RNA prewash buffer twice (400 μL) and RNA wash buffer (700 μL) once before eluting in 90 μL DEPC water. 10 μL 10x Turbo DNAse buffer and 2 μL Turbo DNAse (Invitrogen) were added to the eluant. The mixture was digested at 37 °C for 20 minutes, followed by the addition of 2 μL more DNAse and another 20 minute incubation. Total volume was brought to 200 mL with DEPC water and combined with 200 μL acid-phenol:chloroform  (IAA, Invitrogen), vortexed and centrifuged for 10 minutes at 21,000 g and 4 °C. The top (aqueous) layer was extracted and ethanol precipitated in 20 μL NaOAc (3M), 2 μL GlycoBlue (Invitrogen) and 600 μL cold ethanol. Precipitation mix was incubated at -80 °C for more than 4 hours before centrifuging for 30 minutes at 21,000 g and 4 °C. The pellet was washed  twice with 500 μL cold 70% ethanol, then air dried and resuspended in 50 μL DEPC water. RNA integrity was validated on a 6% TBE-urea acrylamide Novex gel (Invitrogen) and yield was quantified by NanoDrop spectrophotometer. rRNA was removed with the RiboZero rRNA Removal Kit for Bacteria (Illumina).
RNA was fragmented and cDNA libraries were prepared using the KAPA RNA HyperPrep Kit (Roche) and sequenced on an Illumina HiSeq.

treatment_protocol: For the OFF condition (Samples 1-8), 1 mL of cells was diluted into 2 mL M9 containing 75 mM MgSO4 for a final concentration of 50 mM MgSO4. For the ON condition (Samples 11-18), 2 mL of cells were pelleted, washed twice with M9 + 10μM MgSO4, resuspended in M9 + 10 μM MgSO4, and then 1 mL of cells was diluted into 2 mL M9 + 10μM MgSO4. For trans-zeatin induction (Samples 9,10,19,20) cells were grown in 2 mL M9 + 1nM aTc and induced (Samples 19,20) by transferring 1mL cells to 2mL to identical media containing 1.5μM trans-zeatin (for final concentration of 1μM trans-zeatin). Uninduced samples (Samples 9,10) were generated by transferring 1mL cells to 2mL of just M9 + 1nM aTc. For all samples, RNA was harvested 30 minutes after cell induction.

tag: interface mutant

value: mutant pair 15 - PhoQ(SMMNNA)/PhoP(AQALL)

tag: mgso4 treatment

tag: trans-zeatin treatment

value: deltaphoPQ deltalacZYA


title: d14_input

data_processing:
Raw reads were aligned to hg38 by Rsubread package and sort by samtools
bigWig files were produced by command line makeTagDirectory and makeUCSCfile
Genome_build: hg38
Supplementary_files_format_and_content: bam files and bigWig files

extract_protocol:
ChIP for ETS1. Briefly, D0、D2、D5、D9、D14 of hESCs-derived cells were fixed using 1% formaldehyde for 10 min, and 0.125 M glycine was added to stop fixation. Cells were harvested, and DNA was fragmented to 300–500 bp by sonication with a Covaris S220 sonicator. Immunoprecipitation was performed with antibodies conjugated to Dynabeads Protein G beads (1004D, Life Technologies). ChIP DNA was eluted, reverse cross-linked, extracted with phenol/chloroform, and precipitated.
For ChIP-Seq, 1 ng ChIP DNA or input DNA was used to generate sequencing libraries using the Nextera XT DNA sample preparation Kit (FC-131-1024,Illumina). Libraries were sequenced on the NextSeq500 sequencer (Illumina) using the 35nt paired-end sequencing protocol.

tag: tissue

tag: chip antibody

value: none (input)


title: Pcif1-KO input rep1

tag: genotype/variation

tag: passage number


title: pair13_50mM_Mg

description: sample 16

value: mutant pair 13 - PhoQ(ALVSVA)/PhoP(VHSCL)


title: d9_input


title: PhoQwt-PhoP4_1uM_tZ

description: sample 19

value: incompatible pair - PhoQ(wild type)/PhoP4(TPWTL)


title: pair8_50mM_Mg

description: sample 13

value: mutant pair 8 - PhoQ8(ALSMDL)/PhoP(TPWLV)


title: wt_replicate2_50mM_Mg

description: sample 12

value: wild type PhoQ/PhoP


title: ko_0wk_468

value: C57BL/6J Lrp1fl/flOlig1Cre

value: Cortex


title: t14_d14CM


title: AQ4-PhoP4_0uM_tZ

description: sample 10

value: mutant pair - chimera AQ4(SLSLLA)/PhoP4(TPWTL)


title: t09_h_icm


title: AQ4-PhoP4_1uM_tZ

description: sample 20


title: t05_d5cpc


title: PhoQwt-PhoP4_0uM_tZ

description: sample 9


title: d5_ets1


title: t02_d2mesoderm


title: pair1_50mM_Mg

description: sample 17

value: mutant pair 1 - PhoQ(TLSLKA)/PhoP(TPLRH)


title: pair14_50mM_Mg

description: sample 14

value: mutant pair 14 - PhoQ(NLMGRA)/PhoP(GHCSL)


title: d5_input


title: wt_replicate1_50mM_Mg

description: sample 11


title: d9_ets1


title: d0_input


title: d14_ets1


title: pair4_10uM_Mg

description: sample 8

value: mutant pair 4 - PhoQ4(SLSLLA)/PhoP4(TPWTL)


