title: PC3 parental control, replicate 3

description:
Library name: PC3_parental_control_rep3
PC3 parental cells cultured under standard conditions; no drug treatment; RNA extracted for transcriptomic profiling
03_PC3_control_3_S3_001

tag: cell type


title: Grp2, mKG quartile sort, upper quartile

status: Public on Jun 22 2026

type: SRA

city: Cleveland

first: Kenneth

last: Matreyek

state: Ohio

address: 2103 Cornell Rd, Room 5104

department: Pathology

country: USA

institute: Case Western Reserve University

description: Library name: Grp2, wtBxb1, mKG bin 4

library_source: genomic

data_processing:
DNA was sequenced using a NextSeq 550 Mid output (130 million reads) 150 cycle kit (Illumina) at the Case Western Reserve University Genomics Core Facility
Reads were converted to fastq format and demultiplexed by the Case Western Reserve University Genomics Core Facility.  Paired-end reads were combined using PEAR (PMC3933873) using default statistical settings.
Each paired end read was tested for exact matches from the expected library and included as a read for the appropriate library member if applicable.  Read count relative to total number of reads in the sample was used to calculate read frequency for each expected library member.
Read frequencies for quartiles from the same fluorophore sort and the same group were used to calculate a weighted average Sort-Seq score for each fluorophore for expected library member.  Scores were averaged across groups to provide a final Sort-Seq score for each expected library member.  The Sort-Seq scores were used for further analysis in the manuscript.
Assembly: Synthetic Sequences
Supplementary files format and content: "processed_data_counts_frequencies.csv" is a comma separated values data table containing read counts and read frequencies for all observed DNA sequences (library_sequence, column 1).  Each row represents a DNA sequence of interest, and each column represents either a read count ("_counted") or a read frequency ("_freq") for each sample ("IXXXX", corresponding with IXXXX from file names as described in SAMPLES section).

molecule: genomic DNA

organism: synthetic construct

growth_protocol: DMEM with 10% FBS, 1 % Penicillin / Streptomycin, and 2 ug/mL doxycycline.

extract_protocol:
Genomic DNA was extracted using a Dneasy Blood and Tissue Kit (Qiagen, 69506) and DNA concentration was calculated using a Qubit 4 fluorometer (Thermo Fisher, Q33238) using a Qubit Broad Range dsDNA assay kit (Thermo Fisher, Q33265)
Extracted genomic DNA was used as a template to produce a 1564 nucleotide amplicon spanning the genomic integration junction.  Samples were run with gel electrophoresis, the appropriate band was extracted using a Bio-Rad Freeze 'N Squeeze DNA Gel Extraction Spin Column (Bio-Rad, 7326165) and cleaned using a Zymo DNA Clean and Concentrator-5 kit (Zymo Research, D4004).  The resulting DNA was used as the template for to produce a 299 nudleotide amplicon surrounding the DNA sequence of interest using distinct indices for each quartile to be run on the same Illumina NextSeq 550 kit run.  Samples were again run with gel electrophoresis, extracted, and cleaned.  DNA concentration was calculated using a Qubit 4 fluorometer (Thermo Fisher, Q33238) using a Qubit Broad Range dsDNA assay kit (Thermo Fisher, Q33265).  DNA amplicons were pooled to a final concentration of 10-20ng/uL in 21uL of nuclease free water using 15-20ng of DNA from each quartile prior to running the Illumina NextSeq 550 kit.

tag: cell line

value: Cultured Cells

tag: genotype

tag: treatment

value: Recombination into


title: nmr2_2_inf

city: Houston

first: Benson

last: Otarigho

state: Texas

address: 6767 Bertner Avenue

department: Department of Genetics

institute: The University of Texas MD Anderson Cancer Center

library_source: transcriptomic

data_processing:
Fq file dataset were uploaded into Galaxy Tool Version 24.0.rc1  that was used for the analyses
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence,
Sequences were mapped to W274 C elegans genome
The htseq-count were further analyesd for the differentially expressed features from count tables using DESeq2
Assembly: Genome_build: Caenorhabditis elegans genome WS274
Supplementary files format and content: tabular files include htseq-count/values for each Sample

molecule: total RNA

organism: Caenorhabditis elegans

source_name: C elegans whole animal

growth_protocol: Approximately 40 gravid animals were placed for 3 hours on 10-cm NGM plates (seeded with E. coli OP50) in order to have a synchronized population, which developed and grew to L4 larval stage at 20oC. These animals were infected with S. aureus for 4 hours at 25C

extract_protocol:
Total RNA was extracted using the RNeasy Plus Universal Kit (Qiagen, Netherlands). Purification of Total RNA was done using the TURBO DNase.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Animals were washed off the plates with M9 and frozen in QIAzol by ethanol/dry ice and stored at -80 prior to RNA extraction.

