title: METTL8 knockdown polyA-enriched RNA AlkB

city: Chicago

first: Caraline

last: Sepich

state: Illinois

address: 929 E 57th Street

department: Chemistry

institute: University of Chicago

data_processing:
Read1 files from both sequencing experiments were merged using the Linux command cat and then quality checked using FastQC v0.11.5 .
Then, analysis was performed using a pipeline constructed by modifying the framework used by Zhou et al (PMID:PMID: 31548705). Unique molecular identifiers (UMIs) were extracted using UMI-tools. Adapters were trimmed using BBDuk from BBTools version 38.84.
Reads were mapped to the human genome hg38 using hisat2 v2.1.0.
Assembly: hg38
Supplementary files format and content: 240609_m3C_M8KD_mRNA_sites_GEO.csv is a csv file containing a list of putative m3C sites on RNA from the polyA-enriched RNA sequencing experiment. Information is given on the site location, mutation rate in each sample, and coverage for each sample. Calculations were done regarding demethylation rates and changes between control and METTL8 knockdown conditions. Sites were filtered to those in the control libraries with a mutation rate cutoff of 20% and demethylation rate of at least 10%. Information is also included regarding annotations.
Supplementary files format and content: 240609_m3C_M8KD_caRNA_sites_GEO.csv is a csv file containing a list of putative m3C sites on RNA from the chromatin-associated RNA sequencing experiment. Information is given on the site location, mutation rate in each sample, and coverage for each sample. Calculations were done regarding demethylation rates and changes between control and METTL8 knockdown conditions. Sites were filtered to those in the control libraries with a mutation rate cutoff of 20% and demethylation rate of at least 10%. Information is also included regarding annotations.

source_name: HeLa

growth_protocol: Hela cells and derived stable clones were maintained in RPMI supplemented with 10% FBS as previously described.32 For lentivirus production, lentiviral construct TRCN0000236515 and SHC002 (pLKO.1-derived plasmid with target sequence GATCGCCGCTTACAAGTTAAT for METTL8 and CAACAAGATGAAGAGCACCAA as control) together with pCMV-dR8.2 dvpr and pCMV-VSVG were co-transfected into 293TN cells (System Biosciences). Viruses were concentrated using PEG-it Virus precipitation solution (System Biosciences) and used for infecting cells in the presence of TransDux (System Biosciences). Pools of stable transfectants were selected with 2ug/ml puromycin.

extract_protocol:
Total RNA was isolated from HeLa cells using Trizol (Invitrogen) according to the manufacturer’s instructions. Polyadenylated RNA was isolated from total RNA using the Dynabeads mRNA Direct kit (Life Technologies) as per manufacturer instructions. Chromatin-associated RNA was purified as previously described by Liu et al (PMID: 31949099) Small RNA was isolated from total RNA using the RNA Clean and Concentrator kit (Zymo) according to the manufacturer protocol for purification of small and large RNAs into separate fractions, saving the small RNA fraction.
Each RNA sample was fragmented, 3' end repaired, ligated to a 3' adaptor, treated with 5'-exonuclease anddeadenylase, and combined with spike-in probes containing m3C. Then, samples were split into 3 parts: 1 part to be treated withAlkBto demethylate m3C sites, and 2 other untreated technical replicates. Each part then underwent reverse transcription withProtoscriptII, RNase H treatment, linker ligation, PCR, and library purification.

value: HeLa

value: METTL8 knockdown


title: Cgas-/- iBMDMs, 4hr time point, dacA+, biological replicate 3

status: Public on Jul 17 2026

type: SRA

city: Boston

first: Stephanie

last: Ragland

state: MA

address: 1 Blackfan Circle

department: Gastroenterology

country: USA

institute: Boston Children's Hospital

description: Library name: 71

library_source: transcriptomic

data_processing:
Galaxy Server
Assembly: mm10
Supplementary files format and content: tab-delimited text file includes raw counts for each sample

molecule: total RNA

organism: Mus musculus

source_name: iBMDMs

extract_protocol:
RNA was extraced from infected iBMDMs using 300 μL lysis buffer with 1% BME, followed by isolation with the Thermo Fisher Scientific PureLink kit (#12183025). RNA  was treated with Turbo DNase (Thermo #AM190) according to manufacturer's instructions. Illumina RNA-sequencing was performed by Azenta/Genewiz.
NEBNext Ultra II RNA Library Prep Kit for Illumina & NEBNext Poly(A) mRNA Magnetic Isolation Module

tag: cell line

value: iBMDMs

tag: cell type

value: macrophage

tag: genotype

value: Cgas-/-

tag: treatment

value: dacA+

tag: batch

value: same batch for all


title: Grn KO, rimeporide, replicate 4 [HSA-000036448]

city: South San Francisco

first: Thomas

last: Sandmann

email: genomics@dnli.com, sandmann@dnli.com

state: California

address: 161 Oyster Point Blvd

institute: Denali Therapeutics

data_processing:
Data was processed using nf-core/rnaseq v3.11.2 (doi:https://doi.org/10.5281/zenodo.1400710) of the nf-core collection of workflows (Ewelset al., 2020) using Gencode M31 gene annotations. Because the QuantSeq data only captures 3' tags from transcripts, the following custom parameters were supplied: extra_star_align_args: '--alignIntronMax 1000000 --alignIntronMin 20 --alignMatesGapMax 1000000 --alignSJoverhangMin 8 --outFilterMismatchNmax 999 --outFilterType BySJout --outFilterMismatchNoverLmax 0.1 --clip3pAdapterSeq AAAAAAAA' and extra_salmon_quant_args: '--noLengthCorrection'.
Assembly: GRCm39
Supplementary files format and content: File raw_and_normalized_counts.xlsx is a Microsoft Excel file with four worksheets: 1. sample information, 2. gene information, 3. raw count table, 4. normalized count table (log2 counts per million).

molecule: polyA RNA

source_name: brain

growth_protocol: For isolation of primary microglia, brains of neonatal mice (P1-P3) were removed and the cerebral cortices and hippocampus were harvested. Isolated tissues were diced into a fine slurry, suspended in 50mL cold HBSS, and centrifuged at 200xg for 3 minutes. Then, the pelleted tissue was resuspended in warm 0.25% Trypsin-EDTA (Gibco; Cat# 25200056), mixed by inversion, and incubated at 37C for 20 minutes with agitation (100rpm). After 20 minutes, the enzymatic digestion was stopped by adding primary microglia culture medium [DMEM/F12 (Gibco; Cat# 11-330-057), 10% HyClone FBS (Cytiva; Cat# SH30080.03), 1X GlutaMax (Gibco; Cat# 35050061), and penicillin-streptomycin (Gibco; Cat# 15140122)] supplemented with DNase-I (Sigma-Aldrich; Cat# 5025) at 625U/mL culture medium. Dissociated tissue was mixed by inversion 10-12 times and triturated until a homogenous, single-cell suspension was observed. Cells were pelleted by centrifugation at 200xg for 7 minutes, supernatant was removed, and the cells were resuspended in primary microglia culture medium. Cells were filtered through a 70mm strainer to create a single-cell suspension and counted. Poly-D-lysine (Sigma-Aldrich; Cat# P6407)-coated T-75 vented culture flasks were washed twice with PBS and primary microglia were seeded at a density of 6 million cells/flask. After 3 days, the medium was replaced with fresh primary microglia culture medium. Subsequently, half-media changes were performed every other day until the mixed glial culture reached a confluency of 90% (10-14 days). Microglia were then detached from the mixed glial flask using a heated orbital shaker for 2 hours at 240rpm at 37C and 5% CO2. The microglial suspension was centrifuged at 200xg for 8 minutes, resuspended in conditioned medium, counted, and seeded for experiments in 50% conditioned medium with 50% fresh medium. The original mixed glial culture flasks were replenished with fresh medium and maintained in culture with half-media changes every other day. Primary microglia were similarly harvested every 6-8 days for a total duration of 3 weeks. Isolated primary microglia were seeded at assay-dependent concentrations onto poly-D-lysine-coated plates in 50% conditioned medium from the mixed glial culture flask with 50% fresh primary microglia culture medium [DMEM/F12 (Gibco; Cat# 11-330-057), 10% HyClone FBS (Cytiva; Cat# SH30080.03), 1X GlutaMax (Gibco; Cat# 35050061), and penicillin-streptomycin (Gibco; Cat# 15140122)]. After 24 hours, seeded microglia were washed twice with PBS to remove debris, cultured in 50% fresh medium and 50% conditioned medium (collected from mixed glial culture flasks and centrifuged at 1000xg for 3 minutes to remove cellular debris), and used for experiments.

extract_protocol:
RNA was harvested from cells using the RNeasy Micro Kit (Qiagen), following the manufacturer’s protocol. RNA integrity (RIN) and concentration were measured by High Sensitivity RNA Screen Tape Analysis (Agilent) on a 4200 TapeStation System (Agilent). Libraries were generated using the QuantSeq 3’ mRNA-seq Library Prep Kit FWB for Illumina (Lexogen A01173) with the UMI second strand synthesis module in order to identify and remove PCR duplicates, following the protocol defined by the manufacturer. Library quantity and quality were assessed with TapeStation High Sensitivity D1000 Screen Tape (Agilent 5067-5592) using High Sensitivity D1000 reagents (Agilent 5067-5593). Libraries were pooled in equimolar ratios and sent to SeqMatic for sequencing on a NovaSeq S1 (75bp, single-end reads).
Libraries were generated using the QuantSeq 3' mRNA-seq Library Prep Kit FWD for Illumina (Lexogen A01173) with the UMI second strand synthesis module in order to identify and remove PCR duplicates, following the protocol defined by the manufacturer. Library quantity and quality were assessed with TapeStation High Sensitivity D1000 ScreenTape (Agilent 5067-5592) using High Sensitivity D1000 reagents (Agilent 5067-5593).

treatment_protocol: Cells were treated with 2uM rimeporide or equal percentage of DMSO (vehicle) for 72h prior to RNA extraction.

tag: tissue

value: brain

tag: replicate

value: primary microglia

value: Grn KO

value: rimeporide

tag: treatment dose

value: 2 uM

tag: treatment duration

value: 72 hours

tag: rin


title: METTL8 knockdown polyA-enriched RNA input 2

organism: Homo sapiens


title: Ctrl #4

city: Pittsburgh

first: Alyxzandria

last: Gaydosik

state: PA

address: 200 Lothrop St

institute: University of Pittsburgh

description:
Library name: 9166
BC016

library_source: other

data_processing:
Using CellRanger 9.0, FASTQ files were aligned to 10X Genomics reference refdata-gex-GRCh38-2020-A and for fixed FLEX samples the probe set Chromium_Human_Transcriptome_Probe_Set_v1.0.1 was downloaded from 10X Genomics' website and utilized; fixed FLEX samples were also concurrently demultiplexed by probe barcode utilizing cellranger multi.  The resulting feature (in this case, genes) barcode matrices were generated as Hierarchical Data Format (HDF5 or H5) files.
Output Hierarchical Data Format (HDF5 or H5) gene barcode files were processed in R version 4.5.0 using the Seurat package (version 5.2.0)
Assembly: human GRCh38, 10X Genomics refdata-gex-GRCh38-2020-A
Supplementary files format and content: Hierarchical Data Format (HDF5 or H5) containing gene barcode matrices per sample

molecule: other

source_name: muscle

extract_protocol:
For fixed FLEX samples, each samples' FFPE block was cut into several scrolls and prepared into single cell suspensions following 10X Genomics' CG000632 Demonstrated Protocol.  Specifically, the gentleMACS Octo Dissociator for Isolation of Nuclei from FFPE Tissue Sections protocol was employed.  Briefly, scrolls were placed into the gentleMACS C tube and deparaffinized with xylene and rehydrated with graded ethanol washes (100% ethanol, 70% ethanol, 50% ethanol, 100% water), before resuspension in PBS.  Tissue was then dissociated with Liberase TH and the Miltenyi Octo Dissociator.  Nuclei were subsequently filtered through a 30 micron filters and counted with propidium iodided before proceeding to Chromium Fixed RNA Profiling protocols – Probe Hybridization.
Library construction was performed following the manufacturer’s (10X Genomics) official protocols.  For FFPE samples, the FLEX protocol was utilized and, once cell suspensions were generated from FFPE scrolls for each sample, the protocol Chromium Fixed RNA Profiling – Multiplexed Samples with Feature Barcode technology for Protein using Barcode Oligo Capture (CG000673) was utilized to hybridize individual probe barcodes per sample.  Once a probe barcode was associated per sample, library construction proceeded following User Guide CG000527 Chromium Fixed RNA Profiling Reagent Kits for Multiplexed Samples.  Briefly, post-hybridization, the 16 samples containing different probe barcodes were pooled together and unbound probes were washed off.  The samples were then mixed with a Master Mix and loaded into Chromium Chip Q along with gel beads and partitioning oil.  The chip is then loaded into the Chromium X Series instrument to generate the Gel Beads-in-emulsion (GEMs) which are subsequently incubated, amplified, size-selected, indexed, and quantified.  The libraries are standard llumina paired-end constructs, beginning with P5 and ending with P7. The final library includes 16 bp 10x GEM Barcodes (10x Barcode) encoded at the start of TruSeq Read 1 (Read 1T). Finally, the sample index sequences are incorporated as the i5 and i7 index reads.