title: ko_0wk_467

value: KO


title: t00_DOES


title: d2_input


title: d2_ets1


title: pair13_10uM_Mg

description: sample 6


title: d0_ets1


title: pair1_10uM_Mg

description: sample 7


title: pair15_10uM_Mg

description: sample 5


title: pair14_10uM_Mg

description: sample 4


title: pair8_10uM_Mg

description: sample 3


title: wt_replicate1_10uM_Mg

description: sample 1


title: Fto-KO input rep3

description:
input
processed data file: FTO-regulated_RML.txt.gz

data_processing:
Library strategy: m6ACE-seq
Basecalls performed using FASTQ Generation v1.0.0.Fastq sequences were first trimmed of 5' and 3' adapter sequences and poly(A) tails using Cutadapt. The 8-mer 'N7B' (N=A/C/G/T, B=C/G/T) UMI located at the first 8 nucleotides of read 1 was registered and trimmed. Any complementary UMI sequence in read 2 was also trimmed. Reads were mapped to the methylated spike-in using Bowtie2, or to the hg38 assembly transcriptome (Gencode v28 comprehensive gene annotations) using STAR. Aligned pairs that had the same mapping coordinates and UMIs were filtered out as PCR duplicates. Read-start coordinates in hg38-mapped reads that began with an adenosine nucleotide, and had a minimum mean read count of 1 across the triplicate samples were collated. m6A or m6Am sites were identified as read starts that were at least 2-fold enriched in m6ACE libraries than in the corresponding input libraries. This enrichment was calculated using DESeq2 performed on A-only sites across triplicate pairs of m6ACE and corresponding input libraries (FDR<0.1, padj<0.05). Based on read-start patterns observed from m6ACE-seq of methylated spike-ins, we considered identified 'sites' that were 1-4 nucleotides upstream of another identified significant Rm6AC site or 'sites' found within clustered read-starts to be m6ACE-seq false-positives and filtered them out. To identify m6A or m6Am sites that were differentially methylated between sample conditions, we calculated the RML of each site in each sample: The read-start counts at positions -4 to 0 of each site in the m6ACE library were summed and divided by the read-start counts at positions -51 to 0 of the same site in the input library to give 'X'. Similarly, the read-start counts at positions -4 to 0 of the spike-in m6A site in the m6ACE library were summed and divided by the read-start counts at positions -21 to 0 of the same spike-in m6A site in the input library to give 'Y'. X was normalized to Y to give RML. RML values of each site was averaged across triplicates for each sample condition. A site was denoted as differentially methylated if the average RML differs between sample conditions with a log2fold-change (LFC) cutoff of 2.0 (for methylase-KO or demethylase-OE induced RML reduction) or 1.0 (for demethylase-KO induced RML accumulation), as well as a one-tailed T-test p-value cutoff of <0.05.
Genome_build: hg38 (GRCh38)
Supplementary_files_format_and_content: Bedgraph files of first nucleotide coordinates of forward reads. Text files comparing RML of WT versus gene-KO/KD samples.

extract_protocol:
Total RNA was isolated using Trizol-LS.
Poly(A) RNA was purified using Poly(A)Purist Mag kit (Thermofisher AM1922) according to manufacturer?s instructions. Poly(A) RNA was fragmented using RNA fragmentation buffer (Ambion AM8740) and dephosphorylated at its 3? end with T4 PNK (NEB M0201) for 30min at 37oC. 5? phosphorylation was then initiated by adding 1mM ATP and incubating for 30min at 37oC. 3? ligation was then performed as previously described 2, where 5?-adenylated,3-dideoxyC DNA adapters were ligated with truncated T4 RNA ligase 2 (NEB M0242) in 1X ATP-free T4 RNA ligase buffer [50mM Tris pH 7.5, 60µg/ml BSA, 10mM MgCl2, 10mM DTT, 12.5% PEG8000] for 2hr at 25oC. 3?-ligated methylated RNA spike-in (Table S1) was added to ligated Poly(A) RNA and the mixture was denatured for 5min at 65oC before incubating for 2min on ice. This denatured RNA mixture was incubated overnight at 4oC with anti-m6A antibody (Synaptic Systems 202003) in 1X IP buffer [150mM NaCl, 10mM Tris pH 7.4, 0.1% IGEPAL CA-630 (Sigma I8896)] supplemented with RNasin Plus (Promega N2611). In parallel, Protein-A-dynabeads (Life Technologies 10002D) was blocked overnight at 4oC in 1X IP buffer supplemented with 0.5mg/ml BSA (Sigma A7906). The iced antibody-RNA mixture was crosslinked with 0.15J/cm2 254nm UV radiation six times. 1% of the antibody-RNA mixture was set aside as ?input? and the rest (designated as ?m6ACE?) was mixed with decanted BSA-blocked Protein-A-dynabeads for 1.5hr at 4oC. Beads bound with crosslinked RNA were then washed with the following cold buffers in this order: Wash buffer 1 [1M NaCl, 50mM HEPES-KOH pH 7.4, 1% Triton X-100, 0.1% Sodium Deoxycholate, 2mM EDTA], Wash buffer 2 [0.5M NaCl, 50mM HEPES-KOH pH 7.4, 1% IGEPAL, 0.1% Sodium Deoxycholate, 2mM EDTA], Wash buffer 3 [1% Sodium Deoxycholate, 25mM LiCl, 10mM Tris pH 8, 1% Triton X-100, 2mM EDTA], TE [10mM Tris pH 8, 1mM EDTA] and finally 10mM Tris pH 8. m6ACE RNA was subjected to 5? to 3? exonuclease treatment with XRN-1 (NEB M0338) for 1hr at 37oC. The m6ACE RNA-bead mixture was then washed with Wash buffer 1, Wash buffer 2, Wash buffer 3, TE and 10mM Tris pH 8. Both input and m6ACE RNAs were reverse crosslinked in elution buffer [1%SDS, 200mM NaCl, 25mM Tris pH 8, 2mM EDTA, 1mg/ml Proteinase K (Thermo Scientific EO0491)] for 1.5hr at 50oC. RNAs were ethanol-precipitated and ligated to 5?adapters (Table S1) with T4 RNA ligase (Ambion AM2140) for 16hr at 16oC. Reverse transcription primer (Table S1) was annealed (72oC 2min, ice 2min) and reverse transcription was performed with SuperscriptIII (Invitrogen 18080) for 1hr at 50oC, with the reaction stopped by incubating for 15min at 70oC. The cDNA was used for PCR amplification with Phusion High-fidelity PCR mastermix (NEB M0530) and Truseq PCR primers. Finally, primer-dimer and adapter-dimers were removed with AMpure XP beads before undergoing PE75 sequencing on the Illumina Nextseq platforms.

value: Fto-KO

value: anti-m6A (Synaptic Systems 202003)


title: Fto-KO rep2

description:
m6ACE
processed data file: FTO-regulated_RML.txt.gz


title: Fto-KO input rep2


title: pair4_50mM_Mg

description: sample 18


title: wt_replicate2_10uM_Mg

description: sample 2


title: Fto-KO input rep1


title: self-ligation control [degenerate_library_sort_seq]

description: plasmid DNA

data_processing:
Library strategy: PCR amplicon sequencing [degenerate_library_sort_seq]
reads with errors outside of the variable/barcode and mutated nucleotides were discarded to remove low quality reads
The frequency of each mutant in each bin was calculated as a function of the total reads in each bin and the total number of cells sorted into each bin (McClune et al., 2018)
Variants with fewer than 25 total reads were discarded
Gaussian functions were fit to each distribution (in log10YFP units), from both the ON and OFF sorts (SciPy optimize package).
The standard deviation error on the estimated log(YFP) mean was used as a metric to filter poorly fit sequences (sequences were removed if  ONstdev + OFFstdev > 2)
Fold induction was calculated as the ratio of the fit means between the induced (10uM MgSO4) and uninduced  (50mM MgSO4) states.
Supplementary_files_format_and_content: csv file. First line contains column titles

source_name: self-ligation control

growth_protocol: Cells were grown in the indicated conditons for 6hr, then sorted into consecutive bins based on their YFP fluorescence. E. coli libraries were grown overnight in 2xYT media  [degenerate_library_sort_seq]

extract_protocol:
plasmid DNA was extracted with the Quiagen plasmid Miniprep Kit [degenerate_library_sort_seq]
For the two mutagenized regions of the plasmid to be brought into close enough proximity (< 790 bp) for paired-end Illumina sequencing, plasmids were digested with XhoI and then self-ligated (T4 ligase, 4 hr). To isolate only self-ligation products, and not cross-ligation products, ligation reactions were cleaned (Zymo PCR Clean Up) and gel purified to select for the correct size on FlashGels (Lonza). Two PCR reactions were performed, both using KAPA HiFi Hotstart, to add Illumina sequencing adaptors and barcodes. First, ligation reaction products were amplified for 30 cycles (95 °C for 30 s, 65 °C for 15 s, 72 °C for 120 s) with primers CJM642 and CJM643 in an emulsion PCR (Micellula Emulsion PCR) to avoid PCR chimeras. Second, purified PCR product from the first reaction was subjected to a second PCR with barcoding primers for 9 cycles (95 °C for 30 s, 65 °C for 15 s, 72 °C for 60 s). Final products were quantified (NanoDrop), normalized, combined, and sequenced on an Illumina NextSeq.  [degenerate_library_sort_seq]