tag: tissue

value: C elegans whole animal

value: Animals were infected with S. aureus for 4 hours wash with M9 buffer, and frozen (-80 C), total RNA extacted


title: nmr2_1_inf


title: Rice leaf, moderate salinity, rep 1

city: Winnipeg

first: Olivia

last: Wilkins

state: Manitoba

address: 66 Chancellors Cir

department: Biological Sciences

country: Canada

institute: University of Manitoba

description:
Library name: Moderate.Salinity.Rep1
10X Genomics Chromium Next GEM Single Cell 3' Reagents Kit v3.1 (Dual Index)

library_source: transcriptomic single cell

data_processing:
Raw reads were pseudoaligned to the IRGSP1.0 transcriptome (Kawahara et al., 2013) and quantified with scKB (Hsu et al., 2022).
High-quality nuclei were selected with COPILOT (Hsu et al., 2022; minimum 600 UMIs per nucleus) and doublets were removed with DoubletFinder (McGinnis et al., 2019).
Nuclear transcriptomes were loaded into separate Seurat objects with Seurat v5.1.0 (Hao et al., 2024) and counts were normalized and scaled (5000 most variable features).
Nuclei with >10% chloroplast reads were removed. All rRNA, all organellar genes, and nuclear genes with fewer than 100 counts across all nuclei were removed for downstream analyses.
To integrate the samples, integration anchors across all libraries were identified and projected with RPCA (reciprocal principal component analysis) with Seurat's IntegrateLayers().
Nuclei were clustered and manually annotated with tissue systems and cell types (metadata columns TissueSystem and CellAnnotation).
Assembly: IRGSP1.0
Supplementary files format and content: OsLeafStressIntegrated.rds: Fully processed, integrated, and annotated dataset (Seurat v5 R object).
Supplementary files format and content: Gene counts output from COPILOT (Hsu et al., 2022); contains filtered, high-quality cells with doublets removed. Sparse matrices in Market Exchange (MEX) format, consisting of gzipped tsv files of barcodes and genes, and gzipped matrix files.

molecule: polyA RNA

organism: Oryza sativa

source_name: Developing third leaf, ~6cm

growth_protocol: Oryza sativa cv. Nipponbare rice seeds were germinated in moist, dark conditions at 30°C for 96 h prior to planting. Rice seedlings were grown in a Conviron growth chamber with a 12 h day/night cycle, 30°C/25°C day/night temperatures, 700 μmol/m2/s light intensity, and 80% relative humidity. Pots were weighed daily and were re-watered to their original weight with tap water to maintain their set SWC until the third leaves had emerged ~1 cm beyond the sheath collar, for a full length of ~6-7cm.

extract_protocol:
Nuclei were isolated as described in Guillotin et al., (Guillotin et al., 2023) with modifications. Briefly, for each condition, one developing leaf from the shoot apical meristem to the green leaf tip (6-7 cm), was dissected at ZT 2 (Zeitgeber Time) and placed in 300 µL of ice cold lysis buffer [0.3 M sucrose, 1.25% Ficoll, 2.5% Dextran T40, 15 mM Tris-HCl, 20 mM MES, 10 mM MgCl₂, 60 mM KCl, 15 mM NaCl, 0.5 mM spermine and spermidine, 0.05% Triton X-100, 5 mM DTT, 1 mM PMSF, 1% Protease Inhibitor Cocktail, 0.4 u/µl Protector RNase Inhibitor, 0.4% BSA, 1% DEPC] and chopped with a razor blade for four minutes. The macerated tissue was gently ground with a micropestle, then filtered through a 20µm microcentrifuge-top filter and centrifuged at 500 x g for 10 minutes. The pellet of nuclei was resuspended in 300 µL of wash buffer [0.3 M sucrose, 15 mM Tris-HCl, 15 mM MES, 60 mM KCl, 15 mM NaCl, 2 mM EDTA, 0.5 mM spermine and spermidine, 0.05% Triton X-100, 5 mM DTT, 1 mM PMSF, 1% Protease Inhibitor Cocktail, 0.2 u/µl Protector RNase Inhibitor, 0.4% BSA] and centrifuged at 500 x g for 5 minutes. The pellet of nuclei was resuspended in 80 µL of final buffer [0.3 M sucrose, 15 mM Tris-HCl, 15 mM MES, 60 mM KCl, 15 mM NaCl, 0.5 mM spermine and spermidine, 5 mM DTT, 1% Protease Inhibitor Cocktail, 0.2 u/µl Protector RNase Inhibitor, 1% BSA] and filtered through a 10µm microcentrifuge-top filter.
Approximately 16,000 nuclei per sample were prepared for sequencing using the 10X Genomics Chromium Next GEM Single Cell 3’ Reagents Kits v3.1 (Dual Index) protocol without modification on the 10X Genomics Chromium X instrument. Libraries were sequenced at Génome Québec (Montréal, QC, Canada) for sequencing of 100 bp paired-end reads on a NovaSeq 6000 (Illumina, San Diego, CA, USA).