value: muscle

tag: sample group

value: Ctrl


title: Grn KO, rimeporide, replicate 3 [HSA-000036447]


title: Control polyA-enriched RNA AlkB

value: Control (Control shRNA)


title: Cgas-/- iBMDMs, 4hr time point, untreated, biological replicate 3

description: Library name: 69

value: untreated


title: Ctrl #3

description:
Library name: 16413
BC015


title: Cgas-/- iBMDMs, 4hr time point, clearcoli, biological replicate 3

description: Library name: 70

value: clearcoli


title: METTL8 knockdown polyA-enriched RNA input 1

value: HeLa

value: METTL8 knockdown


title: Grn KO, rimeporide, replicate 2 [HSA-000036446]


title: Ctrl #2

description:
Library name: 17920
BC014


title: Grn KO, rimeporide, replicate 1 [HSA-000036445]


title: Ctrl #1

description:
Library name: 14157
BC013


title: WT iBMDMs, 4hr time point, dacA+, biological replicate 3

description: Library name: 67

value: iBMDMs

value: macrophage

value: WT


title: WT, rimeporide, replicate 4 [HSA-000036444]


title: Control polyA-enriched RNA input 1


title: IBM4

description:
Library name: 37029
BC012

value: IBM


title: WT iBMDMs, 4hr time point, clearcoli, biological replicate 3

description: Library name: 66

value: same batch for all


title: DM3

description:
Library name: 9118
BC011

value: DM


title: iMicroglia, sample 2

city: Rockville

first: Marc

last: Ferrer

state: MD

address: 9800 Medical Center Dr.

department: NCATS (National Center for Advancing Translational Sciences)

institute: NIH (National Institutes of Health)

description: Library name: MGL-MOCK-2

data_processing:
Use DESeq2 for filteration and normalization (minCounts=10, minNumSamples=3,sigAdjP=0.05, sigAbsLFC=1), and downstream DEG analysis
Assembly: GRCh38
Supplementary files format and content: csv file includes raw counts for all 9 samples

source_name: Microglia

growth_protocol: For iCell Microglia, each cryovial was placed in a 37 ℃ water bath for exactly 3 minutes before the content was transferred into a 15 mL conical tubes pre-filled with 3 mL microglia thawing media containing iCell Glial Base Medium (FCDI, Cat# M1034) and 1X iCell Neural Supplement C (FCDI, Cat# M1046). Each cryovial was rinsed with an additional 1 mL of microglia thawing media which was added to the cell suspension before gently mixing. Conical tubes containing iCell microglia were centrifuged at 1000 x g for 10 minutes. The supernatant was aspirated, and each cell pellet was resuspended in 5 mL microglia monoculture media containing iCell Glial Base Medium, 1X iCell Microglia Supplement A (FCDI, Cat# M1036), 1X iCell Microglia Supplement B (FCDI, Cat# M1037), and 1X iCell Neural Supplement C. Each tube of resuspended microglia (>1.5M cell/vial) was seeded into 2 wells of a Costar 6-well Clear Flat Bottom Ultra-Low Attachment (ULA) plate (Corning, Cat# 3471) with approximately 2.5 mL cell resuspension/well and cultured for 3 days in the incubator. 3 days later, microglia in each well were collected using the waterfall technique. Briefly, microglia monoculture media was aspirated and re-dispense into each well of the plate tilted at a 45-degree angle 4 to 5 times to wash the adhering cells off the plate. The microglia resuspension was then transferred into a new 50 mL conical tube. 2 mL pre-chilled DPBS (no calcium no magnesium, Gibco, Cat# 14190144) was added back to each well and the plate was placed in 4 ℃ for 10 minutes before bringing back to the BSC and repeat the waterfall technique for cell collection. The process was repeated 3 to 4 times until most microglia have been collected into the conical tube. Conical tubes containing iCell microglia were centrifuged at 1000 x g for 10 minutes. The supernatant was aspirated, and the cell pellet was resuspended in “Tri-culture Seeding Media” containing iCell Base Medium 1 supplemented with 1X Neural Supplement A and 1X iCell Microglia Supplement A (FCDI, Cat# M1036), until further use. For iCell GABANeurons and iCell Astrocytes, cryovials were placed in a 37 ℃ water bath for exactly 3 minutes before the contents of each vial were transferred into separate conical tubes. For iCell GlutaNeurons and iCell DopaNeurons, cryovials were thawed in a 37 ℃ water bath for exactly 2 minutes. Different thawing media were used in the following steps: (a) Thawing media for iCell GABANeurons and iCell Astrocytes: iCell Base Medium 1 (FCDI, Cat# M1010) supplemented with 1X Neural Supplement A (FCDI, Cat# M1032); (b) Thawing media for iCell GlutaNeurons: BrainPhys Neuronal Medium Without Phenol Red (STEMCELL Technologies, Cat# 05791) supplemented with 1X Neural Supplement B (FCDI, Cat# M1029), 1X Nervous System Supplement (FCDI, Cat# M1031), 1% N2 supplement (Thermo, Cat# 17502048), and 0.1% laminin (Invitrogen, Cat# 23017-015); (c) Thawing media for iCell DopaNeurons: iCell Base Medium supplemented with 1X Neural Supplement B and 1X Nervous System Supplement. 1 mL thawing media correspond to each cell type was used to rinse each cryovial then dispense to the conical tubes with cell suspension in drop-wise fashion. Additional 8 mL of thawing media was slowly added to each tube before gently mixing the cell suspension. iCell GABANeurons and iCell Astrocytes were then centrifuged at 300 x g for 5 minutes while iCell GlutaNeurons and iCell DopaNeurons were centrifuged at 400 x g for 5 minutes. The supernatant was discarded, and cell pellets were resuspended in the Tri-culture Seeding Media. Resuspended cells were counted using a Countess Cell Counter (ThermoFisherScientific) before seeding.

extract_protocol:
Spheroids were grown in 384-well round bottom ultra-low attachment microplates as previously described for RNA extraction and submission to Genewiz from Azenta Life Sciences (New Jersey, USA) for whole RNA sequencing. Approximately 75 spheroids were used per sample, samples were collected in triplicates and extracted total RNA was submitted for RNA sequencing (N = 3 per condition from 1 experiment, Genewiz). For RNA extraction, spheroids were collected in 2 mL tubes and centrifuged for 5 min at 500 g and supernatant was discarded. Remaining cell pellet was lysed using TRIzol™ Reagent (Cat# 15596026) according to product input sample requirements of 5-106 cells per 1 mL of TRIzol™ Reagent. The samples were stored frozen at -80 C as a stopping point prior to completion of RNA isolation. Total RNA was purified using Direct-zol™ RNA MiniPrep Kits according to manufacturer’s instructions (Zymo Research, Cat# R2052). Concentration and quality of RNA was determined to match the guidelines of Genewiz from Azenta Life Sciences for RNA sequencing (ultra-low input), and concentration ≥20 ng/μL total RNA resuspended in Nuclease-free water.
Libraries were constructed by Genewiz from Azenta Life Sciences for RNA sequencing with ultra-low input. Sequencing was done with MiSeq, PolyA selected and non-strand specific using paired-end 250 bp (2x250). The data output was 20M read pairs (40M raw reads).

treatment_protocol: No treatments were used other than regular media maintanance.

value: Microglia

value: iPSC derived neural cells

value: iMicroglia

value: Cell lines used in this study includes two donors across cell lines used, based on commercial availability:Donor ID# 01434, Caucasian female (age < 18) with a healthy phenotype and Donor ID# 01279, Caucasian male (aged 50-59) with a healthy phenotype. Wildtype cell lines used includes:iCell Astrocytes (Cat# R1092, Donor ID# 01434, differentiated from fibroblast), iCell GlutaNeurons (Cat# C1033, Donor ID# 01279, differentiated from blood-derived iPSCs, only use if Gluta purity is >80%), iCell GABANeurons (Cat# C1012, Donor ID# 01434, differentiated from fibroblast), iCell DopaNeurons (Cat# C1028, Donor ID# 01279, differentiated from blood-derived iPSCs), and iCell Microglia (Cat# C1110, Donor ID# 01279, differentiated from blood-derived iPSCs


title: WT, rimeporide, replicate 3 [HSA-000036443]

value: brain

value: primary microglia

value: rimeporide

value: 2 uM

value: 72 hours


title: iMicroglia, sample 3

description: Library name: MGL-MOCK-3


title: WT iBMDMs, 4hr time point, untreated, biological replicate 3

description: Library name: 65

value: untreated


title: WT, rimeporide, replicate 2 [HSA-000036442]


title: NM3

description:
Library name: 24124
BC010

value: muscle

value: NM


title: Myd88-/-Trif-/-Lrrc8a-/- iBMDMs, 4hr time point, CV-Cglr2+, biological replicate 2

description: Library name: 64

value: iBMDMs

value: macrophage


title: NM2

description:
Library name: 39232
BC009


title: iMicroglia, sample 1

description: Library name: MGL-MOCK-1


title: Myd88-/-Trif-/-Lrrc8a-/- iBMDMs, 4hr time point, dacA+, biological replicate 2

description: Library name: 63

value: dacA+


title: VTA-like triculture NeuroMIMIC, sample 3

description: Library name: VTAMGL-MOCK-3

source_name: VTA-like NeuroMIMICS

value: VTA-like NeuroMIMICS

value: iDopaminergic neurons, iAstrocytes,iGlutamatergic neurons, iGABAergic neurons, iMicroglia


title: Grn KO, DMSO, replicate 4 [HSA-000036440]

value: Grn KO

value: DMSO

value: 0 uM


title: IBM2

description:
Library name: 10696
BC008

value: IBM


title: Grn KO, DMSO, replicate 3 [HSA-000036439]

value: brain


title: Myd88-/-Trif-/-Lrrc8a-/- iBMDMs, 4hr time point, untreated, biological replicate 2

description: Library name: 61

value: same batch for all


title: Grn KO, DMSO, replicate 2 [HSA-000036438]

value: primary microglia


title: VTA-like triculture NeuroMIMIC, sample 2

description: Library name: VTAMGL-MOCK-2

value: iPSC derived neural cells

value: Cell lines used in this study includes two donors across cell lines used, based on commercial availability:Donor ID# 01434, Caucasian female (age < 18) with a healthy phenotype and Donor ID# 01279, Caucasian male (aged 50-59) with a healthy phenotype. Wildtype cell lines used includes:iCell Astrocytes (Cat# R1092, Donor ID# 01434, differentiated from fibroblast), iCell GlutaNeurons (Cat# C1033, Donor ID# 01279, differentiated from blood-derived iPSCs, only use if Gluta purity is >80%), iCell GABANeurons (Cat# C1012, Donor ID# 01434, differentiated from fibroblast), iCell DopaNeurons (Cat# C1028, Donor ID# 01279, differentiated from blood-derived iPSCs), and iCell Microglia (Cat# C1110, Donor ID# 01279, differentiated from blood-derived iPSCs


title: Myd88-/-Trif-/-Lrrc8a-/- iBMDMs, 4hr time point, clearcoli, biological replicate 2

description: Library name: 62

value: clearcoli


title: ASyS3

description:
Library name: 32732
BC007

value: muscle

value: ASyS


title: VTA-like triculture NeuroMIMIC, sample 1

description: Library name: VTAMGL-MOCK-1


title: Myd88-/-Trif-/-Sting-/- iBMDMs, 4hr time point, CV-Cglr2+, biological replicate 2

description: Library name: 60

value: iBMDMs


title: VTA-like neural co-culture spheroid, sample 3

description: Library name: VTA-MOCK-3

source_name: VTA-like neural spheroid

value: VTA-like neural spheroid

value: iDopaminergic neurons, iAstrocytes,iGlutamatergic neurons, iGABAergic neurons


title: Grn KO, DMSO, replicate 1 [HSA-000036437]

value: 72 hours


title: Myd88-/-Trif-/-Sting-/- iBMDMs, 4hr time point, dacA+, biological replicate 2

description: Library name: 59

value: macrophage

value: dacA+


title: WT, DMSO, replicate 4 [HSA-000036436]

value: WT

value: DMSO


title: DM1

description:
Library name: 6813
BC004

value: DM


title: VTA-like neural co-culture spheroid, sample 1

description: Library name: VTA-MOCK-1


title: Myd88-/-Trif-/-Sting-/- iBMDMs, 4hr time point, clearcoli, biological replicate 2