treatment_protocol: After sorint, before plasmid extraction, E. coli libraries were grown overnight in 2xYT media  [degenerate_library_sort_seq]

tag: replicate

tag: agent

tag: facs gate


title: Fto-KO rep3


title: raw library [degenerate_library_sort_seq]

description: plasmid DNA

source_name: raw library


title: rep3 50mM MgSO4 presort [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep3 50mM MgSO4 presort

value: replicate 3

value: 50mM MgSO4

value: presort


title: Fto-KO rep1


title: Alkbh5-KO input rep3

description:
input
processed data file: ALKBH5-regulated_RML.txt.gz


title: rep2 50mM MgSO4 presort [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep2 50mM MgSO4 presort

value: replicate 2


title: rep3 10uM MgSO4 presort [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep3 10uM MgSO4 presort

value: 10microM MgSO4


title: rep1 50mM MgSO4 presort [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep1 50mM MgSO4 presort

value: replicate 1


title: Alkbh5-KO input rep1


title: Alkbh5-KO rep3

description:
m6ACE
processed data file: ALKBH5-regulated_RML.txt.gz


title: Alkbh5-KO input rep2


title: rep2 10uM MgSO4 presort [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep2 10uM MgSO4 presort


title: Alkbh5-KO rep1

source_name: Human embryonic kidney cells


title: rep3 50mM MgSO4 gate 7 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep3 50mM MgSO4 gate 7


title: rep3 50mM MgSO4 gate 8 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep3 50mM MgSO4 gate 8


title: rep1 10uM MgSO4 presort [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep1 10uM MgSO4 presort


title: Mettl16-KD rep3

description:
m6ACE
processed data file: METTL16-dependent_RML.txt.gz


title: Mettl16-KD rep2


title: Mettl16-KD input rep3

description:
input
processed data file: METTL16-dependent_RML.txt.gz


title: rep3 50mM MgSO4 gate 5 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep3 50mM MgSO4 gate 5


title: Alkbh5-KO rep2


title: Mettl3-KO input rep3

description:
input
processed data file: METTL3-dependent_RML.txt.gz


title: Mettl3-KO rep3

description:
m6ACE
processed data file: METTL3-dependent_RML.txt.gz


title: rep3 50mM MgSO4 gate 3 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep3 50mM MgSO4 gate 3


title: Mettl3-KO input rep2


title: rep3 50mM MgSO4 gate 2 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep3 50mM MgSO4 gate 2


title: rep3 50mM MgSO4 gate 4 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep3 50mM MgSO4 gate 4


title: rep2 50mM MgSO4 gate 8 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep2 50mM MgSO4 gate 8


title: Mettl3-KO rep2


title: rep3 50mM MgSO4 gate 6 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep3 50mM MgSO4 gate 6


title: Mettl3-KO input rep1


title: rep2 50mM MgSO4 gate 6 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep2 50mM MgSO4 gate 6


title: Mettl16-KD input rep1


title: rep2 50mM MgSO4 gate 7 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep2 50mM MgSO4 gate 7


title: Mettl16-KD input rep2


title: WT input rep3

description:
input
processed data file: all .txt files

value: Wild type


title: Mettl3-KO rep1


title: Mettl16-KD rep1


title: WT rep3

description:
m6ACE
processed data file: all .txt files


title: WT input rep2


title: rep2 50mM MgSO4 gate 4 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep2 50mM MgSO4 gate 4


title: rep2 50mM MgSO4 gate 5 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep2 50mM MgSO4 gate 5


title: WT rep2


title: rep2 50mM MgSO4 gate 3 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep2 50mM MgSO4 gate 3


title: WT input rep1


title: WT rep1


title: rep2 50mM MgSO4 gate 2 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep2 50mM MgSO4 gate 2


title: rep2 50mM MgSO4 gate 1 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep2 50mM MgSO4 gate 1


title: rep3 50mM MgSO4 gate 1 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep3 50mM MgSO4 gate 1


title: rep1 50mM MgSO4 gate 7 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep1 50mM MgSO4 gate 7


title: rep1 50mM MgSO4 gate 6 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep1 50mM MgSO4 gate 6


title: rep1 50mM MgSO4 gate 8 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep1 50mM MgSO4 gate 8


title: rep1 50mM MgSO4 gate 4 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep1 50mM MgSO4 gate 4


title: rep1 50mM MgSO4 gate 5 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep1 50mM MgSO4 gate 5


title: rep1 50mM MgSO4 gate 3 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep1 50mM MgSO4 gate 3


title: control individual JR73 replicate 1

city: New York

first: Oliver

middle: H

last: Tam

state: New York

address: 435 E 30th St

department: Institute for Systems Genetics

institute: NYU Grossman School of Medicine

data_processing:
Base calling performed with bcl2fastq conversion software v2.17
Reads were aligned to hg19 genome using STAR v2.5.2b with the following configurations: --outFilterMultimapNmax 100; --outFilterMismatchNoverReadLmax 0.04
Genes and TE were quantified using TEcount (from TEtranscripts v2.0.3) with the following configurations: hg19 genic GTF from refGene (Jan 2018), custom hg19 TE GTF generated from repeatMasker; --stranded reverse
Genome_build: hg19
Supplementary_files_format_and_content: Individual count tables were generated as output from TEcount

source_name: Control motor cortex

extract_protocol:
RNA was extracted from each of the flash-frozen patient samples using the Ambion PureLink RNA Mini kit. RNA was assessed using the Bioanalyzer to ensure RNA Integrity (RIN) values of > 5.5.
RNA-seq libraries were prepared from 500 ng of total RNA using the Illumina Tru-seq Stranded RNA kit.  Samples were barcoded to multiplex 8 samples per batch, randomly mixing ALS and Control samples in each library preparation and sequencing batch.