treatment_protocol: Stable gravimetric soil water content (SWC) was achieved by mixing oven-dried soil with tap water or NaCl made with tap water, as follows: 3 g water/g soil for the well-watered control; 1.5 g water/g soil for mild drought; 1 g water/g soil for moderate drought; 3 g of 100 mM NaCl/g soil for the mild salinity; 3 g of 200 mM NaCl/g soil for the moderate salinity. Mild salinity had a consistent electrical conductivity (EC) of 1.25 mS/cm throughout the experiment, while moderate salinity had an EC of 2.5 mS/cm; well-watered control had an EC of 0.5 mS/cm.

value: Developing third leaf, ~6cm

tag: cultivar

value: Nipponbare

value: Moderate salinity; 3 g 200 mM NaCl / g soil

tag: batch


title: Grp2, mKG quartile sort, upper middle quartile

description: Library name: Grp2, wtBxb1, mKG bin 3


title: PC3 parental control, replicate 2

city: Bethesda

first: Yves

last: Pommier

address: 9000 Rockville Pike

institute: National Cancer Institute

description:
Library name: PC3_parental_control_rep2
PC3 parental cells cultured under standard conditions; no drug treatment; RNA extracted for transcriptomic profiling
02_PC3_control_2_S2_001

data_processing:
Raw FASTQ files were processed using the RENEE RNA-seq analysis pipeline. Quality control was performed using FastQC. Reads were aligned to the human reference genome (hg38) using STAR (v2.7). Gene-level quantification was performed using RSEM.
Assembly: hg38 (GRCh38)
Supplementary files format and content: Processed data files include gene-level expression matrices in FPKM and/or raw counts format for all samples. Files contain one row per gene and one column per sample, with gene symbols and expression values.

organism: Homo sapiens

source_name: prostate cancer (cell line)

growth_protocol: PC3 and LNCaP cells were cultured under standard conditions. PC3 cells were maintained in F-12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. LNCaP cells were cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin–streptomycin. Cells were grown at 37 °C in a humidified 5% CO₂ incubator and routinely tested for mycoplasma contamination.

extract_protocol:
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA integrity was assessed using the Agilent TapeStation system. 800 ng of high-quality RNA (poly(A)+) from each sample was used for library preparation.
Poly(A) mRNA was enriched using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB E7490). Sequencing libraries were constructed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB E7765) with sample purification beads and NEBNext Unique Dual Index primers (E6440S). Libraries were sequenced on an Illumina NextSeq 2000 instrument using paired-end 100 bp reads.

treatment_protocol: For short-term drug exposure, cells were treated with Acetalax at 10 µM for 4 hours. Control samples received no treatment. Chronic exposure (CE) derivatives were generated by culturing parental cells under gradually increasing Acetalax concentrations over multiple passages until stable resistance was established. CE cells were cultured in drug-free medium for two weeks prior to RNA extraction.