description: Library name: 58


title: WT, DMSO, replicate 3 [HSA-000036435]

value: brain

value: 0 uM


title: VTA-like neural co-culture spheroid, sample 2

description: Library name: VTA-MOCK-2


title: ASyS1

description:
Library name: 35145
BC003


title: NM1

description:
Library name: 32635
BC002

value: NM


title: WT, DMSO, replicate 1 [HSA-000036433]

value: primary microglia


city: Memphis

first: Wojciech

last: Rosikiewicz

state: TN

address: 262 Danny Thomas Place

department: Center for Applied Bioinformatics

institute: St. Jude Childrens Research Hospital

description: Library name: Kasumi-1_RAD21_4bp-correction.Miller-Plate33-F04-TATCATTCGA-AGCATG

library_source: genomic

data_processing:
samples were demultiplexed using index sequences
fastq files were generated
fastq riles were merged with Paired-End reAd mergeR (PEAR)
analysis using CRIS.py
Supplementary files format and content: .txt files; top exact read counts and indel sizes and matches to test_seqs

molecule: genomic DNA

source_name: cell line

extract_protocol:
crude lysis with QuickExtract buffer
targeted amplicon sequencing using 2-step PCR

treatment_protocol: nucleofection of RNPs

value: cell line

value: Myeloid leukemia of Down syndrome (ML-DS) cell line


description: Library name: Kasumi-1_RAD21_4bp-correction.Miller-Plate33-F02-GTGACATGAA-AGCATG


title: IBM1

description:
Library name: 29125
BC001

value: muscle

value: IBM


title: Myd88-/-Trif-/-Cgas-/- iBMDMs, 4hr time point, dacA+, biological replicate 2

description: Library name: 55

value: iBMDMs

value: same batch for all


title: BMDM, GrnKO, DMSO, replicate 4 [HEX000747_BMDM]

data_processing:
Data was processed using nf-core/rnaseq v3.11.2 (doi:https://doi.org/10.5281/zenodo.1400710) of the nf-core collection of workflows (Ewelset al., 2020) using Gencode M31 gene annotations. Because the QuantSeq data only captures 3' tags from transcripts, the following custom parameters were supplied: extra_star_align_args: '--alignIntronMax 1000000 --alignIntronMin 20 --alignMatesGapMax 1000000 --alignSJoverhangMin 8 --outFilterMismatchNmax 999 --outFilterType BySJout --outFilterMismatchNoverLmax 0.1 --clip3pAdapterSeq AAAAAAAA' and extra_salmon_quant_args: '--noLengthCorrection'.
Assembly: GRCm39
Supplementary files format and content: File raw_and_normalized_counts.xlsx is a Microsoft Excel file with four worksheets: 1. sample information, 2. gene information, 3. raw count table, 4. normalized count table (log2 counts per million),

source_name: Bone-marrow-derived macrophage

growth_protocol: Animals were sacrificed and the femur and tibia bones were collected into cold HBSS (Gibco; Cat# 14175095). Bones from multiple mice (6 for WT only studies) were pooled for each individual biological replicate and sterilized with 70% isopropanol, washed three times with HBSS, and cracked in HBSS using a mortar and pestle. The cell suspension containing bone marrow cells was filtered through a 70mm strainer to remove cracked bone pieces and centrifuged at 300xg for 5 minutes. The supernatant was removed, and the pellet was resuspended in ACK Lysing Buffer (ThermoFisher; Cat# A1049201) for 2 minutes at room temperature to remove red blood cells. Then, BMDM culture medium [RPMI-1640 (Gibco; Cat# 61870036), 10% HyClone FBS (Cytiva; Cat# SH30080.03), and penicillin-streptomycin (Gibco; Cat# 15140122)] was added to stop the ACK lysis reaction. Cells were pelleted at 300xg for 5 minutes and the supernatant was discarded. Then, the cell pellet was resuspended in BMDM medium, counted, and plated onto 15cm non-tissue culture treated petri dishes at a density of 20 million cells/plate in BMDM medium containing murine M-CSF (Life Technologies; Cat# PMC2044) at 50ng/mL. Four days after seeding the cells, an additional 50ng/mL fresh M-CSF was added to each plate. On day 7, differentiated BMDMs were washed once with PBS and harvested in BMDM medium using a cell scraper. Cells were pelleted at 300xg for 5 minutes, resuspended in BMDM medium, and counted. Finally, cells were centrifuged at 300xg for 5 minutes, resuspended in Recovery Cell Culture Freezing Medium (Gibco; Cat# 12648010) at a density of 10 million cells/mL, and cryopreserved for later use. Differentiated BMDMs were thawed, counted, and seeded at 75k cells/well onto poly-D-lysine-coated plates in BMDM culture medium [RPMI-1640 (Gibco; Cat# 61870036), 10% HyClone FBS (Cytiva; Cat# SH30080.03), and penicillin-streptomycin (Gibco; Cat# 15140122)] with supplemental M-CSF (50ng/mL). Fresh BMDM medium was changed 24 hours after seeding of the cells.

tag: strain

value: Grn KO

value: Bone-marrow-derived macrophage


description: Library name: Kasumi-1_RAD21_4bp-correction.Miller-Plate33-F01-CGATCGATGG-AGCATG


title: HeLa_METTL8_OE_small_RNA_AlkB

description: Library name: m3C-16

data_processing:
Reads were trimmed using the 'trim' command in HomerTools31, and low-quality reads were subsequently removed. The remaining high-quality reads were then mapped to spike-in sequences, the mitochondrial genome (NC_012920.1), and tRNA sequences (Homo sapiens GRCh38/hg38 from GtRNAdb, https://gtrnadb.ucsc.edu/) using BWA32 with the following parameters: 'bwa aln -t 4 -n 0.06 -q 20' for downstream analysis. m3C sites were defined as sites with an average mutation rate greater than 0.2 in input samples and less than 0.05 in AlkB-treated samples
Assembly: high-quality reads were then mapped to spike-in sequences, the mitochondrial genome (NC_012920.1), and tRNA sequences (Homo sapiens GRCh38/hg38 from GtRNAdb, https://gtrnadb.ucsc.edu/) using BWA32
Supplementary files format and content: mutation rates at tRNA m3C sites in each sample

growth_protocol: Hela cells and derived stable clones were maintained in RPMI supplemented with 10% FBS as previously described (Mol Cell. 2020 Nov 19;80(4):633-647.e7).

extract_protocol:
Total RNA was isolated from HeLa cells using Trizol (Invitrogen) according to the manufacturer’s instructions. Small RNA was isolated from total RNA using the RNA Clean and Concentrator kit (Zymo) according to the manufacturer protocol for purification of small and large RNAs into separate fractions, saving the small RNA fraction.
300 ng of small RNA was mixed with 3 μL of 1 ng/μL mixture of spike-in m3C probes (DH-718-5-33mer: 5'-GGA GAC GGU CGG Gm3CC CAG AUA UUC GUA UCU GUC-3’ and DP-518-6-27mer: 5'-UAC ACU CAG m3CUC GGA CUA AAG CUG) and split into two for 3’ RNA ligation. RNA in 9 μL RNase-free water was mixed with 1 μL of 11.25 μM 3 '-adaptor (5-’rApp-NN NNN ATC ACG AG ATC GGA AGA GCA CAC-3’) and incubated at 70 °C for 2 minutes. Then, after chilling on ice for 2 minutes, 2 μL of RNase-free water, 2.5 μL of 10x T4Rnl1 buffer (NEB #B0216S), 7.5 μL of 50% PEG800 (NEB #B1004S), 1 μL of SuperaseIn (Invitrogen, AM2694) and 2 μL of T4 RNA ligase 1 truncated KQ (NEB #0373) were added and mixed well. The reaction mixture was incubated at 25 °C 1 h followed by 16 °C for 12 h. Then, RNase-free water was added to the total volume of 47 μL and 2 μL of 5 '-deadenylase (NEB #M0331) was added, mixed and incubated at 30 °C for 1 h. Next, 1 μL of RecJf (NEB, M0264S) was added, mixed and incubated at 37 °C for 1 h. After that, two ligation mixtures coming from the same sample were pooled together, purified by RNA Clean and Concentrator kit (Zymo) and eluted with 31 μL RNase-free water. Then, sample was split into four parts: 3 x 3 μL were used as three technical replicates and 20 μL was treated with AlkB as described previously (Nat Cell Biol. 2021;23(7):684-691). After treatment with AlkB, reverse transcription was performed as follows: To RNA in 8.5 μL RNase-free water, 1 μL of 3.75 μM (input) or 1 μL of 11.25 μM (AlkB-treated) SR RT primer (5 '-AGA CGT GTG CTC TTC CGA TCT, IDT) was added and incubated at 65 °C for 5 minutes then chilled on ice for 2 min. Then, 4 μL of 5x PSII buffer, 2 μL of 0.1 M DTT, 2 μL of 10 mM dNTPs, 0.5 μL of RNase inhibitor (NEB, M0314) and 1 μL of PSII (NEB M0368L) were added, mixed and incubated at 42 °C for 1h followed by overnight incubation at 35 °C for 16 hours. Next, 1 μL of RNase H buffer and 1 μL of RNase H were added, mixed and incubated at 37 °C for 30 min. Then, sample volume was adjusted to 50 μL with Nuclease-free water and purified by RNA Clean and Concentrator kit (Zymo) and eluted with 6.5 μL of Nuclease-free water. cDNA ligation was performed as follows: 1 μL of 10 μM 5’ linker (5 '-/Phos/NNN NNG ATC GTC GGA CTG TAG AAC TCT GAA C/3SpC3, IDT) and 1 μL of DMSO were mixed with 5.5 μL of cDNA and incubated at 70 °C for 2 min. and then chilled on ice for 2 min. Next, 2 μL of 10x RNA ligation buffer (NEB #B0216S), 0.2 μL of 100 mM ATP, 9 μL of 50% PEG8000 (NEB, B1004S), 0.6 μL of 50 mM hexaamminecobalt (III) chloride [Co(NH3)6Cl3] (Sigma-Aldrich 481521), 0.4 μL of 5’-deadenylase (NEB, M0331) and 1.5 μL of T4Rnl1 high concentration (NEB, M0437M) were mixed and incubated at 25 °C for 16 hours. Then, 7 volumes of DCC binding buffer were added and samples were purified by DNA Clean and Concentrator kit (Zymo) and eluted with 20 μL Nuclease-free water. qPCR was then conducted to test the cycles needed for library amplification: 10 μL FastStart Essential DNA Green Master Mix (Roche), 1 μL index 1 primer (NEB small RNA kit, E7330S), 1 μL SR primer (NEB, E7330S), 1 μL cDNA template, and 7 μL RNase-free water on a LightCycler96 (Roche). PCR was performed for each sample using the appropriate number of cycles per sample by combining 12.5 μL 2x LongAmp Taq Mix (NEB small RNA kit), 0.625 μL SR primer (NEB small RNA kit), 1.25 μL RNase-free water, 10 μL cDNA, and 0.625 μL of an index primer unique to each sample (NEB). Samples were pre-incubated at 94 °C for 30s, followed by cycling for a number of cycles determined by qPCR at 94 °C for 15 seconds, 62 °C for 30 seconds, and 70 °C for 15 seconds, and finishing with a 70 °C, 5-minute final extension. Libraries were purified using a 3% low melting agarose gel using 6x loading buffer (NEB #B7024) and the Quick Load pBR322 DNA-MspI Digest NEB #N3032 ladder. Samples were run at 90 V for 1-1.5 hours and purified by MinElute Gel Extraction Kit (Qiagen). Samples were sequenced on the NextSeq2000 system (Illumina).

treatment_protocol: For lentivirus production, lentiviral constructs TRCN0000236515 and SHC002 (pLKO.1-derived plasmid with target sequence GATCGCCGCTTACAAGTTAAT for METTL8 and CAACAAGATGAAGAGCACCAA as control) together with pCMV-dR8.2 dvpr and pCMV-VSVG were co-transfected into 293TN cells (System Biosciences). Viruses were concentrated using PEG-it Virus precipitation solution (System Biosciences) and used for infecting cells in the presence of TransDux (System Biosciences). Pools of stable transfectants were selected with 2 μg/ml puromycin.