treatment_protocol: Flash frozen autopsy tissue

value: motor cortex

tag: subject id

tag: diagnosis

value: Non-neurological control


title: control individual JR70 replicate 1


title: control individual JR73 replicate 2


title: control individual JR65 replicate 2


title: control individual JR70 replicate 2


title: rep1 50mM MgSO4 gate 2 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep1 50mM MgSO4 gate 2


title: control individual JR65 replicate 1


title: control individual JR67 replicate 1


title: rep3 10uM MgSO4 gate 8 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep3 10uM MgSO4 gate 8


title: rep1 50mM MgSO4 gate 1 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep1 50mM MgSO4 gate 1


title: rep3 10uM MgSO4 gate 7 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep3 10uM MgSO4 gate 7


title: control individual JR44 replicate 1


title: control individual JR44 replicate 2


title: rep3 10uM MgSO4 gate 6 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep3 10uM MgSO4 gate 6


title: rep3 10uM MgSO4 gate 4 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep3 10uM MgSO4 gate 4


title: sALS patient JR27 replicate 2

source_name: sALS Motor cortex

value: sALS


title: sALS patient JR27 replicate 1


title: control individual JR39 replicate 2


title: sALS patient JR18 replicate 2


title: control individual JR67 replicate 2


title: rep2 10uM MgSO4 gate 7 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep2 10uM MgSO4 gate 7


title: sALS patient JR34 replicate 1


title: sALS patient JR34 replicate 2


title: rep2 10uM MgSO4 gate 8 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep2 10uM MgSO4 gate 8


title: sALS patient JR61 replicate 2


title: sALS patient JR61 replicate 1


title: sALS patient JR60 replicate 2


title: sALS patient JR18 replicate 1


title: rep2 10uM MgSO4 gate 6 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep2 10uM MgSO4 gate 6


title: rep2 10uM MgSO4 gate 3 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep2 10uM MgSO4 gate 3


title: rep3 10uM MgSO4 gate 2 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep3 10uM MgSO4 gate 2


title: rep3 10uM MgSO4 gate 3 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep3 10uM MgSO4 gate 3


title: sALS patient JR47 replicate 2

source_name: sALS/FTD Motor cortex

value: sALS/FTD


title: rep2 10uM MgSO4 gate 4 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep2 10uM MgSO4 gate 4


title: rep3 10uM MgSO4 gate 5 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep3 10uM MgSO4 gate 5


title: rep2 10uM MgSO4 gate 1 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep2 10uM MgSO4 gate 1


title: rep2 10uM MgSO4 gate 2 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep2 10uM MgSO4 gate 2


title: rep3 10uM MgSO4 gate 1 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep3 10uM MgSO4 gate 1


title: sALS patient JR36 replicate 2


title: rep1 10uM MgSO4 gate 5 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep1 10uM MgSO4 gate 5


title: rep2 10uM MgSO4 gate 5 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep2 10uM MgSO4 gate 5


title: rep1 10uM MgSO4 gate 6 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep1 10uM MgSO4 gate 6


title: rep1 10uM MgSO4 gate 8 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep1 10uM MgSO4 gate 8


title: sALS patient JR35 replicate 2

source_name: sALS Motor cortex


title: sALS patient JR47 replicate 1


title: sALS patient JR60 replicate 1


title: control individual JR39 replicate 1

source_name: Control motor cortex


title: sALS patient JR35 replicate 1


title: rep1 10uM MgSO4 gate 2 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep1 10uM MgSO4 gate 2


title: rep1 10uM MgSO4 gate 3 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep1 10uM MgSO4 gate 3


title: sALS patient JR33 replicate 1


title: rep1 10uM MgSO4 gate 1 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep1 10uM MgSO4 gate 1


title: sALS patient JR33 replicate 2


title: sALS patient JR30 replicate 1


title: rep1 10uM MgSO4 gate 7 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep1 10uM MgSO4 gate 7


title: sALS patient JR48 replicate 2


title: sALS patient JR36 replicate 1


title: sALS patient JR29 replicate 2


title: sALS patient JR29 replicate 1


title: sALS patient JR9 replicate 1


title: CD8 Dendritic Cells KO1

first: Brian

middle: Yee Hong

last: Lam

address: Box 289 Addenbookes Hospital, Hills Road

department: Metabolic Research Laboratories

country: United Kingdom

institute: University of Cambridge

description: CD8 Dendritic Cells Atg16l1 -/- replicate 1

data_processing:
STAR v 2.5.0a was used to map reads onto the Mouse GRCm38 genome and determine gene-level counts based on Ensembl V90 gtf
Genome_build: GRCm38
Supplementary_files_format_and_content: gene-level read counts in tab-delimited text

source_name: spleen Dendritic Cells CD8

growth_protocol: Dendritic cell subsets were FACS-purified (MHCIIhighCD11chighCD11b+CD8-: DC CD11b; MHCIIhighCD11chighCD11b-CD8+) from single suspension obtained after DC enrichment (mouse CD11c MicroBeads UltraPure, followed by AUTOMacs positive selection, Milteny) from digested spleens (collagenase D, dispase I, DNAse I in RPMI, 45 min at 37°C) from individual mice

extract_protocol:
Cells samples were extensively washed in PBS, resuspended in RLT lysis buffer, snapfrozen and kept at -80°C.  RNA extraction and residual DNA removal upon DNase digestion was performed with the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions.
7 ng of total RNA, after quality check with Agilent Bioanalyser 2100 system, was made into sequencing libraries using Takara’s the SMARTer Stranded Total RNA-Seq Kit v2 – Pico Input Mammalian, following manufacturer’s instruction,

tag: strain

value: spleen

tag: age

value: 16 weeks

tag: genotype


title: CD8 Dendritic Cells KO2

description: CD8 Dendritic Cells Atg16l1 -/- replicate 2


title: TARDBP eCLIP, replicate 2

description: immunoprecipitated RNA

data_processing:
Base calling performed with bcl2fastq conversion software v2.17
Reads were aligned to hg19 genome using STAR v2.5.2b with the following configurations: --outFilterMultimapNmax 100; --outFilterMismatchNoverReadLmax 0.04
Multi-mappers were re-aligned using CLAM v1.1.3 with default parameters
Peaks were called using CLAM v1.1.3 with the following parameters: custom GTF with genes and TE annotations for hg19; --min-clip-cov 10
Peaks identified in both replicates were selected, and peaks merged if they are bookended
Genome_build: hg19
Supplementary_files_format_and_content: bed files were generated from CLAM peakcalling

source_name: SH-SY5Y cell line

growth_protocol: SH-SY5Y cells (CRL-2266, ATCC) were grown in DMEM/F-12 Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12  (ThermoFisher, 11320033) supplemented with 10% FBS and 1% penicillin–streptomycin, and cultured at 37 °C with 5% CO2.

extract_protocol:
Cell pellets were lysed in 1 mL lysis buffer, followed by RNAse I digestion and immunoprecipitation with 10 µg of anti-TDP-43 antibody. Barcoded RNA adapter was ligated to the 3′ end, run on standard protein gels and transferred to nitrocellulose membranes. A region 75 kDa (~150 nt of RNA) above the protein size was isolated and treated with proteinase K.
RNA was reverse transcribed and treated with ExoSAP-IT. A second DNA adapter was then ligated to the cDNA fragment 3′ end, and the products were PCR-amplified to generate library for sequencing.

treatment_protocol: 20 million of SH-SY5Y (CRL-2266, ATCC) cells were UV-crosslinked (254 nm, 400 mJ/cm2) in PBS and cell pellets were stored at-80 C. Each sample was prepared as an independent biological replicate and included non-crosslinked cells as a control.

tag: cell line

tag: antibody

value: anti-TDP-43 antibody (Proteintech)


title: control shRNA 2, replicate 2

description: Control2 rep2

extract_protocol:
Total RNA was extracted using Trizol (ThermoFisher) according to the manufacturer’s instructions. One microgram of total RNA was subjected to rRNA removal using the Ribo-Zero Gold rRNA Removal kit (Illumina).
Strand specific RNA-seq libraries were constructed using NEBNext Ultra II Directional Library Prep Kit (NEB).