value: prostate cancer (cell line)

value: epithelial cancer cell line


title: WT 3

growth_protocol: Approximately 40 gravid animals were placed for 3 hours on 10-cm NGM plates (seeded with E. coli OP50) in order to have a synchronized population, which developed and grew to L4 larval stage at 20oC

value: Wild type (N2)

value: Animals wash with M9 buffer, and frozen (-80 C), total RNA extacted


title: PC3 parental control, replicate 1

description:
Library name: PC3_parental_control_rep1
PC3 parental cells cultured under standard conditions; no drug treatment; RNA extracted for transcriptomic profiling
01_PC3_control_1_S1_001


title: Grp2, mKG quartile sort, lower middle quartile

description: Library name: Grp2, wtBxb1, mKG bin 2


title: Rice leaf, mild salinity, rep 2

description:
Library name: Mild.Salinity.Rep2
10X Genomics Chromium Next GEM Single Cell 3' Reagents Kit v3.1 (Dual Index)

value: Mild salinity; 3 g 100 mM NaCl / g soil


title: Rice leaf, mild salinity, rep 1

description:
Library name: Mild.Salinity.Rep1
10X Genomics Chromium Next GEM Single Cell 3' Reagents Kit v3.1 (Dual Index)


title: WT 2


title: Grp2, mKG quartile sort, lower quartile

description: Library name: Grp2, wtBxb1, mKG bin 1


title: Rice leaf, moderate drought, rep 2

description:
Library name: Moderate.Drought.Rep2
10X Genomics Chromium Next GEM Single Cell 3' Reagents Kit v3.1 (Dual Index)

value: Moderate drought; 1 g water / g soil


title: miRNA-Seq, PA1-Dicer mutation D1709N homozygous bio repl. 2

city: Edinburgh

first: Jeroen

last: Witteveldt

state: midlothian

address: The King's Buildings, Ashworth Laboratories

department: IIIR

country: United Kingdom

institute: The University of Edinburgh

description: Library name: PA1-D1709N-HOM2

data_processing:
Small RNA-Seq data were processed as follows. FastQC and MultiQC were used to evaluate the quality of the raw reads . Reaper was used to remove the 3’ adapter sequences, and distinct sequences were collapsed using tally, selecting those between 20-25nt, and keeping track of the counts of each read. Unique reads were mapped against the human genome (GRCh38), using bowtie1 v1.3.1 allowing 1 mismatch and reporting all possible locations (-v 1 --all --best –strata). A python script was then used to distribute each read’s counts evenly between all mapping locations. The remaining analyses were conducted within the R environment for statistical computing. FeatureCounts was used to quantify overlapping reads. Differential expression tests were performed within the edgeR framework, with quasi-dispersion estimates, and using glmTreat to test against a minimum fold-change threshold of 2 and a Benjamini-Hochberg corrected FDR < 0.01. Normalisation factors were calculated manually using tRNAs as the reference annotation. Plotting used a combination of base R, ggplot2, ggside and ggrepel. A custom genome annotation was developed, compiling protein-coding, lncRNA and tRNA annotation from Gencode r49, microRNAs from miRBase r22.1 and MirGeneDB r3.0, mirtrons from mirtronDB v2.
Assembly: GRCh38/hg38 Gencode39
Supplementary files format and content: fold change

source_name: ovary

growth_protocol: Human PA-1 cells were cultured in Minimum Essential Medium (MEM) with GlutaMAX (Gibco) supplemented with 20% heat-inactivated fetal bovine serum (FBS,Gibco), 50ug/ml Gentamicin (Gibco) and 0.1mM MEM Non-Essential Amino Acids (Gibco) at 37°C with 5% CO2.

extract_protocol:
The Qiagen miRNeasy kit was used to isolate the total RNA fraction
Libraries were prepared from 500 ng of each total RNA sample using the QIAseq miRNA library kit 96 (QIAGEN, #331505) and the QIAseq miRNA 96 Index Kit IL UDI-E (#331655) according to the provided protocol. Mature miRNAs possess both a 3’ hydroxyl group and a 5’ phosphate. A pre-adenylated DNA adapter was ligated to the 3’ ends of all miRNAs and then an RNA adapter was ligated to the 5’ end of mature miRNAs. After both ligations, the ligated miRNAs were reverse-transcribed into cDNA. The reverse transcription primer contains an integrated UMI and binds to a region of the 3’ adapter where it can facilitate conversion of the ligated miRNAs into cDNA while assigning a UMI to every miRNA molecule. During this step a universal sequence is also added that is recognized by the sample indexing primers during library amplification. Libraries were purified using QIASeq miRNA NGS Beads (QMN beads). Purified libraries were then amplified for 13 cycles of PCR with unique 10bp dual index (UDI) sequences to allow multiplexing on a single sequencing run. Amplified indexed libraries were purified and size selected with QMN beads to enrich for library fragments from miRNA.