value: HeLa

value: METTL8 overexpression


title: Myd88-/-Trif-/-Cgas-/- iBMDMs, 4hr time point, clearcoli, biological replicate 2

description: Library name: 54

value: macrophage

value: clearcoli


description: Library name: CMY_RAD21_correction.Miller-Plate65-D06-TCGGTCCGTA-TGTTCG

value: CMY


title: HeLa_METTL8_OE_small_RNA_input_#3

description: Library name: m3C-15


title: Myd88-/-Trif-/-Cgas-/- iBMDMs, 4hr time point, untreated, biological replicate 2

description: Library name: 53

value: untreated


title: BMDM, GrnKO, DMSO, replicate 2 [HEX000747_BMDM]

value: DMSO


description: Library name: CMY_RAD21_correction.Miller-Plate65-D05-AAGGTGGTAA-TGTTCG


title: HeLa_METTL8_OE_small_RNA_input_#2

description: Library name: m3C-14


title: BMDM, GrnKO, DMSO, replicate 1 [HEX000747_BMDM]


title: Myd88-/-Trif-/- iBMDMs, 4hr time point, dacA+, biological replicate 2

description: Library name: 51

value: dacA+


description: Library name: CMY_RAD21_correction.Miller-Plate65-D03-GGTATTGCTG-TGTTCG


title: Myd88-/-Trif-/- iBMDMs, 4hr time point, CV-Cglr2+, biological replicate 2

description: Library name: 52


title: BMDM, WT, DMSO, replicate 4 [HEX000747_BMDM]

value: wildtype


description: Library name: CMY_RAD21_correction.Miller-Plate65-D02-GAGACGGTCT-TGTTCG

value: cell line


title: HeLa_Ctrl_OE_small_RNA_AlkB

description: Library name: m3C-12

value: overexpression control


title: BMDM, WT, DMSO, replicate 3 [HEX000747_BMDM]


title: HeLa_Ctrl_OE_small_RNA_input_#3

description: Library name: m3C-11


title: Myd88-/-Trif-/- iBMDMs, 4hr time point, untreated, biological replicate 2

description: Library name: 49


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-C02-GTGGCGTAGA-CTAGTC

value: Myeloid leukemia of Down syndrome (ML-DS) cell line


title: Sting-/- iBMDMs, 4hr time point, CV-Cglr2+, biological replicate 2

description: Library name: 44

value: Sting-/-


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-C01-GTTGACCGCG-CTAGTC


title: BMDM, WT, DMSO, replicate 1 [HEX000747_BMDM]


title: BMDM, GrnKO, DMSO, replicate 3 [HEX000747_BMDM]


title: Sting-/- iBMDMs, 4hr time point, dacA+, biological replicate 2

description: Library name: 43

value: iBMDMs


title: HeLa_METTL8_OE_small_RNA_input_#1

description: Library name: m3C-13


title: Healthy blood plasma cfDNA Male 10 P5plain P7plain primers control 2 (Nanopore)

city: Aurora

first: Srinivas

last: Ramachandran

state: CO

department: Biochemistry and Molecular Genetics

institute: University of Colorado School of Medicine

description:
Library name: Nanopore_P5plnP7pln_Control_2
No_Enrich_ALL.bw

data_processing:
Illumina cfDNA datasets were processed with Cutadapt in paired-end mode to remove Illumina adapters. Only reads at least 35 bp after removal of adapters were retained. Reads were trimmed down to a maximum of 130 bp using Cutadapt. Trimmed reads were aligned to the human reference genome (T2T CHM13v2.0/hs1) using bowtie2 (with parameters “--local --very-sensitive-local  --no-unal --no-mixed --no-discordant -I 10 -X 500”). Samtools and bedtools were used for processing aligned reads from SAM to BED files. Duplicate reads were discarded for further analysis if the reads had the same start and end coordinates. For Nanopore data, base-calling was performed with Dorado. Since P5/P7 adapters were ligated before ONT library preparation, we could use them as anchors to precisely define read lengths. Hence, we wrote custom python script to trim P5 and P7 adapters from ONT reads. Reads were also aligned to the CHM13 T2T hs1 v2 human reference genome using minimap2. Alignments with SAM flags of 0 and 16 were selected and used for constructing BigWig files.
For Illumina libraries, BigWig files were created using centers of fragments between 120 and 160 bp. Selected fragment centers were extended by 30 bp upstream and downstream, and coverage was calculated  at 10 bp windows genome-wide, which was calculated as the number of extended fragment centers that mapped at that window, normalized by the factor N: N = 3117275501/(Total number of mapped reads). 3117275501 was a number chosen arbitrarily. For Nanopore reads, BigWig files were created for fragments (with an upper limit of 100 kb in length). Coverage was calculated at 100 bp windows genome-wide, which is number of fragments that map at a window normalized by a factor N: 3117275501/(Total number of mapped reads). 3117275501 was a number chosen arbitrarily.
Assembly: Telomere to Telomere (T2T) CHM13 hs1 v2
Supplementary files format and content: bw

source_name: plasma

extract_protocol:
Human plasma (1 to 10 ml) was thawed from −80°C storage. Plasma samples were spun at maximum speed [21,000 rcf (relative centrifugal force)] at 4°C for 5 to 10 min to pellet any cell debris. The blood plasma supernatant was transferred to new tubes. For 1 mL blood plasma cfDNA extractions the following steps were followed. 1 mL of blood plasma was transferred to a 5 mL conical and 10 µL of proteinase K (20 mg/mL) was added to each donor sample. 650 µL of Digestion Buffer (5M GITC, 25% Tween 20, 50 mM Tris pH 8.0, and 25 mM EDTA) was added to the blood plasma and mixed well by pipetting. The samples incubated at 56°C for 1 hour. 3.3 mL of Binding Buffer (3.5M GITC, 45% Isopropanol, 2.5% Tween 20, 10 mM Tris pH 8.0, and 1mM EDTA) was added to each sample. 40 µL of 400 nm SPHERO Silica Superparamagnetic beads were added to each blood plasma sample and mixed well by vortexing. The bead-plasma slurry incubated for 10 minutes at RT. The tubes were placed on a magnetic rack and when the supernatant was clear (~ 5 min) the blood plasma supernatant was discarded. The tubes were then removed from the magnetic rack, the beads were resuspended and washed with 500 µL of Wash Buffer 1 (3M GITC, 30% Isopropanol, 5% Tween 20, 40 mM Bis-Tris pH 6.0, and 2 mM EDTA). The tubes were placed back on the magnetic rack and once the slurry was clear the supernatant was discarded. The tubes were removed again and were resuspended and washed with 500 µL of Wash Buffer 2 (50 mM Tris pH 8.0, 0.5 mM EDTA, and 80% EtOH). The tubes were placed back on the magnetic rack and once the slurry was clear the supernatant was discarded. The tubes were removed and resuspended with 100 µL of 100% EtOH. The new sample slurries were moved to 1.5 mL tubes. The tubes were then placed on a 1.5 mL magnetic rack and when the slurry cleared the supernatant was discarded. The tubes were spun down briefly and returned to the magnetic rack to remove the remaining EtOH. With the tube caps open they were air dried at 37°C for 2 minutes in a bead bath. 30 µL of nuclease free H2O were added to each sample and carefully mixed by pipetting. The tubes incubated at RT for 5 minutes and were returned to the magnetic rack. Once the supernatant was clear the DNA was eluted to a new tube for each sample. DNA fragments were then checked for quality using the Invitrogen Qubit dsDNA HS Assay kit.
UltraPrep cfDNA Isolation: cfDNA extraction was performed using the UltraPrep method. Briefly, human plasma (1 to 10 mL) was thawed from −80°C and spun at 21,000 rcf (relative centrifugal force), 4°C for 5 to 10 min to pellet any cell debris. The blood plasma supernatant was transferred to new tubes. 1 mL of blood plasma was transferred to a 5 mL conical tube, and 10 µL of proteinase K (20 mg/mL) was added to each donor sample. 650 µL of Digestion Buffer (5M guanidine thiocyanate (GITC), 25% Tween 20, 50 mM Tris pH 8.0, and 25 mM ethylenediaminetetraacetic acid (EDTA)) was added to the plasma and mixed well by pipetting. The samples were incubated at 56°C for 1 hour. 3.3 mL of Binding Buffer (3.5M GITC, 45% isopropanol, 2.5% Tween 20, 10 mM Tris pH 8.0, and 1 mM EDTA) was added to each sample. 40 µL of 400 nm SPHERO Silica Superparamagnetic beads (Spherotech) were added to each blood plasma sample and mixed well by vortexing. The bead-plasma slurry was incubated for 10 minutes at room temperature (RT). The tubes were placed on a magnetic rack, and when the supernatant was clear (~ 5 minutes), it was discarded. The tubes were then removed from the magnetic rack, the beads were resuspended and washed with 500 µL of Wash Buffer 1 (3M GITC, 30% isopropanol, 5% Tween 20, 40 mM bis-tris pH 6.0, and 2 mM EDTA). The tubes were placed back on the magnetic rack, and once the slurry was clear, the supernatant was discarded. The beads were resuspended and washed with 500 µL of Wash Buffer 2 (50 mM Tris pH 8.0, 0.5 mM EDTA, and 80% ethanol). The tubes were placed back on the magnetic rack, and once the slurry was clear, the supernatant was discarded. The beads were resuspended with 100 µL of 100% EtOH. The new sample slurries were moved to 1.5 mL tubes. The tubes were then placed on a 1.5 mL magnetic rack, and when the slurry cleared, the supernatant was discarded. The tubes were spun down briefly and returned to the magnetic rack to remove the remaining EtOH. With the tube caps open, they were air dried at 37°C for 2 minutes in a bead bath. 30 µL of nuclease-free H2O was added to each sample and carefully mixed by pipetting. The tubes were incubated at RT for 5 minutes and returned to the magnetic rack. Once the supernatant was clear, the DNA was eluted into a new tube for each sample. Extracted DNA was quantified using the Invitrogen Qubit dsDNA HS Assay. Sequencing Libraries: cfDNA libraries were generated using the SRSLY PicoPlus Base Kit (cat. CBS-K250B-96). 1 to 10 ng of cfDNA per sample was combined with Nuclease-Free (NF) H2O and SRSLY ss Enhancer in a 0.5 mL Axygen PCR tube (Fisher Scientific cat. 14-222-290) on ice. Following incubation on ice for 2 minutes, tubes were moved into a 0.5 mL PCR block for a heat shock at 98°C (lid 105°C) for 3 minutes to allow denaturation. The tubes were then incubated on ice for 2 minutes and the PCR block was set to 37°C (lid temp 45°C). A phosphorylation master mix was created by combining the SRSLY Master Mix, SRSLY Adapter A, and SRSLY Adapter B, all provided from the SRSLY Base Kit. To each sample, a phosphorylation master mix was added for a final volume of 50 μL. Once the tubes were mixed well via vortex, they were incubated for 1 hour in a PCR block at 37°C (lid temp 45°C). cfDNA was isolated after incubation using AMPure XP Beads (Beckman Coulter cat. A63881) at a ratio of 0.54x, mixed with 100% Isopropanol at a ratio of 0.24x, and NF-H2O.  A 0.5 mL magnetic rack was used to capture and wash cfDNA with 80% EtOH. cfDNA-beads were suspended in 15 μL of NF-H2O, and cfDNA was eluted into new 0.5 mL Axygen PCR Tubes. Purified cfDNA libraries were amplified via PCR with a master SRSLY PCR Mix (provided in the base kit) and NF-H2O. Uniquely barcoded primers, P5 and P7, with a final concentration of 0.25 μM each, were added to each sample. Samples were initially incubated at 98°C (lid 105°C) for 3 minutes, followed by 10 cycles of amplification [98°C for 20 seconds, 65°C for 30 seconds, 72°C for 30 seconds], 72°C for 1 minute, then a final hold at 12°C. After PCR, cfDNA libraries were captured and washed with AMPure XP beads at a ratio of 0.95x. The final elution was in 20 μL 0.1X TE buffer and transferred to 1.5 mL Eppendorf LoBind tubes (cat. 022431021).  Eluted libraries were quantified via Qubit (dsDNA Quantitation, High Sensitivity kit cat. Q32854) and quality checked using an Agilent Tapestation (D1000 tape cat. 5067-5582).  Sequencing libraries were stored at -20°C until shipped for sequencing on the Novaseq. All libraries contained dual barcodes and were subjected to 150 bp paired-end sequencing. PCR enrichment of telomere sequences from SRSLY cfDNA libraries: cfDNA libraries until the PCR amplification step were generated as described above. To enrich for telomeric sequences for long-read sequencing, the libraries were amplified using CloneAmp HiFi PCR premix, which accurately amplifies long amplicons. Either standard P5/P7 primers or primers that recognize the junction between Illumina adapters and telomeric repeats were used for amplification (primer sequences below). We used three primer sets: 1. P5 standard primer sequence and P7 standard primer sequence 2. P5 standard primer sequence and P7 + telomere repeat sequence 3. P7 standard primer sequence and P5 + telomere repeat sequence The PCR conditions were (lid temp 105°C), 98°C 10 seconds, 52°C 15 seconds, 72°C 5 seconds, repeated for 12 total cycles, and a 10°C holding temperature. The cfDNA PCR product was purified using a 2X AMPure bead clean. Eluted libraries were quantified using Qubit (dsDNA Quantitation, High Sensitivity kit cat. Q32854) and quality-checked using an Agilent Tapestation (D1000 tape cat. 5067-5582).  Sequencing libraries were stored at -20°C until utilized for Nanopore sequencing. P5 standard primer sequence: 5’ TTTCCCTACACGACGCTCTT 3’ P7 standard primer sequence: 5’ GTTCAGACGTGTGCTCTTCC 3’ P5 + telomere repeat sequence: 5’ TCTTCCGATCTCCCTAACCCTA 3’ P7 + telomere repeat sequence: 5’ CTCTTCCGATCTCCCTAACCC 3’ Oxford Nanopore Long Read Sequencing: The Nanopore Ligation Sequencing DNA SQK-LSK 110 and SQK-LSK 114 kits were used to generate cfDNA Nanopore sequencing libraries, which were sequenced on R9.4.1 MinION and R10.4.1 PromethION flow cells, respectively.