treatment_protocol: 1.5-2 million SH-SY5Y cells per well of a 6-well plate were “spinfected” with 3 mL of virus containing media and 8µg/mL polybrene for 1 hour at 800g.   Cells stably expressing shRNAs were selected with 2 µg/mL of puromycin 48 hours post-infection for 3 days. Uninfected cells at similar density were used as a control for puromycin selection

tag: hairpin

value: SHC016 (Sigma Aldritch)


title: CD8 Dendritic Cells WT2

description: CD8 Dendritic Cells Atg16l1 + replicate 2


title: CD8 Dendritic Cells WT3

description: CD8 Dendritic Cells Atg16l1 + replicate 3


title: control shRNA 2, replicate 1

description: Control2 rep1


title: CD8 Dendritic Cells WT1

description: CD8 Dendritic Cells Atg16l1 + replicate 1


title: rep1 10uM MgSO4 gate 4 [degenerate_library_sort_seq]

description: plasmid DNA

source_name: rep1 10uM MgSO4 gate 4


title: control shRNA 1, replicate 2

description: Control1 rep2

value: SHC007 (Sigma Aldritch)


title: CD11b Dendritic Cells KO2

description: CD11b Dendritic Cells Atg16l1 -/- replicate 2

source_name: spleen Dendritic Cells CD11b


title: TARDBP eCLIP, replicate 1

description: immunoprecipitated RNA


title: sALS patient JR30 replicate 2

source_name: sALS/FTD Motor cortex


title: PIG_Trachea

description: Sample was prepared by cell dissociation from a fresh newborn pig trachea. The whole clean trachea was incubated with 0.1% protease type XIV from Streptomyces griseus (Sigma-Aldrich) in supplemented HBSS at 4°C degrees overnight. Epithelial cells were gently detached from the turbinate by washing with HBSS pipetting up and down and then transferred to a 50 ml Falcon tube. Cells were centrifuged (150g for 5 min at 4°C), after removing of the supernatant the cells were resuspended in 1 ml of HBSS, 50 units of DNase I (EN0523 Thermo Fisher Scientific) per 250 µL were directly added and cells were further incubated at room temperature for 10 min. Cells were centrifuged (150g for 5 min at 4°C) and resuspended in 1 ml supplemented HBSS containing 10% FCS, centrifuged again (150g for 5 min at 4°C) and resuspended in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a Scienceware® Flowmi™ Cell Strainer (40 µm porosity), centrifuged (150 g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed with Scepter™ 2.0 Cell Counter (Millipore) and Countess™ automated cell counter (Thermo Fisher Scientific). Cell viability was checked with Countess™ automated cell counter (Thermo Fisher Scientific). All steps except the DNAse I incubation were performed on ice. For the cell capture by the 10X genomics device, the cell concentration was adjusted to 500 cells/µl in HBSS aiming to capture 5000 cells.

data_processing:
The Cell Ranger Single Cell Software Suite 2.0 was used to perform sample demultiplexing, barcode processing, and single cell 3’ gene counting (https://support.10xgenomics.com/single-cell/software/pipelines/latest/what-is-cell-ranger).
Cell Ranger command used to produce output directory for each sample was executed using transcriptome based on human genome release hg19, mouse genome mm10 and pig genome Sscrofa11.2
Supplementary_files_format_and_content: Cellranger raw gene / barcode hdf5 matrix file.

organism: Sus scrofa

source_name: Pig trachea epithelial cells

tag: cell type

value: airway epithelial cells

tag: differentiation day

tag: cell origin

value: Fresh tissue

tag: donor


title: CD11b Dendritic Cells KO1

description: CD11b Dendritic Cells Atg16l1 -/- replicate 1

tag: tissue


title: TARDBP shRNA knockdown replicate 2

description: KD rep 2


title: sALS patient JR9 replicate 2


title: control shRNA 1, replicate 1

description: Control1 rep1


title: CD11b Dendritic Cells WT3

description: CD11b Dendritic Cells Atg16l1 + replicate 3


title: TARDBP eCLIP size matched input

description: immunoprecipitated RNA


title: CD11b Dendritic Cells WT1

description: CD11b Dendritic Cells  Atg16l1 + replicate 1


title: Turbinate

description: Sample was prepared by cell dissociation from fresh tissue. The whole turbinate was incubated with 0.1% protease type XIV from Streptomyces griseus (Sigma-Aldrich) in supplemented HBSS at 4°C degrees overnight. Epithelial cells were gently detached from the turbinate by washing with HBSS pipetting up and down and then transferred to a 50 ml Falcon tube. Cells were centrifuged (150g for 5 min at 4°C), after removing of the supernatant the cells were resuspended in 1 ml of HBSS, 50 units of DNase I (EN0523 Thermo Fisher Scientific) per 250 µL were directly added and cells were further incubated at room temperature for 10 min. Cells were centrifuged (150g for 5 min at 4°C) and resuspended in 1 ml supplemented HBSS containing 10% FCS, centrifuged again (150g for 5 min at 4°C) and resuspended in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a  Flowmi 40 µm cell strainer (Belart), centrifuged (150 g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed and 5000 cells were used for single-cell sequencing.

source_name: Human nasal turbinate epithelial cells


title: Nasal_Brushing

description: Sample was prepared by cell dissociation from a nasal brushing which was performed with a 2 mm cytology brush (Medi-Globe) in the inferior turbinate zone. The brush was soaked in a 5 mL eppendorf containing 1 mL of dissociation buffer which was composed of Hypothermosol (BioLife Solutions) 10 mg/mL protease from Bacillus Licheniformis (Sigma-Aldrich, reference P5380) and 0.5 mM EDTA. The tube was shaken vigorously and centrifuged for 2 min at 150 g. The brush was removed, cells pipetted up and down 5 times and then incubated cells on ice for 30 min, with gentle trituration with 21G needles 5 times every 5 min. Protease was inactivated by adding 200 μL of HBSS/2% BSA. Cells were centrifuged (400g for 5 min at 4°C). Supernatant was discarded leaving 10 μL of residual liquid on the pellet. Cells were resuspended in 500 μL of wash buffer (HBSS/0.05% BSA) and 2.250 mL of Ammonium Chloride 0.8% was added to perform red blood cell lysis. After a 10 min incubation, 2 mL of wash buffer were added, and cells were centrifuged (400g for 5 min at 4°C). Supernatant was discarded leaving 10 μL of residual liquid on the pellet, cells were resuspended in 1000 μL of wash buffer centrifuged (400g for 5 min at 4°C). Supernatant was discarded leaving 10 μL of residual liquid on the pellet, cells were resuspended in 1000 μL of wash buffer and passed through a Flowmi 40 µm cell strainer (Belart), then centrifuged  (400g for 5 min at 4°C). Supernatant was discarded leaving 10 μL of residual liquid on the pellet, cells were resuspended in 100 μL of wash buffer. Cell counts and viability were performed with Countess™ automated cell counter (Thermo Fisher Scientific). For the cell capture by the 10X genomics device, the cell concentration was adjusted to 500 cells/µl in HBSS aiming to capture 5000 cells. All steps were performed on ice.