treatment_protocol: Cells did not receive any specific treatments

value: ovary

value: teratocarcinoma of the ovary


title: WT 1


title: miRNA-Seq, PA1-Dicer mutation D1709N homozygous bio repl. 1

description: Library name: PA1-D1709N-HOM1


title: Grp2, mCherry quartile sort, upper quartile

description: Library name: Grp2, wtBxb1, mCherry bin 4


title: Rice leaf, moderate drought, rep 1

description:
Library name: Moderate.Drought.Rep1
10X Genomics Chromium Next GEM Single Cell 3' Reagents Kit v3.1 (Dual Index)


title: nmr2 3


title: nmr2 2


title: miRNA-Seq, PA1-Dicer mutation S1344L homozygous

description: Library name: PA1-S1344L-HOM


title: Rice leaf, mild drought, rep 2

description:
Library name: Mild.Drought.Rep2
10X Genomics Chromium Next GEM Single Cell 3' Reagents Kit v3.1 (Dual Index)

value: Mild drought; 1.5 g water / g soil


title: Grp2, mCherry quartile sort, lower middle quartile

description: Library name: Grp2, wtBxb1, mCherry bin 2


title: miRNA-Seq, PA1-wild type

description: Library name: PA1-WT


title: Grp2, mCherry quartile sort, upper middle quartile

description: Library name: Grp2, wtBxb1, mCherry bin 3


title: smallRNA-Seq, PA1-Dicer heterozygous

description: Library name: PA1-Dicer-HET


title: Grp2, mCherry quartile sort, lower quartile

description: Library name: Grp2, wtBxb1, mCherry bin 1


title: Rice leaf, mild drought, rep 1

description:
Library name: Mild.Drought.Rep1
10X Genomics Chromium Next GEM Single Cell 3' Reagents Kit v3.1 (Dual Index)


title: nmr2 1


title: Rice leaf, well-watered, rep 3

description:
Library name: Well.Watered.Rep3
10X Genomics Chromium Next GEM Single Cell 3' Reagents Kit v3.1 (Dual Index)

value: Well-watered; 3 g water / g soil


title: Mature Replciate 4

city: Philadelphia

first: Jianing

last: Yang

state: Pennsylvania

address: 3400 Civic Blvd

department: Neuroscience

institute: University of Pennsylvania

description: Library name: Sample 8

data_processing:
STAR version 2.7.6a, --outFilterMismatchNmax 33 --seedSearchStartLmax 33 --alignSJoverhangMin 8
Assembly: BDGP.46
Supplementary files format and content: ReadsPerGene.out.tab

organism: Drosophila melanogaster

source_name: Female Central brain

growth_protocol: Th>Tdtomato and negative control +>TdTomato  female flies aged 0-1 days or 6-10 days were reared at 25C under a 12h:12h light:dark cycle.

extract_protocol:
Brains were dissected in cold Schneider's media. Brains were first enzymatically dissociated in Papain (100U/ml) suspended in 1X PBS and supplemented with 0.18U/ml LiberaseTM cocktail (Roche). Dissociation was carried out for 50 min at room temperature with nutating. Dissociated brains were rinsed three times in cold Schneider’s to stop dissociation. Schneider’s medium was replaced by 2% Bovine Serum Albumin in 1XPBS.  Mechanical trituration was performed by first pipetting the brains up and down 100% in P200 pipette tips pre-coated with fetal bovine serum; then, dissociated tissue was passed 20 times through a 3cc gauge needle pre-coated with fetal bovine serum. The suspension was then transferred to FACS tubes kept on ice. DAPI was added to label dead cells.  In parallel, +>TdTomato brains were treated in parallel to gate TdTomato signal during FACS sorting. Sorting was performed using an Aria cytometer equipped with a  450 nm UV and 582 nm lasers and a 100 um nozzle.
RNA was prepared by Admera using the SMARTseq V4 with Nextera XT

value: Female Central brain

value: dopaminergic neurons

value: Th>Tdtomato

tag: age

value: Mature


title: smallRNA-Seq, PA1-Dicer mutation D1709N compound heterozygous

description: Library name: PA1-D1709N-DCR


title: Grp1, miRFP670 quartile sort, upper quartile

description: Library name: Grp1, eeBxb1, miRFP670 bin 4

value: Cultured Cells

value: Recombination into


title: Mature Replicate 3

description: Library name: Sample 7


title: smallRNA-Seq, PA1-Dicer mutation D1709N homozygous bio repl. 2

description: Library name: PA1-D1709N-HOM2


title: Rice leaf, well-watered, rep 2

description:
Library name: Well.Watered.Rep2
10X Genomics Chromium Next GEM Single Cell 3' Reagents Kit v3.1 (Dual Index)