value: plasma

tag: age

value: 25 yo

tag: sample type

value: Blood plasma cfDNA


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-B12-ATCTTATGCG-CTAGTC


title: Myd88-/-Trif-/- iBMDMs, 4hr time point, clearcoli, biological replicate 2

description: Library name: 50

value: same batch for all


title: HeLa_shMETTL8_small_RNA_AlkB

description: Library name: m3C-8

value: HeLa

value: METTL8 knockdown


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-C03-AGCTCAGGGT-CTAGTC


title: Sting-/- iBMDMs, 4hr time point, clearcoli, biological replicate 2

description: Library name: 42

value: macrophage

value: clearcoli


title: MSU, 7 hours, replicate 2 [HSA-000022209]

source_name: BMDM

treatment_protocol: Cells were treated with DMSO, 25nM BafA1, 6uM U18666A, 2uM VPS34IN1, 50uM E64 + 50uM leupeptin + 50uM pepstatin (E/LP), or 200ug/mL monosodium urate (MSU) for 1, 7, or 22h to RNA extraction. Total DMSO concentration was equal across all different treatments.

value: BMDM

value: WT

value: MSU

value: 200 ug/ml

value: 7 hours


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-B11-CATCCCTTCT-CTAGTC

value: cell line

value: CMY


title: BMDM, WT, DMSO, replicate 2 [HEX000747_BMDM]

value: wildtype

value: Bone-marrow-derived macrophage

value: DMSO


title: Healthy blood plasma cfDNA Male 10 P5plain P7plain primers control 1 (Nanopore)

description:
Library name: Nanopore_P5plnP7pln_Control_1
No_Enrich_ALL.bw

value: plasma

value: 25 yo


title: Sting-/- iBMDMs, 4hr time point, untreated, biological replicate 2

description: Library name: 41

value: iBMDMs

value: Sting-/-

value: untreated

value: same batch for all


title: HeLa_shMETTL8_small_RNA_input_#3

description: Library name: m3C-7

value: HeLa

value: METTL8 knockdown


title: HeLa_Ctrl_OE_small_RNA_input_#2

description: Library name: m3C-10

value: overexpression control


title: Healthy blood plasma cfDNA Male 07 P5Telomere 7nt adapter P5plain P7plain primers (Nanopore)

description:
Library name: Nanopore_P5TelAdapter
Tel_Adapter_NP5E2_ALL.bw

value: Blood plasma cfDNA


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-B10-CAAATAGAAC-CTAGTC

value: Myeloid leukemia of Down syndrome (ML-DS) cell line


title: 3276416_3D11_RabIgG_001

description:
Library name: 3276416_3D11_RabIgG_001
Spike-in species: Drosophila melanogaster

data_processing:
Raw sequencing reads were quality-filtered and adapter-trimmed using Trim Galore (v0.4.4) with cutadapt (Q20 quality cutoff).
Trimmed reads were aligned to a combined human-Drosophila hybrid reference genome (GRCh38.p12 + dm6) using BWA (v0.7.12-r1039). Alignments were converted to BAM format using samtools (v1.2), and duplicate reads were marked using biobambam2 (v2.0.87).
Spike-in normalization factors were calculated based on the fraction of reads mapping to the Drosophila (dm6) genome relative to all mapped reads. For downstream analyses, only reads mapping to the human genome were retained. Alignments were filtered to uniquely mapped reads (MAPQ ≥1), excluding low-quality, secondary, and supplementary alignments.
Filtered BAM files were name-sorted and converted to paired-end fragment representations (BEDPE) using bedtools (v2.17.0), retaining properly paired fragments. Genome-wide coverage tracks were generated from fragments shorter than 2 kb by extracting the central 80 bp of each fragment (midpoint ±40 bp). Coverage was computed using bedtools genomecov and converted to bigWig format using UCSC bedGraphToBigWig. Coverage tracks were normalized to 10 million fragments and additionally scaled by the spike-in normalization factor.
Peak calling was performed using MACS2 with matched IgG controls. Broad histone targets (H3K4me1) were called with SICER (v1.1; p‑value 1e‑5). Peaks overlapping ENCODE blacklist regions were removed using bedtools.
Assembly: hg38
Supplementary files format and content: Processed data include peak files in narrowPeak BED, for targets with narrow peak profile, and BED format for broad targets; BigWig files containing sample level normalized genome-wide signal tracks and condition level signals (samples average).
Library strategy: CUT&Run

source_name: CMY

growth_protocol: Cmy cells and isogenic clones were cultured in RPMI (Gibco) supplemented with heat-inactivated fetal bovine serum (10%) and 1× penicillin/streptomycin at 37 °C with 5% CO₂. Cells were passaged every 2–5 days and confirmed to be mycoplasma-free and authenticated prior to experiments.

extract_protocol:
Nuclei were isolated from intact cells and chromatin-bound proteins were selectively released using micrococcal nuclease (MNase) digestion following the Epicypher CUTANA CUT&RUN protocol. MNase digestion time was modified to 1.5 hours.
CUT&RUN libraries were prepared using the CUTANA CUT&RUN Library Prep Kit (Epicypher; Cat#14-1001) according to the manufacturer’s protocol following antibody-directed MNase digestion. Libraries were indexed, pooled, and sequenced on an Illumina NovaSeq platform.

treatment_protocol: No chemical treatment was applied prior to CUT&RUN. Genome-edited cell lines were generated by CRISPR-Cas9 nucleofection and clonally expanded prior to chromatin profiling.

value: RAD21 WT

tag: antibody

value: Epicypher 13-0042K


title: 3276421_CMY_H3K27ac_003

description:
Library name: 3276421_CMY_H3K27ac_003
Spike-in species: Drosophila melanogaster

value: CMY

value: RAD21 Y3*

value: Active Motif 39133


title: HeLa_shMETTL8_small_RNA_input_#2

description: Library name: m3C-6


title: Cgas-/- iBMDMs, 4hr time point, CV-Cglr2+, biological replicate 2

description: Library name: 40

value: macrophage

value: Cgas-/-


title: 3276422_2B10_H3K27ac_001

description:
Library name: 3276422_2B10_H3K27ac_001
Spike-in species: Drosophila melanogaster


title: HeLa_shMETTL8_small_RNA_input_#1

description: Library name: m3C-5


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-B09-AAGCCGTACA-CTAGTC

value: cell line


title: Healthy blood plasma cfDNA Male 07 P5Telomere P7plain primers (Nanopore)

description:
Library name: Nanopore_P5TelP7pln
Crich_NP5_ALL.bw

value: plasma

value: 25 yo


title: WA09_invivo_DAPI-_4

city: Rochester

first: Steven

last: Goldman

state: NY

address: 601 Elmwood Ave

department: Center for Translational Neuromedicine

institute: University of Rochester Medical Center

description:
Library name: Sample_WA09_Shiv
Mouse Human Chimera

library_source: transcriptomic single cell

data_processing:
Alignment and gene counting were accomplished using STARsolo with the following flags: “--twopassMode Basic --outSAMtype BAM Unsorted --readFilesCommand zcat --soloType CB_UMI_Simple --soloCBwhitelist 3M-february-2018.txt --soloUMIlen 12 --soloUMIfiltering MultiGeneUMI --soloCBmatchWLtype 1MM_multi_pseudocounts --limitSjdbInsertNsj 2000000”. Samples produced using 10X Genomics scRNA 2.0 chemistry were aligned using the following STARsolo flags: “--twopassMode Basic --outSAMtype BAM Unsorted --readFilesCommand zcat --soloType CB_UMI_Simple --soloCBwhitelist 737K-august-2016.txt --soloCBstart 1 --soloCBlen 16 --soloUMIstart 17 --soloUMIlen 10 --soloBarcodeReadLength 0 --soloUMIfiltering MultiGeneUMI --soloCBmatchWLtype 1MM_multi_pseudocounts --limitSjdbInsertNsj 2000000.”
Assembly: A custom chimeric reference comprised of concatenating GRCh38 ensembl 106 to GRCm39 ensembl 106 and filtering for transcript types: “protein_coding”, “lncRNA”, or “miRNA”.
Supplementary files format and content: Contains two 10X sparse matrixes with gene counts per cell. One for the mouse fraction and one for the human fraction. The mouse fraction of invivo cultures consist of the small subset of genes with homology great enough that the chimeric aligner cannot distinguish them.

organism:
Homo sapiens
Mus musculus

extract_protocol:
For invitro samples unsorted PSC and GPC stage cells were incubated in Accutase (Millipore Sigma Cat. A6964) diluted 50% in DPBS for 6 minutes at 37 degrees. Cells were resuspended in DMEM and filtered for FACS. . Cells were selected based on forward and side scatter in addition to negative gating of 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific, D1306) staining to remove dead cells and debri (1 µg ml−1). Cells were then captured for scRNA-seq on a 10X Genomics chromium controller. For Invivo samples the mouse brains were removed and immersed in ice-cold HBSS and their corpora callosa dissected and chopped into small pieces. This tissue was then incubated in a papain/DNase dissociation solution at 37 °C for 50 min. Papain was inactivated with the addition of ovomucoid dissolved in EBSS. A single-cell suspension was then achieved via repeated pipetting. Cells were pelleted, resuspended into MEM, and filtered for FACS. Cells were selected based on forward and side scatter in addition to negative gating of 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific, D1306) staining to remove dead cells and debri (1 µg ml−1). Cells were then captured for scRNA-seq on a 10X Genomics chromium controller.
Libraries were constructed according to manufacturers instructions for either single cell 3' v2 or single cell 3' v3.1 from 10X genomics as noted in the metadata table.

tag: Stage

value: In Vivo

tag: sort

value: DAPI-

tag: 10x chemistry


title: Cgas-/- iBMDMs, 4hr time point, dacA+, biological replicate 2

description: Library name: 39

tag: cell line

value: iBMDMs

tag: cell type

value: dacA+

value: same batch for all


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-B08-GAATCGTAAA-CTAGTC


title: Cgas-/- iBMDMs, 4hr time point, clearcoli, biological replicate 2

description: Library name: 38

tag: genotype

tag: treatment

value: clearcoli


title: HeLa_shCtrl_small_RNA_AlkB

description: Library name: m3C-4

value: HeLa

value: shRNA control


title: C27_invivo_DAPI-_Shi

description:
Library name: Sample_C27_Shi
Mouse Human Chimera


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-B07-TTGTTAGTTT-CTAGTC

tag: tissue

value: Myeloid leukemia of Down syndrome (ML-DS) cell line

tag: batch


title: HeLa_shCtrl_small_RNA_input_#3

description: Library name: m3C-3


title: Cgas-/- iBMDMs, 4hr time point, untreated, biological replicate 2

description: Library name: 37

value: untreated


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-B06-CGGACAAGCA-CTAGTC

value: CMY


title: WA09_invivo_DAPI-_3

description:
Library name: Sample_34
Mouse Human Chimera
included is a technical replicate sequenced on a HiSeq 2500


title: HeLa_shCtrl_small_RNA_input_#2

description: Library name: m3C-2


title: WA09_invivo_DAPI-_2

description:
Library name: Sample_33
Mouse Human Chimera
included is a technical replicate sequenced on a HiSeq 2500


title: WT iBMDMs, 4hr time point, CV-Cglr2+, biological replicate 2

description: Library name: 36

value: WT


title: HeLa_shCtrl_small_RNA_input_#1

description: Library name: m3C-1


title: HeLa_Ctrl_OE_small_RNA_input_#1

description: Library name: m3C-9

value: overexpression control


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-B05-TTTCTAGGCA-CTAGTC


title: WA09_invivo_DAPI-_1

description:
Library name: Sample_32
Mouse Human Chimera
included is a technical replicate sequenced on a HiSeq 2500