source_name: Human nasal brushing epithelial cells


title: Bronchial_Biopsy

description: Sample was prepared by cell dissociation from a fresh bronchial biopsy. The biopsy was soaked in 1 mL dissociation buffer which was composed of DPBS, 10 mg/mL protease from Bacillus Licheniformis (Sigma-Aldrich, reference P5380) and 0.5 mM EDTA. After 1 h, the biopsy was finely minced with a scalpel, and returned to dissociation buffer. Cells pipetted up and down 5 times and then incubated cells on ice for 90 min, with gentle trituration with 21G needles 5 times every 5 min. Protease was inactivated by adding 200 μL of HBSS/2% BSA. Cells were centrifuged (400g for 5 min at 4°C). Supernatant was discarded leaving 10 μL of residual liquid on the pellet. Cells were resuspended in 500 μL of wash buffer (HBSS/0.05% BSA) and 2.250 mL of Ammonium Chloride 0.8% was added to perform red blood cell lysis. After a 10 min incubation, 2 mL of wash buffer were added, and cells were centrifuged (400g for 5 min at 4°C). Supernatant was discarded leaving 10 μL of residual liquid on the pellet, cells were resuspended in 1000 μL of wash buffer centrifuged (400g for 5 min at 4°C). Supernatant was discarded leaving 10 μL of residual liquid on the pellet, cells were resuspended in 1000 μL of wash buffer and passed through a Flowmi 40 µm cell strainer (Belart), then centrifuged  (400g for 5 min at 4°C). Supernatant was discarded leaving 10 μL of residual liquid on the pellet, cells were resuspended in 100 μL of wash buffer. Cell counts and viability were performed with Countess™ automated cell counter (Thermo Fisher Scientific). For the cell capture by the 10X genomics device, the cell concentration was adjusted to 500 cells/µl in HBSS aiming to capture 5000 cells. All steps were performed on ice.

source_name: Human bronchial biopsy epithelial cells


title: S44_Adult_Lung_EC

city: New York City

first: Balvir

last: Kunar

address: 1300 York Ave

department: Regenerative Medicine; Physiology, Biophysics, & Systems Biology

institute: Weill Cornell Medical College

data_processing:
Base calling and sequencing reads de-multiplexing were performed using bcl2fastq v2.17.
Sequencing reads were checked for quality (FastQC v0.11.5), and trimmed/filtered using Trimmomatic v0.36.
Read alignment to the transcriptome sequence reference of the UCSC mm10 genome build were performed using TopHat2 v2.1.0.
Mapped reads were assembled into transcripts, and abundance measures (FPKM values) quantified using Cufflinks v2.2.1.
Genome_build: mm10
Supplementary_files_format_and_content: Single tab-delimited data-matrix with GENE_ID, GENE_DESCRIPTION, and SAMPLE_NAMES. All numerical inputs represent FPKM values.

source_name: endothelial cell

extract_protocol:
At least 100 ng of total RNA from both freshly harvested and cultured cells was isolated (phenol-chloroform separation of TRIzol LS) and purified using Qiagen’s RNeasy Mini Kit.
RNA library preps were prepared and multiplexed using Illumina’s TruSeq RNA Library Preparation Kit v2 (non-stranded and poly-A selection) and 10 nM of cDNA was used as input for high-throughput sequencing via Illumina’s HiSeq 4000 producing 51 bp paired-end reads.

treatment_protocol: All cell populations were purified via FACS. Purity mask was applied.

value: wild type

tag: source

value: lung

tag: culture status

value: freshly isolated from tissue


title: S52_Adult_Lung_EC


title: S38_Adult_Lung_EC


title: HAECs_Pneumacult_ALI28

description: Sample was prepared 28 days after air-liquid interface in Pneumacult medium. HAECs were cultivated as described in Revinsky et al. (Nature Communications, 2018). Cells were incubated with 0.10% protease type XIV from Streptomyces griseus in HBSS for 4 hours at 4°C degrees. Cells were  detached from the Transwells by pipetting and then transferred to a microtube. 50 units of DNase I per 250µl, were directly added and cells were further incubated at room temperature for 10 min. Cells were washed in 500 µL HBSS 10% Fetal Bovine Serum then in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a Scienceware® Flowmi™ Cell Strainer (40µm porosity), then centrifuged (150g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed and 5000 cells were used for single-cell sequencing.

source_name: Human airway epithellial cells differentiated in Pneumacult medium for 28 days

extract_protocol:
Samples were run on the Chromium machine from 10x Genomics following the  User Guide for v2 Chemistry (https://support.10xgenomics.com/single-cell/sample-prep/doc/user-guide-chromium-single-cell-3-reagent-kit-v2-chemistry).
Single cell RNA-Seq libraries were made following the User Guide for v2 Chemistry (https://support.10xgenomics.com/single-cell/sample-prep/doc/user-guide-chromium-single-cell-3-reagent-kit-v2-chemistry). Library was sequenced as follows: 26bp Read 1 (16bp Chromium barcode and 10bp UMI), 57bp Read 2 (transcript), and 8bp i7 sample barcode for each scRNA-Seq library.

value: airway epithelial cells  (HAECs)

value: Primary culture


title: SAM24342202 Intestine Small Inflammation KIKO

city: South San Francisco

first: Rohit

last: Reja

state: CA

address: 1 DNA Way

institute: Genentech

data_processing: Reads were aligned to the GRCm38 genome using GSNAP and genes were counted using the HTSeqGenie Bioconductor package. Gene models were from our internal database and available in the manuscript supplementary material. "nRPKM" normalization was performed. This is similar to "RPKM" normalization but includes an extra step for sizeFactor adjustment and is described in the Methods of the manuscript. Supplementary files use Entrez Gene IDs, counts and nRPKM statistics.

source_name: RNA from e16.5 mouse intestine

extract_protocol:
Total RNA was extracted using a RNeasy kit with on-column DNase digestion (Qiagen)
and 0.5ug used for library preparation with a TruSeq RNA Sample Preparation Kit v2 (Illumina).
The libraries were multiplexed and then sequenced on a HiSeq4000 (Illumina) to generate 30 million
single end 50 bp reads.

tag: developmental stage

tag: primary tissue

value: Intestine Small

tag: tissue diagnosis

value: Inflammation

tag: group

value: KIKO:Casp8 C362A knockin MLKL knockout (Casp8 KI MLKL KO)


title: HAECs_Pneumacult_ALI47

description: Sample was prepared 47 days after air-liquid interface in Pneumacult medium. HAECs were cultivated as described in Revinsky et al. (Nature Communications, 2018). Cells were incubated with 0.10% protease type XIV from Streptomyces griseus in HBSS for 4 hours at 4°C degrees. Cells were  detached from the Transwells by pipetting and then transferred to a microtube. 50 units of DNase I per 250µl, were directly added and cells were further incubated at room temperature for 10 min. Cells were washed in 500 µL HBSS 10% Fetal Bovine Serum then in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a Scienceware® Flowmi™ Cell Strainer (40µm porosity), then centrifuged (150g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed and 2000 cells were used for single-cell sequencing.