value: Developing third leaf, ~6cm

value: Nipponbare


title: Grp1, miRFP670 quartile sort, upper middle quartile

description: Library name: Grp1, eeBxb1, miRFP670 bin 3


title: Mature Replicate 2

description: Library name: Sample 6


title: Rice leaf, well-watered, rep 1

description:
Library name: Well.Watered.Rep1
10X Genomics Chromium Next GEM Single Cell 3' Reagents Kit v3.1 (Dual Index)


title: Grp1, miRFP670 quartile sort, lower middle quartile

description: Library name: Grp1, eeBxb1, miRFP670 bin 2


title: smallRNA-Seq, PA1-Dicer mutation D1709N homozygous bio repl. 1

description: Library name: PA1-D1709N-HOM1


title: C10 tumor Exp 2

type: RNA

first: Jia

last: Nie

state: Maryland

address: Building 37, Room 3016

department: Center for Cancer Research

hyb_protocol: Then FFPE tissue sections were loaded onto a Xenium slide (12 x 24 mm), followed by deparaffinization, and decrosslinking to make the mRNA accessible. Subsequently, probe hybridization, ligation and amplification were performed following the Xenium In Situ Gene Expression User Guide (CG000582).

scan_protocol: Xenium slide containing FFPE tumor tissue sections were then loaded on Xenium Analyzer instrument (software version 1.7.4.0) for imaging and high-throughput, automated in situ analysis.

data_processing: Segmentation and transcript aggregation into segmented cells was performed with Xenium Ranger 1.7,and subsequently analyzed with Seurat v5.

organism: Mus musculus

source_name: C10 subcutaneous tumor exp_2

extract_protocol: C10 tumors on day 10 were dissected and were subjected to formalin-fixed and paraffin-embedded (FFPE) processing. Then 5 μm FFPE tissue sections were cut.

value: RAG1–/– and tKO mice

value: PBS or Smarta treatment

value: C10 subcutaneous tumor


title: smallRNA-Seq, PA1-Dicer mutation S1344L homozygous

description: Library name: PA1-S1344L-HOM


title: Mature Replicate 1

description: Library name: Sample 5


title: C10 tumor Exp 1

source_name: C10 subcutaneous tumor exp_1


title: Juvenile Replciate 4

description: Library name: Sample 4

value: Juvenile


title: smallRNA-Seq, PA1-wild type

description: Library name: PA1-WT


title: Grp1, mKG quartile sort, upper quartile

description: Library name: Grp1, eeBxb1, mKG bin 4


title: Larvae, 6dpf, DMSO-treated (Ctrl), 3

city: Hamburg

first: Olga

last: Tsoy

state: Hamburg

address: Alber-Einstein Ring 8-10

country: Germany

institute: Universität Hamburg

description: Library name: H03_22Jul4

data_processing:
RNA-Seq raw reads for NFP-treated zebrafish larvae and isolated glomeruli after both 5 and 6 dpf and compared to the control were mapped to the zebrafish reference genome GRCz11 with the genome annotation from Ensembl version 108 using STAR version 2.7.5c with 2-pass mode
Gene read counts were obtained by the featureCounts tool included in the Subread 2.0.0
Read counts were further normalized and used for differential gene expression by DESeq2 version 1.38.3
For isoform switch analysis, we prepared transcript counts using salmon version 1.9.0 with the transcript annotation based on Ensembl version 108 and ran the R package IsoformSwitchAnalyzeR version 1.20.0 with the DESeq2 algorithm
For alternative splicing analysis, we used leafcutter version 0.2.9, rMATS version 4.1.2, and Whippet version 0.11.1 (with julia 1.6.6) with the default parameters. We defined significantly differentially used events as events with a p-value < 0.05 (for leafcutter and rMATS) or a confidence > 0.95 (for Whippet).
Assembly: GRCz11
Supplementary files format and content: tab-delimired text file includes raw gene counts for each sample
Supplementary files format and content: tab-delimired text file includes TPM transcript counts for each sample