title: WT iBMDMs, 4hr time point, dacA+, biological replicate 2

description: Library name: 35

value: macrophage


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-B04-CTTCATCGGA-CTAGTC

value: cell line


title: RBM20_WT_Rep3_bB

city: Heidelberg

first: Lars

last: Steinmetz

state: Baden Wurttemberg

address: Meyerhofstraße 1

department: Genome Biology Unit

country: Germany

institute: EMBL

description: Library name: RBM20_WT_Rep3_bB

data_processing:
Sequencing data were processed using the RNA-sequencing module from snakePipes72 , with the flags --trim and -m "alignment-free,alignment". Reads were mapped to the Homo sapiens reference genome (Ensembl assembly GRCh38) and transcriptome annotation release 114 using STAR. Gene counts were generated using feature coutns. DESeq2 was done to perform differential gene expression, comparisons were made using the model formula ~batch + condition to account for batch-specific effects of cardioid differentiation.
Assembly: GRCh38
Supplementary files format and content: counts.tsv counts per gene for all samples
Supplementary files format and content: KO_deseq2.results.tsv,RQ_deseq2.results.tsv,PL_deseq2.results.tsv, differential expression gene

source_name: Cardioid

growth_protocol: On D-1, 12,000 hiPSCs were seeded per well of a 96-well Clear Round Bottom Ultra-Low Attachment Microplate (Costar, 7007) in 200 µl Essential 8 Flex Medium supplemented with 5 µM Y-27632 ROCK inhibitor and 1% Penicillin-Streptomycin. After seeding, plates were centrifuged at 200g for 5 minutes and stored at 37°C and 5% CO2. Differentiation from D0 to D11 was performed as previously described70 with slight alterations. Media components are listed in Supplementary Table 1. Briefly, induction was done at D0 using FLyABCH(Ins) medium (CDM medium supplemented with 3 ng/ml FGF2, 5 µM LY294002, 4 ng/ml Activin A, 10 ng/ml BMP4, and 4 µM CHIR99021). 40h later, the medium was changed to BFIC59Ra medium (CDM medium supplemented with 8 ng/ml FGF2, 10 ng/ml BMP4, 10 µg/ml insulin, 2 µM c-59, and 0.05 µM retinoic acid) for four days. Then, the medium was changed to BFI medium (CDM medium supplemented with 8 ng/ml FGF2, 10 ng/ml BMP4, and 10 µg/ml insulin) for two days. From D2 to D7, the medium was refreshed every day removing roughly 190 µl by aspiration and adding the same volume of fresh medium. First beating was consistently observed on D6. From D8, cardioids were cultured in CDM-I medium (CDM medium supplemented with 10 µg/ml insulin) for four days with medium changes every other day. Finally, cardioids were matured using metabolic maturation medium71. From this stage, the medium was changed every two to four days. On the day of harvest, cardioids were collected in batches of at least five cardioids in a microcentrifuge tube per genotype and differentiation round, and the medium was aspirated. Cardioids were then snap frozen and stored at -70°C until downstream applications.

extract_protocol:
Pooled cardioids were homogenized in PBS with addition of RNAse inhibitor (Invitrogen), spun down, and the pellet was then resuspended in 100 µl of TRIzol LS Reagent (Thermo Fisher Scientific). Next, 20 µl of chloroform was added to the TRIzol-sample, and phase separation was achieved at room temperature by vigorous shaking followed by centrifugation at 15,000 × g for 5 minutes. From each sample, 40 µl of the aqueous phase were transferred to a new Eppendorf tube and mixed with 75.5 µl of isopropanol and 1:150 of glycoblue (Invitrogen). The samples were then stored at −80°C for 24–36 hours to dehydrate. RNA was pelleted by centrifuging at 4°C at maximum speed for 15 minutes. The supernatant was discarded, and the pellet was washed with 70% ethanol, air-dried, and resuspended in 12 µl of nuclease-free water.
Libraries for total RNA-sequencing were prepared from D30 cardioids. Total RNA was isolated by douncing cardioids to generate cell suspensions, followed by RNA extraction using Trizol LS according to the manufacturer’s instructions (Thermo Fisher). RNA samples were subjected to RNase H-based rRNA depletion using the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat). Ribo-depleted RNA was then used for library preparation with the NEBNext Ultra™ II Directional RNA Library Prep Kit for Illumina. Paired-end sequencing (101 bp reads) was performed on the Illumina NextSeq2000 platform.

value: Cardioid

value: iPSCS WTC11

value: iPSCs derived cardioids

value: wildtype

value: B


title: WT iBMDMs, 4hr time point, clearcoli, biological replicate 2

description: Library name: 34

value: iBMDMs


title: WA09_invitro_GPC_DAPI-_1

description: Library name: Sample_unsorted

value: In Vitro - GPC Stage

value: DAPI-


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-B03-AAACTGTTGC-CTAGTC


title: RBM20_WT_Rep2_bA

description: Library name: RBM20_WT_Rep2_bA

value: A


title: WT iBMDMs, 4hr time point, untreated, biological replicate 2

description: Library name: 33

value: same batch for all


title: WA09_invitro_GPC_CD140+_1

description: Library name: Sample_CD-140


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-B02-CCTGTAGCAC-CTAGTC

value: Myeloid leukemia of Down syndrome (ML-DS) cell line


title: Myd88-/-Trif-/-Lrrc8a-/- iBMDMs, 4hr time point, CV-Cglr1+, biological replicate 1

description: Library name: 32


title: RBM20_WT_Rep1_bC

description: Library name: RBM20_WT_Rep1_bC

value: C


title: WT_185

city: Baltimore

first: Jin-Young

last: Koh

address: 670 W Baltimore Street

department: Otorhinolaryngology-Head & Neck Surgery

institute: University of Maryland, Baltimore

description:
Library name: Sample 12
Smart-Seq2 protocol

data_processing:
The Illumina raw sequences were aligned to the mm10 genome (GRCm38) using STAR 2.5.2b (parameters: --alignIntronMax 20000 --outFilterMismatchNoverLmax 0.05).
Sorted BAM files were created from the STAR SAM files using samtools 1.31.
Transcripts were assembled and their expression was quantified using Cufflinks 2.2.1 (parameters: --GTF-guide Mus_musculus.GRCm38.86.gtf --num-threads 5).
Assembly: mm10
Supplementary files format and content: Cufflinks transcripts GTF file for each sample

source_name: cochlear lateral wall

extract_protocol:
After euthanasia by CO2, the temporal bones were removed and placed into DPBS. The temporal bone was removed and opened under a dissecting microscope to access the cochlear membranous labyrinth. Once the cochlea was accessible, the stria vascularis tissue was extracted from the temporal bone and incubated for 5min at 37°C with collagenase from clostridium histolyticum solution at a working concentration of 0.3mg/500µl. The cell dissociation was completed by gently pipetting up and down. The disrupted stria vascularis tissues were placed under an inverted microscope equipped with 20x and 40x objectives and two 3D micromanipulators, each driving a pulled-glass micropipette attached to a nitrogen gas-powered Pico-Injector to control aspiration pressure. The dissociated cells were examined under the inverted microscope and were aspirated into the first pulled-glass micropipette in a slow and controlled manner then washed in fresh DPBS to remove residual collagenase solution and debris.
Nextera XT DNA sample preparation kits, including Tn5 enzymes for tagmentation, were used together with Nextera XT index kits for multiplexing.

value: cochlear lateral wall

value: root cell


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-B01-ATGTAGCACC-CTAGTC

value: CMY


title: RBM20_RQ_Rep3_bA

description: Library name: RBM20_RQ_Rep3_bA


title: WT_198

description:
Library name: Sample 11
Smart-Seq2 protocol

value: spindle cell


title: Myd88-/-Trif-/-Lrrc8a-/- iBMDMs, 4hr time point, dacA+, biological replicate 1

description: Library name: 31

value: macrophage

value: dacA+


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-A12-AGACGATCCA-CTAGTC

value: cell line


title: Myd88-/-Trif-/-Lrrc8a-/- iBMDMs, 4hr time point, clearcoli, biological replicate 1

description: Library name: 30

value: clearcoli


title: RBM20_RQ_Rep2_bC

description: Library name: RBM20_RQ_Rep2_bC

value: Cardioid

value: iPSCS WTC11

value: iPSCs derived cardioids


title: WT_173

description:
Library name: Sample 10
Smart-Seq2 protocol


title: RBM20_RQ_Rep1_bB

description: Library name: RBM20_RQ_Rep1_bB

value: B


title: Myd88-/-Trif-/-Lrrc8a-/- iBMDMs, 4hr time point, untreated, biological replicate 1

description: Library name: 29

value: iBMDMs

value: untreated


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-A11-CGGTTCTCCA-CTAGTC


title: WT_172

description:
Library name: Sample 9
Smart-Seq2 protocol


title: RBM20_PL_Rep3_bC

description: Library name: RBM20_PL_Rep3_bC


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-A10-GATAGTATTG-CTAGTC


title: Myd88-/-Trif-/-Sting-/- iBMDMs, 4hr time point, CV-Cglr1+, biological replicate 1

description: Library name: 28

value: same batch for all


title: WT_169

description:
Library name: Sample 8
Smart-Seq2 protocol


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-A09-GGGATGTGGA-CTAGTC

value: Myeloid leukemia of Down syndrome (ML-DS) cell line


title: Myd88-/-Trif-/-Sting-/- iBMDMs, 4hr time point, dacA+, biological replicate 1

description: Library name: 27


title: RBM20_PL_Rep2_bB

description: Library name: RBM20_PL_Rep2_bB


title: Veh 3

city: Cincinnati

first: Theresa

last: Alenghat

state: Ohio

address: 3333 Burnet Avenue

department: Division of Immunobiology

institute: Cincinnati Children's Hospital Medical Center

description: Library name: sample 3

data_processing:
Sequence reads were aligned to the mm10 genome using the Illumina sequence analysis pipeline.
Differentially expressed genes between Vehicle control and RA-treated stem cell orgnaoids were identified using DSeq2.
Assembly: mm10
Supplementary files format and content: text files with Dseq2 output for each sample

source_name: stem cell enteroid

growth_protocol: Organoids were dervied from the ileum (enteroids) of WT C57Bl/6 and maintained at 37C, 5% CO2, in organoid growth media.

extract_protocol:
Organoids were harvested and RNA was isolated using the RNeasy Kit (Qiagen) according to manufacturer's instructions
Directional polyA RNAseq according to standard Illumina protocols

treatment_protocol: Stem cell-enriched organoids were cultured in organoid growth media supplemented with 10 μM CHIR99021 for 96 hours, followed by treatment with 10 μM retinoic acid or vehicle control for 24 hours

value: stem cell enteroid

value: organoid

value: intestinal stem cells

value: Vehicle/DMSO


title: Myd88-/-Trif-/-Lrrc8a-/- iBMDMs, 4hr time point, untreated, biological replicate 3

description: Library name: 93


title: WT_161

description:
Library name: Sample 7
Smart-Seq2 protocol

value: cochlear lateral wall

value: spindle cell


title: RBM20_PL_Rep1_bA

description: Library name: RBM20_PL_Rep1_bA

value: A


title: RBM20_KO_Rep3_bB

description: Library name: RBM20_KO_Rep3_bB


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-A08-CCCTTGTGTA-CTAGTC

value: cell line

value: CMY


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-A07-CTCTCGCGAA-CTAGTC


title: Myd88-/-Trif-/-Sting-/- iBMDMs, 4hr time point, clearcoli, biological replicate 1

description: Library name: 26

value: macrophage

value: clearcoli


title: WT_215

description:
Library name: Sample 6
Smart-Seq2 protocol


title: Myd88-/-Trif-/-Sting-/- iBMDMs, 4hr time point, untreated, biological replicate 1

description: Library name: 25


title: WT_209

description:
Library name: Sample 5
Smart-Seq2 protocol

value: pendrin cell


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-A06-GCGTTAGGAT-CTAGTC


title: Myd88-/-Trif-/-Cgas-/- iBMDMs, 4hr time point, CV-Cglr1+, biological replicate 1

description: Library name: 24


title: RBM20_KO_Rep2_bC

description: Library name: RBM20_KO_Rep2_bC

value: Cardioid

value: iPSCS WTC11

value: iPSCs derived cardioids

value: C


title: WT_143

description:
Library name: Sample 4
Smart-Seq2 protocol

value: root cell


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-A05-GTGCAAATTC-CTAGTC

value: Myeloid leukemia of Down syndrome (ML-DS) cell line


title: RBM20_KO_Rep1_bA

description: Library name: RBM20_KO_Rep1_bA


title: Myd88-/-Trif-/-Cgas-/- iBMDMs, 4hr time point, dacA+, biological replicate 1

description: Library name: 23

value: iBMDMs

value: dacA+

value: same batch for all


title: WT_075

description:
Library name: Sample 3
Smart-Seq2 protocol

value: cochlear lateral wall


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-A04-CAAGACAGCA-CTAGTC


title: Mettl16 mutant IP rep3

city: Shang Hai

first: song

last: penghui

address: No. 2005, Songhu Road, Yangpu District

country: China

institute: FuDan University

description: Library name: Mettl16_1-9_IP_3

data_processing:
fastp software (https://github.com/OpenGene/fastp) was used to remove the reads that contained adaptor contamination, low quality bases and undetermined bases with default parameter. And sequence quality of IP and Input samples were verified using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and RseQC (http://rseqc.sourceforge.net/). Then we used HISAT2 (http://daehwankimlab.github.io/hisat2) to map reads to the reference genome drosophila melanogaster (Version: v107). Peak calling and diff peak analysis were performed by R package exomePeak (https://www.bioconductor.org/packages/3.3/bioc/html/exomePeak.html), and peaks were annotated by intersection with gene architecture using R package ANNOVAR (http://www.openbioinformatics.org/annovar/). HOMER (http://homer.ucsd.edu/homer/motif) was used for de novo and known motif finding followed by localization of the motif with respect to peak summit. And StringTie (https://ccb.jhu.edu/software/stringtie) was used to perform expression level for all transcripts and genes from input libraries by calculating FPKM (total exon fragments /mapped reads (millions) × exon length (kB)). The differentially expressed transcripts and genes were selected with log2 (fold change) ≥ 1 or log2 (fold change) ≤ -1 and p value < 0.05 by R package edgeR (https://bioconductor.org/packages/edgeR).
Assembly: genome drosophila melanogaster (Version: v107)
Supplementary files format and content: input FPKM all genes.csv and all bigwig files
Library strategy: MeRIP-Seq

organism: Drosophila melanogaster

source_name: adult flies whole body

extract_protocol:
Total RNA was isolated and purified using TRIzol reagent from ~50 adult males of Mettl161-9 and w1118.
Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA), and the RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA). Poly (A) RNA was purified from 50μg total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) by two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 86℃ 7min. The cleaved RNA fragments were incubated for 2h at 4℃ with m6A-specific antibody (No. 202003, Synaptic Systems, Germany) in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630). The IP RNA was reverse-transcribed to cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which was next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA）. An A-base was added to the blunt ends of each strand, preparing for ligation to the indexed adapters. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. At last, we performed paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 platform(LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.