source_name: Human airway epithellial cells differentiated in Pneumacult medium for 47 days


title: HAECs_Pneumacult_ALI12

description: Sample was prepared 12 days after air-liquid interface in Pneumacult medium. HAECs were cultivated as described in Revinsky et al. (Nature Communications, 2018). Cells were incubated with 0.10% protease type XIV from Streptomyces griseus in HBSS for 4 hours at 4°C degrees. Cells were  detached from the Transwells by pipetting and then transferred to a microtube. 50 units of DNase I per 250µl, were directly added and cells were further incubated at room temperature for 10 min. Cells were washed in 500 µL HBSS 10% Fetal Bovine Serum then in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a Scienceware® Flowmi™ Cell Strainer (40µm porosity), then centrifuged (150g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed and 5000 cells were used for single-cell sequencing.

source_name: Human airway epithellial cells differentiated in Pneumacult medium for 12 days


title: SAM24342211 Intestine Small Normal KO

value: Normal

value: Casp8 wildtype MLKL knockout (Casp8 WT MLKL KO)


title: HAECs_Pneumacult_ALI7

description: Sample was prepared 7 days after air-liquid interface in Pneumacult medium. HAECs were cultivated as described in Revinsky et al. (Nature Communications, 2018). Cells were incubated with 0.10% protease type XIV from Streptomyces griseus in HBSS for 4 hours at 4°C degrees. Cells were  detached from the Transwells by pipetting and then transferred to a microtube. 50 units of DNase I per 250µl, were directly added and cells were further incubated at room temperature for 10 min. Cells were washed in 500 µL HBSS 10% Fetal Bovine Serum then in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a Scienceware® Flowmi™ Cell Strainer (40µm porosity), then centrifuged (150g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed and 5000 cells were used for single-cell sequencing.

source_name: Human airway epithellial cells differentiated in Pneumacult medium for 7 days


title: sALS patient JR48 replicate 1

source_name: sALS Motor cortex

value: motor cortex

tag: subject id

tag: diagnosis

value: sALS


title: HAECs_BEGM_ALI17

description: Sample was prepared 17 days after air-liquid interface in BEGM medium. HAECs were cultivated as described in Revinsky et al. (Nature Communications, 2018). Cells were incubated with 0.10% protease type XIV from Streptomyces griseus in HBSS for 4 hours at 4°C degrees. Cells were  detached from the Transwells by pipetting and then transferred to a microtube. 50 units of DNase I per 250µl, were directly added and cells were further incubated at room temperature for 10 min. Cells were washed in 500 µL HBSS 10% Fetal Bovine Serum then in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a Scienceware® Flowmi™ Cell Strainer (40µm porosity), then centrifuged (150g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed and 1500 cells were used for single-cell sequencing.

source_name: Human airway epithellial cells differentiated in BEGM medium for 17 days


title: SAM24342216 Intestine Small Normal dKO

value: Casp8 MLKL double knockout (Casp8 KO MLKL KO)


title: HAECs_BEGM_ALI62

description: Sample was prepared 62 days after air-liquid interface in BEGM medium. HAECs were cultivated as described in Revinsky et al. (Nature Communications, 2018). Cells were incubated with 0.10% protease type XIV from Streptomyces griseus in HBSS for 4 hours at 4°C degrees. Cells were  detached from the Transwells by pipetting and then transferred to a microtube. 50 units of DNase I per 250µl, were directly added and cells were further incubated at room temperature for 10 min. Cells were washed in 500 µL HBSS 10% Fetal Bovine Serum then in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a Scienceware® Flowmi™ Cell Strainer (40µm porosity), then centrifuged (150g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed and 1000 cells were used for single-cell sequencing. Cell concentration measurements were performed and 2000 cells were used for single-cell sequencing.

source_name: Human airway epithellial cells differentiated in BEGM medium for 62 days


title: SAM24342212 Intestine Small Normal KO


title: TARDBP shRNA knockdown replicate 1

description: KD rep 1

source_name: SH-SY5Y cell line


title: SAM24342210 Intestine Small Inflammation KIKO


title: HAECs_BEGM_ALI12

description: Sample was prepared 12 days after air-liquid interface in BEGM medium. HAECs were cultivated as described in Revinsky et al. (Nature Communications, 2018). Cells were incubated with 0.10% protease type XIV from Streptomyces griseus in HBSS for 4 hours at 4°C degrees. Cells were  detached from the Transwells by pipetting and then transferred to a microtube. 50 units of DNase I per 250µl, were directly added and cells were further incubated at room temperature for 10 min. Cells were washed in 500 µL HBSS 10% Fetal Bovine Serum then in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a Scienceware® Flowmi™ Cell Strainer (40µm porosity), then centrifuged (150g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed and 1500 cells were used for single-cell sequencing.

source_name: Human airway epithellial cells differentiated in BEGM medium for 12 days


title: CD11b Dendritic Cells WT2

description: CD11b Dendritic Cells Atg16l1 + replicate 2

source_name: spleen Dendritic Cells CD11b

tag: strain

value: spleen

tag: age

value: 16 weeks

tag: genotype


title: HAECs_BEGM_ALI22

description: Sample was prepared 22 days after air-liquid interface in BEGM medium. HAECs were cultivated as described in Revinsky et al. (Nature Communications, 2018). Cells were incubated with 0.10% protease type XIV from Streptomyces griseus in HBSS for 4 hours at 4°C degrees. Cells were  detached from the Transwells by pipetting and then transferred to a microtube. 50 units of DNase I per 250µl, were directly added and cells were further incubated at room temperature for 10 min. Cells were washed in 500 µL HBSS 10% Fetal Bovine Serum then in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a Scienceware® Flowmi™ Cell Strainer (40µm porosity), then centrifuged (150g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed and 1500 cells were used for single-cell sequencing.

source_name: Human airway epithellial cells differentiated in BEGM medium for 22 days


title: HAECs_BEGM_ALI7

description: Sample was prepared 7 days after air-liquid interface in BEGM medium. HAECs were cultivated as described in Revinsky et al. (Nature Communications, 2018). Cells were incubated with 0.10% protease type XIV from Streptomyces griseus in HBSS for 4 hours at 4°C degrees. Cells were  detached from the Transwells by pipetting and then transferred to a microtube. 50 units of DNase I per 250µl, were directly added and cells were further incubated at room temperature for 10 min. Cells were washed in 500 µL HBSS 10% Fetal Bovine Serum then in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a Scienceware® Flowmi™ Cell Strainer (40µm porosity), then centrifuged (150g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed and 1500 cells were used for single-cell sequencing.

source_name: Human airway epithellial cells differentiated in BEGM medium for 7 days


title: HAECs_BEGM_ALI4

description: Sample was prepared 4 days after air-liquid interface in BEGM medium. HAECs were cultivated as described in Revinsky et al. (Nature Communications, 2018). Cells were incubated with 0.10% protease type XIV from Streptomyces griseus in HBSS for 4 hours at 4°C degrees. Cells were  detached from the Transwells by pipetting and then transferred to a microtube. 50 units of DNase I per 250µl, were directly added and cells were further incubated at room temperature for 10 min. Cells were washed in 500 µL HBSS 10% Fetal Bovine Serum then in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a Scienceware® Flowmi™ Cell Strainer (40µm porosity), then centrifuged (150g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed and 1500 cells were used for single-cell sequencing.