organism: Danio rerio

source_name: Larvae

extract_protocol:
For total RNA, whole larvae samples were processed using Tri-Reagent (Sigma-Aldrich) according to manufacturer's instructions, followed by DNA-digestion and purification steps using the RNase-Free DNase I Kit and RNA Clean-Up and Concentration kit according to manufacturer’s instructions (both Norgen Biotek Corp., Thorold, ON, Canada). The quality of the extracted RNA was assessed using the Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA).  All samples exhibited a mean RNA integrity score (RIN) of 10. Total RNA of isolated glomeruli was derived by combination of Tri-Reagent (Sigma Aldrich) and Norgen Single Cell RNA Purification kit (Norgen Biotek Corp., Thorold, ON, Canada) according to manufacturer´s instructions with an elution volume of 10µl. The quality of the extracted RNA was assessed using the Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA).  All samples exhibited a mean RNA integrity score (RIN) of 7.4 ± 0.3 SD.
PolyA library was prepared based on 500ng total RNA per sample using the TruSeq stranded mRNA kit (Illumina, San Diego, CA, USA).  Subsequently, for sequencing 200pM of the final library were loaded on the NovaSeq 6000 System, (2 x 100 bp pair-end reads). A sequencing depth of ~ 200 Mio Cluster per sample was obtained.

value: Larvae

value: Whole Larvae

value: DMSO


title: Juvenile Replicate 3

description: Library name: Sample 3


title: PA1-Dicer mutation D1709N compound heterozygous

description: Library name: PA1-D1709N-HET

data_processing:
Total RNA-sequencings were processed with the following pipeline. Gene based read counts and TPMs were retrieved for each sample after mapping over human genome GRCh38/hg38 using Gencode39 annotation (Ensembl based) with STAR v2.7.9a58 with default parameters. Statistical inference of regulated genes of one comparison of grouped DICER vs WT samples were calculated with the DESeq2 v1.3459 R library by setting the contrast parameter and weighted equally to the log2 fold changes of the four DICER mutants. Differentially regulated genes were retrieved with an FDR cut-off of 0.05 obtained with the Benjamini & Hochberg correction.
Assembly: GRCh38/hg38 Gencode39
Supplementary files format and content: fold change

extract_protocol:
The Qiagen miRNA easy kit was used to isolate the total RNA fraction
500ng of total-RNA was added to the ribosomal RNA (rRNA) depletion reaction using the NEBNext rRNA depletion kit (Human/mouse/rat). This step uses specific probes that bind to the rRNA in order to cleave it. rRNA-depleted RNA was then DNase treated and purified using Agencourt RNAClean XP beads (Beckman Coulter Inc, #66514). RNA was then fragmented using random primers before undergoing first strand and second strand synthesis to create cDNA. Fragmentation time was dependent upon the quality of each total RNA sample. cDNA was end-repaired before ligation of sequencing adapters, and adapter-ligated libraries were enriched by 10 cycles of PCR using NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) (NEB, #E6440 or E6442). Final libraries had an average peak size of 332bp.