value: adult flies whole body


title: WT_028

description:
Library name: Sample 2
Smart-Seq2 protocol


title: Mettl16 mutant IP rep2

description: Library name: Mettl16_1-9_IP_2


title: Myd88-/-Trif-/-Cgas-/- iBMDMs, 4hr time point, clearcoli, biological replicate 1

description: Library name: 22


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-A03-AAGCAAAGCG-CTAGTC


title: Myd88-/-Trif-/-Cgas-/- iBMDMs, 4hr time point, untreated, biological replicate 1

description: Library name: 21

value: untreated


title: Myd88-/-Trif-/- iBMDMs, 4hr time point, CV-Cglr1+, biological replicate 1

description: Library name: 20


title: Diet_3_ZT0

city: Strasbourg

first: Frank

last: Jühling

address: 3 Rue Koeberlé

country: France

institute: Université de Strasbourg, Inserm, Institut de Recherche sur Les Maladies Virales et Hépatiques UMR_S1110

data_processing:
Sequence reads were mapped to GrCM38/mm10 using the STAR alignment tool v2.7.9a (Dobin et al., 2013).
Read count extraction for the gencode vM25 primary annotation was performed using htseq-count.
Read count normalization was performed using DESeq2.
Assembly: GrCM38/mm10_e100
Supplementary files format and content: tab-delimited text files include raw read counts and normalized read counts for each sample

source_name: liver

growth_protocol: 8 weeks old mice were fed with chow diet or CDA-HFD (Research Diet; ref. A06071302) for 12 weeks. The on and off times of light were defined as Zeitgeber time with (ZT0) being the start of the light period and (ZT12) the start of the dark period. Animals were sacrificed at ZT0 (7 am), ZT6 (1 pm), ZT12 (7 pm), and ZT18 (1 am).

extract_protocol:
Total RNA was extracted using Promega ReliaPrep™ RNA extraction kit
Total RNA were used for Illumina HiSeq 4000 (BCF platform, Vienna, Austria)

treatment_protocol: No treatment

value: liver

tag: zeitgeber time

value: HSC

value: choline-deficient amino-acid adapted high-fat diet (CDA-HFD, Research Diet #A 06071302) for 6 months

value: 20 weeks


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-A01-ATAGAGCCAG-CTAGTC

value: cell line

value: CMY


title: Myd88-/-Trif-/- iBMDMs, 4hr time point, dacA+, biological replicate 1

description: Library name: 19

value: macrophage


title: contro IP rep3

description: Library name: w1118_IP_3


description: Library name: CMY_CIITA_KO.Miller-Plate106-F03-TCGCTAAGTG-CTAGTC


title: contro IP rep2

description: Library name: w1118_IP_2


title: Myd88-/-Trif-/- iBMDMs, 4hr time point, clearcoli, biological replicate 1

description: Library name: 18

value: clearcoli


title: Diet_3_ZT12


description: Library name: CMY_CIITA_KO.Miller-Plate106-F02-CAGTCCTGGG-CTAGTC


title: contro IP rep1

description: Library name: w1118_IP_1


description: Library name: CMY_CIITA_KO.Miller-Plate106-F01-CCCGCAACAT-CTAGTC


title: Mettl16 mutant input rep3

description: Library name: Mettl16_1-9_input_3

data_processing:
fastp software (https://github.com/OpenGene/fastp) was used to remove the reads that contained adaptor contamination, low quality bases and undetermined bases with default parameter. And sequence quality of IP and Input samples were verified using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and RseQC (http://rseqc.sourceforge.net/). Then we used HISAT2 (http://daehwankimlab.github.io/hisat2) to map reads to the reference genome drosophila melanogaster (Version: v107). Peak calling and diff peak analysis were performed by R package exomePeak (https://www.bioconductor.org/packages/3.3/bioc/html/exomePeak.html), and peaks were annotated by intersection with gene architecture using R package ANNOVAR (http://www.openbioinformatics.org/annovar/). HOMER (http://homer.ucsd.edu/homer/motif) was used for de novo and known motif finding followed by localization of the motif with respect to peak summit. And StringTie (https://ccb.jhu.edu/software/stringtie) was used to perform expression level for all transcripts and genes from input libraries by calculating FPKM (total exon fragments /mapped reads (millions) × exon length (kB)). The differentially expressed transcripts and genes were selected with log2 (fold change) ≥ 1 or log2 (fold change) ≤ -1 and p value < 0.05 by R package edgeR (https://bioconductor.org/packages/edgeR).
Assembly: genome drosophila melanogaster (Version: v107)
Supplementary files format and content: input FPKM all genes.csv and all bigwig files


title: Diet_2_ZT0


title: Sting-/- iBMDMs, 4hr time point, dacA+, biological replicate 1

description: Library name: 11

value: Sting-/-


title: Mettl16 mutant input rep2

description: Library name: Mettl16_1-9_input_2


title: Diet_2_ZT18


description: Library name: CMY_CIITA_KO.Miller-Plate106-E11-AGCGCGCTTT-CTAGTC


title: Mettl16 mutant input rep1

description: Library name: Mettl16_1-9_input_1


title: Diet_2_ZT12


title: Sting-/- iBMDMs, 4hr time point, clearcoli, biological replicate 1

description: Library name: 10


description: Library name: CMY_CIITA_KO.Miller-Plate106-E10-GCGCTGTTGT-CTAGTC


title: control input rep3

description: Library name: w1118_input_3


title: Sting-/- iBMDMs, 4hr time point, untreated, biological replicate 1

description: Library name: 9


title: Diet_2_ZT6


title: control input rep2

description: Library name: w1118_input_2


title: Cgas-/- iBMDMs, 4hr time point, CV-Cglr1+, biological replicate 1

description: Library name: 8

value: Cgas-/-


description: Library name: CMY_CIITA_KO.Miller-Plate106-E09-ACCGACTCAC-CTAGTC

value: Myeloid leukemia of Down syndrome (ML-DS) cell line


title: Diet_1_ZT0


description: Library name: CMY_CIITA_KO.Miller-Plate106-E08-CAGTCTTAAC-CTAGTC


title: Cgas-/- iBMDMs, 4hr time point, dacA+, biological replicate 1

description: Library name: 7

value: iBMDMs


description: Library name: CMY_PPP1R12C_KO.Miller-Plate106-A02-CGTGCCTTAA-CTAGTC


title: WT_023

description:
Library name: Sample 1
Smart-Seq2 protocol

value: root cell


title: control input rep1

description: Library name: w1118_input_1


title: Mettl16 mutant IP rep1

description: Library name: Mettl16_1-9_IP_1


title: Diet_1_ZT18


description: Library name: CMY_CIITA_KO.Miller-Plate106-E07-AACCTCGAGG-CTAGTC


title: Healthy blood plasma cfDNA Male 07 P5plain P7Telomere primers (Nanopore)

description:
Library name: Nanopore_P5plnP7Tel
Grich_NP4_ALL.bw

value: plasma

value: 25 yo

value: Blood plasma cfDNA


title: Cgas-/- iBMDMs, 4hr time point, clearcoli, biological replicate 1

description: Library name: 6

value: same batch for all


title: Diet_3_ZT18

value: liver


title: Diet_1_ZT12

value: HSC


title: Diet_1_ZT6

value: choline-deficient amino-acid adapted high-fat diet (CDA-HFD, Research Diet #A 06071302) for 6 months


description: Library name: CMY_CIITA_KO.Miller-Plate106-E05-AGTGATGACC-CTAGTC


title: Cgas-/- iBMDMs, 4hr time point, untreated, biological replicate 1

description: Library name: 5

value: untreated


title: NESt tumour scRNA-seq, MYCN(T58A) induced in vivo, three reps

city: London

first: Diego

last: Chillon Pino

address: Great Ormond Street Hospital for Children NHS

department: Cancer development and genomics

country: United Kingdom

institute: UCL

description:
Library name: NESt_scRNA
10x Genomics Chromium GEM-X Flex (v2); sample-multiplexed by probe barcode [confirm BC id for rep1]; shares pooled FASTQs with the other two samples

data_processing:
Alignment to GRCh38, probe-barcode sample demultiplexing and per-sample filtered feature-barcode matrix generation with Cell Ranger v9.0.1 (cellranger multi) using the Chromium Human Transcriptome Probe Set v1.1.0.
Per-dataset quality filtering (Seurat v5.5.0): cells retained within median +/- 5 SD for RNA reads and detected genes, and median +/- 3 SD for mitochondrial percentage (lower bounds floored at 0 for mito%/reads, 30 for genes).
Doublet removal with scDblFinder v1.24.10; only singlets retained.
SCTransform variance-stabilising normalisation; PCA and non-linear dimensional reduction (dims 1-30); Louvain clustering (resolution selected by adjusted random index and cluster-tree stability); batch integration with harmony v2.0.3.
Cluster markers by Wilcoxon rank-sum test (features in >1% of cluster cells, log2FC > 0.1); trajectory/pseudotime with monocle3 v1.4.26.
Assembly: GRCh38
Supplementary files format and content: Per-sample 10x MEX filtered feature-barcode matrices (barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz), demultiplexed by probe barcode.

source_name: brain tumour (intracranial xenograft)

growth_protocol: Human embryonic hindbrain-derived NES cells (Sai2 line) were cultured in DMEM/F12 with 2 mM L-glutamine, N2 supplement, B27, penicillin-streptomycin (1:100), EGF (10 ng/mL) and FGF2 (10 ng/mL). Flasks were double-coated with poly-L-ornithine (0.1 mg/mL) then laminin (2 µg/mL). Cells were split ~1:3 every ~3 days with TrypLE Express; medium was refreshed daily. Maintained at 37 °C, 5% CO2.

extract_protocol:
Tumours were snap-frozen in liquid nitrogen at endpoint and stored at -80 C. Frozen tumours were thawed and dissociated to a single-cell suspension following the 10x Genomics GEM-X Flex Sample Preparation Kit protocol.
Single-cell libraries were prepared with the 10x Genomics Chromium GEM-X Flex Gene Expression Kit (v2 chemistry; probe-based detection of polyadenylated transcripts in fixed cells). The three tumour samples were multiplexed by probe barcode on a single Chromium chip (~5,000 cells/sample target) and sequenced on an Illumina NovaSeq platform (~20,000 reads/cell).

treatment_protocol: In vitro: MYCN(T58A) expression was induced in NES ON cultures by doxycycline; NES CTRL cultures were maintained without doxycycline. In vivo (NESt): recipient NSG mice received doxycycline feed (20 g/kg) from >=7 days before intracranial implantation and throughout tumour growth.