source_name: Human airway epithellial cells differentiated in BEGM medium for 4 days


title: HAECs_BEGM_ALI2

description: Sample was prepared 2 days after air-liquid interface in BEGM medium. HAECs were cultivated as described in Revinsky et al. (Nature Communications, 2018). Cells were incubated with 0.10% protease type XIV from Streptomyces griseus in HBSS for 4 hours at 4°C degrees. Cells were  detached from the Transwells by pipetting and then transferred to a microtube. 50 units of DNase I per 250µl, were directly added and cells were further incubated at room temperature for 10 min. Cells were washed in 500 µL HBSS 10% Fetal Bovine Serum then in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a Scienceware® Flowmi™ Cell Strainer (40µm porosity), then centrifuged (150g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed and 1500 cells were used for single-cell sequencing.

source_name: Human airway epithellial cells differentiated in BEGM medium for 2 days


title: SAM24342206 Intestine Small Normal dKO


title: SAM24342203 Intestine Small Inflammation KIKO


title: SAM24342214 Intestine Small Normal dKO


title: HEK-293T_siHNRNPH1_rep3

first: Thomas

middle: Joshua

last: Meyer

state: Maryland

address: 41 Center Drive, Building 41, Room B620

department: National Cancer Institute

institute: National Institutes of Health

description: HEK-293T cells treated with siHNRNPH1, replicate 3

data_processing:
RNA libraries were prepared and sequenced on a NextSeq 500 Sequencer
RNA-Seq reads were aligned to the hg19 reference genome using TopHat v2.0.13
FPKM values was calculated using Cufflinks and ucsc genome browser gene level coordinates.
RSEM tools were utilized for the analysis of the expression level of protein-coding EWSR1 transcript variants from targeted RNA-Seq data.
Reads counts across the signature junction of each transcript variant were extracted and calculated using an in-house splicing junction analysis tools developed in Python (v 3.6.0).
Genome_build: hg19

source_name: HEK-293T cells, 48h siHNRNPH1

growth_protocol: Both cell lines were grown in RPMI-1640 (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Invitrogen).

extract_protocol:
RNA was extracted with Maxwell® 16 LEV simplyRNA Cells Kit. RNA quantity was measured using the Nanodrop.
Poly(A)-selected RNA from three biological replicates and sequencing libraries were created using TruSeq Stranded mRNA LT Sample Preparation Kit as described by manufacturer’s instructions (Illumina, San Diego, CA). Then, we incorporated a capture step using fosmid clones of full genomic coverage for EWSR1 (WIBR2-3436F12; GRCh37/hg19 chr22:29662533-29698996) or EWS-FLI1 (WIBR2-3436F12 and WIBR2-1520I3; GRCh37/hg19 chr11:128640224-128682737). Capture bait was prepared by Klenow extension of biotinylated random hexamers priming on purified fosmid DNA template. The prepared bait was bound to streptavidin-coated Dynabeads C1 (Invitrogen) and hybridized to denatured, pooled libraries for 48 hours. The enriched library was recovered after a series of stringency washes by PCR using primers homologous to library end-sequences (Forward: 5'-AATGATACGGCGACCACC-3’, Reverse: 5’-CAAGCAGAAGACGGCATA-3’). Libraries were quantified by qPCR (Kapa Biosystems, Wilmington, MA).

treatment_protocol: To generate enriched EWSR1 and EWS-FLI1 profiles for each cells, total RNA was purified from siRNA-transfected each cells. We transfected cells in six-well plates (1.35E-5 cells per well) using RNAi-max and 20 nM siRNA. Cells were harvested 48 hours post siRNA-transfection.

tag: cell line

value: Human Embryonic Kidney

tag: treatment


title: SAM24342205 Intestine Small Normal KO


title: SAM24342215 Intestine Small Normal dKO


title: HEK-293T_siHNRNPH1_rep1

description: HEK-293T cells treated with siHNRNPH1, replicate 1

tag: cell type


title: SAM24342213 Intestine Small Normal KO


title: SAM24342207 Intestine Small Normal dKO


title: SAM24342208 Intestine Small Inflammation KIKO


title: HEK-293T_siNeg_rep3

description: HEK-293T cells treated with siNeg, replicate 3

source_name: HEK-293T cells, 48h siNeg

value: siNeg


title: HEK-293T_siNeg_rep2

description: HEK-293T cells treated with siNeg, replicate 2


title: TC32_siHNRNPH1_rep3

description: TC32 cells treated with siHNRNPH1, replicate 3

source_name: TC32 cells, 48h siHNRNPH1

value: Ewing sarcoma


title: TC32_siHNRNPH1_rep2

description: TC32 cells treated with siHNRNPH1, replicate 2


title: SAM24342209 Intestine Small Inflammation KIKO

source_name: RNA from e16.5 mouse intestine


title: TC32_siNeg_rep3

description: TC32 cells treated with siNeg, replicate 3

source_name: TC32 cells, 48h siNeg


title: TC32_siHNRNPH1_rep1

description: TC32 cells treated with siHNRNPH1, replicate 1


title: TC32_siNeg_rep1

description: TC32 cells treated with siNeg, replicate 1


title: hNSC

city: Incheon Metropolitan City

first: Yong Seung

last: Lee

address: SimGokro 100gil 25

department: College of Medicine

country: South Korea

institute: Catholic Kwandong University

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence(using fastx_trimmer v.0.0.14), then mapped to hg19 whole genome using tophat2 v.2.0.9
Read count were calculated using edgeR.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include read count values for each Sample

source_name: neural stem cells

growth_protocol: HDF cells were cultured with DMEM including 10% FBS, and 1% penicillin/streptomycin at 37 oC in an atmosphere of 5% CO2. Culture of hNSC was suspension cultured in StemPro® NSC SFM (Gibco) supplemented with 2 mM GlutaMAX™-I Supplement (Gibco), 6 U/mL heparin (Sigma-Aldrich, St. Louis, MO, USA), and 200 µM ascorbic acid (Sigma) at 37 oC in an atmosphere of 5% CO2.  And n/Entr cells were cultured with ultrasound treated hNSC culture medium

extract_protocol:
RNA was isolated using Trizol reagent. The construction of library was performed using SENSE 3’ mRNA-Seq Library Prep Kit from 1 ug of total RNA.
RNA libraries were prepared for sequencing using LEXOGEN Quant-Seq Library Prep Kit standard protocols.

treatment_protocol: The HDF cells were directly exposed to ultrasound stimulus (20 KHz, 1.0 W/cm2) for 5 sec. And then, 2 x 105 cells of ultrasound treated cells were cultured with ultrasound-treated media (20 KHz, 5.0 W/cm2, for 10min) on the 35mm petri dish.

value: brain

tag: passage


title: TC32_siNeg_rep2

description: TC32 cells treated with siNeg, replicate 2


title: 1D n/Entr

source_name: dermal fibroblast cells

value: skin


title: p1 n/Entr