value: brain tumour (intracranial xenograft)

value: Sai2 (NES MYCN(T58A)-derived)

value: MYCN(T58A), doxycycline-induced

value: doxycycline (in vivo dox diet)


description: Library name: CMY_CIITA_KO.Miller-Plate106-E06-ATCCTCTCCT-CTAGTC

value: cell line

value: CMY


title: Parse Biosciences WT sublibrary 8

city: Cary

first: Sofia

last: Benavides

state: NC

address: 1134 Dominion Oak Cir

institute: UNC Chapel Hill

description:
Library name: Sublibrary 8
Parse Biosciences

data_processing:
Preprocessing was performed using Scanpy (v1.9.3) following a previously established workflow. Cells expressing fewer than 300 genes were excluded, as were genes expressed in fewer than 5 cells. To further refine the dataset, cells expressing more than 8,000 genes or with total gene counts exceeding 20,000—likely representing doublets—were removed. Additionally, cells with mitochondrial gene expression exceeding 15% of the total counts were filtered out to exclude unhealthy or dying cells. Clustering was performed using the Leiden algorithm as implemented in Scanpy, enabling robust detection of transcriptionally distinct populations. Clusters were annotated using the top DEGs, canonical markers, and reference data from previously published single-cell RNA sequencing datasets. No size cutoff was used to remove small clusters.
Differentially expressed genes distinguishing each cluster were identified using Wilcoxon rank testing implemented in Scanpy.
Assembly: Human Hg38
Supplementary files format and content: Tab separated values files and matrix files

source_name: Primary

extract_protocol:
12 mL of fresh peripheral blood was collected using vacutainer EDTA tubes, diluted with 12 mL of DPBS supplemented with EDTA (2mM) and FBS (1%), and then gently layered over Lymphoprep (STEMCELL Technologies, USA) in a 50 mL conical tube. Samples were centrifuged at 800g for 15 min at room temperature with breaks off.  After centrifugation, PBMCs in the white foamy layer were carefully transferred to a new tube. After being washed with DPBS twice, samples were then centrifuged at 250g for 10 min, and the pellets were resuspended in cell culture media (RPMI supplemented with 5% human serum). Given their shorter lifespan in cell culture, PMNs were recovered from the red blood cells layer following 1 x RBS lysis buffer digestion procedure and washed twice with 1X PBS and resuspended in cell culture media. Single-cell suspensions were fixed following the Parse Biosciences Evercode Fixation Kit protocol and stored at −80°C until further processing. Fixed cells were rehydrated and pooled into sublibraries according to the Evercode split-pool combinatorial barcoding workflow. Each sublibrary underwent sequential rounds of combinatorial barcoding, including three rounds of in situ barcoding followed by a final indexing step (i5/i7 dual indexing) during library preparation.After barcoding, cDNA was generated and amplified according to the Parse Biosciences Evercode Whole Transcriptome protocol. Final sequencing libraries were constructed using standard Illumina-compatible adapters and quality-controlled prior to sequencing.Sequencing was performed on an Illumina platform, and raw reads were processed using the Parse Biosciences analysis pipeline to assign reads to individual cells based on combinatorial barcode identities.
The single-cell libraries were constructed according to the Parse WT kit manufacturer’s protocol. 4166 cells per sample (for a total of 100,000 cells from 24 samples) were barcoded using the reverse transcription barcoding approach. Barcoded cDNA was amplified and verified via TapeStation for size distribution following fragmentation, end repair, A-tailing, and adaptor ligation to complete library construction. All libraries were sequenced as paired-end 150-bp reads using the Illumina NovaSeq X Plus 300 platform with a reading depth of 83-104M reads per sample. Libraries were prepared with the whole transcriptome 100K kit from Parse Biosciences, which involves barcoding and then pooling cells from all samples prior to generation of eight sublibraries. Because of the design of the kit, sequencing reads of all samples are distributed across all eight sublibraries. For further details see attached `README.txt` file.

treatment_protocol: PBMC cell counts were performed on the automated cell counter, and 250.000 cells/well were seeded into 24-well plates, incubated at 37 °C in a 5% CO2 incubator for 1 hour, and then treated with 100 nM Cortisol, 10 nM Norepinephrine, or 0.0001% DMSO (vehicle control) overnight. Neutrophils were seeded into 24-well plates at a seeding density of 250,000 cells/well, incubated at 37C in a 5% CO2 incubator for 1 hour, and then treated with 100 nM cortisol, 10 nM norepinephrine, or 0.0001% DMSO for 4 hours.

value: Primary

value: PBMC, Neutrophils

value: mixed (DMSO, CORT, NE)


title: WT iBMDMs, 4hr time point, CV-Cglr1+, biological replicate 1

description: Library name: 4

data_processing:
Galaxy Server
Assembly: mm10
Supplementary files format and content: tab-delimited text file includes raw counts for each sample

source_name: iBMDMs

value: iBMDMs

value: macrophage

value: WT


title: Myd88-/-Trif-/- iBMDMs, 4hr time point, untreated, biological replicate 1

description: Library name: 17

value: same batch for all


title: Diet_3_ZT6

value: liver

value: 20 weeks


description: Library name: CMY_CIITA_KO.Miller-Plate106-E12-CCATGGTAGT-CTAGTC

value: Myeloid leukemia of Down syndrome (ML-DS) cell line


title: Sting-/- iBMDMs, 4hr time point, CV-Cglr1+, biological replicate 1

description: Library name: 12

value: Sting-/-


title: Control_3_ZT0

value: HSC

value: Control


description: Library name: CMY_CIITA_KO.Miller-Plate106-E04-CGCATGGTTG-CTAGTC


title: Parse Biosciences WT sublibrary 7

description:
Library name: Sublibrary 7
Parse Biosciences


title: WT iBMDMs, 4hr time point, dacA+, biological replicate 1

description: Library name: 3

value: dacA+


title: Parse Biosciences WT sublibrary 6

description:
Library name: Sublibrary 6
Parse Biosciences


description: Library name: CMY_CIITA_KO.Miller-Plate106-E03-TCCTTTCAGG-CTAGTC


title: Control_3_ZT18


title: WT iBMDMs, 4hr time point, clearcoli, biological replicate 1

description: Library name: 2

value: clearcoli


title: Control_2_ZT0


description: Library name: CMY_CIITA_KO.Miller-Plate106-E02-ACTGCTTTCT-CTAGTC

value: cell line


title: WT iBMDMs, 4hr time point, untreated, biological replicate 1

description: Library name: 1

value: untreated


title: Parse Biosciences WT sublibrary 5

description:
Library name: Sublibrary 5
Parse Biosciences

value: Primary


title: Control_2_ZT18


description: Library name: CMY_CIITA_KO.Miller-Plate106-E01-TCTTGGCTAA-CTAGTC

value: CMY


title: Parse Biosciences WT sublibrary 4

description:
Library name: Sublibrary 4
Parse Biosciences

value: PBMC, Neutrophils

value: mixed (DMSO, CORT, NE)


title: Chow_5

description: Sample 3378

data_processing:
Raw reads were mapped to mouse genome GRCm39 using STAR 2.7.11b.
Counts were calculated while mapping applying STAR option --quantMode GeneCounts and the gencode.vM35 annotation.
Assembly: GRCm39
Supplementary files format and content: raw_counts.txt: tab-delimited text file includes gene expression raw counts for each sample

growth_protocol: Forty male C57BL/6J mice were fed with standard chow diet (n=10) or with a choline-deficient amino-acid adapted high-fat diet (CDA-HFD) (n=30) for a total of 18 weeks.

extract_protocol:
All the mice were anesthetized and sacrificed after a total of 18 weeks. Liver pieces were  snap frozen and kept at -80 °C . A small fraction was processed using gentleMACS™ Dissociator (Miltenyi Biotec) and TRIzol®. Total RNA was purified using Direct-zol RNA Purification Kit (Zymo Research)
NovaSeq X Plus (Novogene)

treatment_protocol: After 12 weeks of diet, the mice fed with CDA-HFD were randomized in 2 groups, receiving daily IP injections of either SR9009 (100mg/kg/day) or vehicle control (Kolliphor ELP 15% in injectable NaCl) for 6 weeks (n=15 per group).

value: chow diet


title: Control_2_ZT12


title: Parse Biosciences WT sublibrary 3

description:
Library name: Sublibrary 3
Parse Biosciences


description: Library name: CMY_CIITA_KO.Miller-Plate106-D12-GCCTTGCCAT-CTAGTC


title: Parse Biosciences WT sublibrary 2

description:
Library name: Sublibrary 2
Parse Biosciences


description: Library name: CMY_CIITA_KO.Miller-Plate106-D11-ATTTCTTTAA-CTAGTC


title: Chow_4

description: Sample 3380


title: Control_2_ZT6


title: Chow_3

description: Sample 3377


title: Control_1_ZT0


description: Library name: CMY_CIITA_KO.Miller-Plate106-D10-ATCGATCACG-CTAGTC


title: Parse Biosciences WT sublibrary 1

description:
Library name: Sublibrary 1
Parse Biosciences


title: Chow_2

description: Sample 3376


title: Control_1_ZT18


description: Library name: CMY_CIITA_KO.Miller-Plate106-D09-AAGTTCTTGA-CTAGTC


title: HK2 cells, igg

city: Guangzhou

first: Lijun

last: Dong

address: 1023-1063 Shatai South Road, Baiyun District

institute: Southern Medical University

description: Library name: Sample 9

data_processing:
Raw sequencing reads were assessed for quality using FastQC, and adapter sequences and low-quality bases were trimmed using Trimmomatic.
Clean reads were aligned to the human reference genome (GRCh38/hg38) using the splice-aware aligner STAR. Gene-level read counts were quantified using featureCounts based on the corresponding Ensembl gene annotation (GTF file).
To identify transcripts significantly enriched in DDX18 immunoprecipitates, differential enrichment analysis was performed by comparing IP samples with matched input (or IgG) controls using DESeq2.
Transcripts with an adjusted p-value < 0.05 and |log₂ fold-change| ≥ 1 were considered significant candidate interactors.
Downstream functional annotation, including Gene Ontology (GO) and KEGG pathway enrichment analysis, was performed to explore the biological relevance of the identified transcripts.
Assembly: GRCh38/hg38
Supplementary files format and content: bigWig, narrowPeak(except for input sample)

source_name: kidney

extract_protocol:
Following lysis of HK-2 cells with lysis buffer, DDX18 was immunoprecipitated from the cell lysates using an anti-DDX18 antibody.Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer’s instructions.
RNA libraries for RNA-seq were prepared using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® following manufacturer's protocols.

value: kidney

value: HK2 cells

value: renal tubular epithelial cells

value: igg

value: hypoxia/reoxygenation


title: SR9009_5

description: Sample 3366

value: liver


description: Library name: CMY_CIITA_KO.Miller-Plate106-D08-GGGCAAGGTT-CTAGTC

data_processing:
samples were demultiplexed using index sequences
fastq files were generated
fastq riles were merged with Paired-End reAd mergeR (PEAR)
analysis using CRIS.py
Supplementary files format and content: .txt files; top exact read counts and indel sizes and matches to test_seqs

value: Myeloid leukemia of Down syndrome (ML-DS) cell line


title: Control_1_ZT12

value: HSC

value: Control

value: 20 weeks


title: HK2 cells, DDX18A_input

description: Library name: Sample 8


title: Vehicle_5

description: Sample 3356

value: CDA/HFD


title: Control_1_ZT6


title: HK2 cells, DDX18_input

description: Library name: Sample 7


title: Vehicle_4

description: Sample 3345


title: T1-NONTARGETplusIMA1-48H_S18

city: Philadelphia

first: Ronald

middle: P

last: DeMatteo

address: 3400 Spruce Street

department: Surgery

institute: University of Pennsylvania Perelman School of Medicine

description:
Library name: T1-NONTARGETplusIMA1-48H_S18
Imatinib sensitive GIST-T1 cells

data_processing:
Reads were aligned to the human genome (GRCh38) using STAR aligner(v2.7.3a) with default parameters. Gene-count matrices were produced by featureCounts (v2.0.1).
Assembly: GRCh38
Supplementary files format and content: Sample by gene matrix of read counts

growth_protocol: Cells grown in RPMI media + 10% FBS + 1% antibiotics. Media replaced with in anti-biotic free RPMI media with 10% FBS 24h prior to transfection

extract_protocol:
RNA isolated using Rneasy Plus Mini Kit (Qiagen)
Pooled libraries were then sequenced with paired-end 2x100 bp format

treatment_protocol: Lipofectamine and siRNA transfection reagents diluted in Opti-MEM medium and added to each well. Cells harvested 48h after transfection.

value: Human GIST

value: KIT exon 11 del560-79

value: Non-target control + imatinib


description: Library name: CMY_CIITA_KO.Miller-Plate106-D06-GTAATGTTTG-CTAGTC

value: cell line

value: CMY


title: HK2 cells, DDX18A_3

description: Library name: Sample 6


title: SR9009_4

description: Sample 3357


title: T1-NONTARGETplusIMA1-48H_S17

description:
Library name: T1-NONTARGETplusIMA1-48H_S17
Imatinib sensitive GIST-T1 cells


description: Library name: CMY_CIITA_KO.Miller-Plate106-D05-TGCAAATTCA-CTAGTC