value: ovary

value: teratocarcinoma of the ovary

value: D1709N compound heterozygous


title: Grp1, mKG quartile sort, upper middle quartile

description: Library name: Grp1, eeBxb1, mKG bin 3


title: Glomeruli, 5dpf, NFP-treated, 3

description: Library name: H02_22Jul4

source_name: Glomeruli

value: Glomeruli

value: Isolated Glomeruli

value: NFP


title: Juvenile Replicate 2

description: Library name: Sample 2

value: Female Central brain

value: dopaminergic neurons

value: Th>Tdtomato


title: PA1-Dicer mutation D1709N homozygous rep2

description: Library name: PA1-D1709N-HOM-biol rep2

value: D1709N homozygous


title: Grp1, mKG quartile sort, lower middle quartile

description: Library name: Grp1, eeBxb1, mKG bin 2

value: Cultured Cells

value: Recombination into


title: Grp1, miRFP670 quartile sort, lower quartile

description: Library name: Grp1, eeBxb1, miRFP670 bin 1


title: Glomeruli, 5dpf, NFP-treated, 2

description: Library name: H01_22Jul4


title: Juvenile Replicate 1

description: Library name: Sample 1


title: PA1-Dicer mutation D1709N homozygous rep1

description: Library name: PA1-D1709N-HOM-biol rep1


title: Larvae, 6dpf, NFP-treated, 3

description: Library name: G03_22Jul4


title: Grp1, mKG quartile sort, lower quartile

description: Library name: Grp1, eeBxb1, mKG bin 1


title: Sublibrary2 pool of all samples

city: Chicago

first: Daniel

last: Cavanaugh

state: IL

address: 1050 W Sheridan Rd., Life Sciences Building Room 215

department: Biology

institute: Loyola University Chicago

description: Library name: Sublibrary2

data_processing:
FASTQ files were processed for barcode correction, read alignment, read deduplication, and transcript quantification using Parse Biosciences TrailmakerTM pipeline module (v1.5.1). The reference genome was generated by adding GAL4 and Cas9.P2 sequences to the Drosophila melanogaster genome (Ensembl BDGP6.32 release 109).
Further processing of transcripts was done in the TrailmakerTM insights module. Nuclei with <1000 transcripts, >0.2% mitochondrial content, and >0.2 probability of being a doublet (determined by scDblFinder) were filtered out. Outliers in the distribution of the number of transcripts vs. number of genes were removed, as determined by fitting a spline regression model with default settings. Data normalization, principal component analysis (PCA), and data integration were performed using Seurat and Harmony. Initial clusters were identified with the Leiden method at a 0.4 resolution, and a Uniform Manifold Approximation and Projection (UMAP) embedding was calculated.
The resulting data were further processed in R (v4.5.1) with the Seurat package (v5.2.0). Ambient RNA contamination was filtered out using automatic estimations from SoupX with the original unfiltered data and the clustered data from TrailmakerTM used as input. Nuclei were removed if their log transformed transcript count was >3 absolute deviations from the median log transformed transcript count, and genes were removed if they were expressed in <3 nuclei. After this filtering, data were processed with the standard Seurat workflow, using the following functions with default values unless otherwise stated. Raw counts were normalized with NormalizeData, which divides transcript counts of a single gene from each nucleus by the total number of counts of all genes for that nucleus, multiples them by a scaling factor, and performs a natural log transformation of the result. Highly variable genes were determined with FindVariableFeatures and normalized transcript counts were scaled with ScaleData so that PCA could be performed using RunPCA, and the first 10 PCs were used for downstream processing. New clusters were determined with FindNeighbors and FindClusters, using the Leiden algorithm at a 0.2 resolution. Finally, non-linear dimensional reduction was performed with RunUMAP.
Assembly: Dm6
Supplementary files format and content: RDS file contains single-cell Seurat object with relevant metadata for analysis in R.

molecule: nuclear RNA

source_name: Abdomen

growth_protocol: Flies were raised in incubators held at 25°C and entrained for 7d under 12:12 LD cycles and transferred to DD conditions on the day of tissue collection.

extract_protocol:
Nuclei isolation was performed using protocols adapted from McLaughlin et al., 2022. Frozen fly abdomens were quickly thawed on ice and resuspended in 950 μl of homogenization buffer (HB) consisting of: 250 mM sucrose, 10 mM Tris (pH 8.0), 25 mM KCl, 5 mM MgCl2, 0.1% Triton X-100, 1x protease inhibitor cocktail (Promega G6521), 0.1 mM DTT, 200 U/mL RNAsin Plus (Promega N2615), and 20 U/mL heat-activated DNAse I (Thermo Scientific EN0521), in nuclease free water. Resuspended abdomens were transferred to a prechilled (4°C) Wheaton dounce homogenizer (1 mL capacity; Fisher Scientific 06-434) on ice and allowed to settle at the bottom of the homogenizer. To free nuclei, the loose pestle was first used to shear the tissue with 20 half-turns while trapping the abdomens at the bottom of the dounce. This was followed by 20 strokes with the loose pestle and 40 strokes with the tight pestle. The homogenized tissue was then filtered through a SWiSH 30 micron mini cell strainer (Stellar Scientific TC70-SWM-30) and centrifuged for 10 min at 500 x g at 4°C in a fixed-angle table-top centrifuge. Following isolation, the Evercode Nuclei Fixation V3 kit (Parse Biosciences ECFN3300) was used to fix and store nuclei following manufacturer instructions.
Single nuclei barcoding and library preparation with the Evercode WT Mini V3 kit (Parse Biosciences ECWT3100) was performed by the Northwestern University Center for Genetic Medicine NUseq Core facility according to manufacturer instructions.

treatment_protocol: Nuclei from abdominal tissues of male control (to-GAL4>UAS-Cas9.P2) and experimental (to-GAL4>UAS-Cas9.P2; UAS-gRNA-per) flies were sampled at two circadian timepoints: CT0 and CT12.

value: Abdomen

value: 7 days

value: Male

value: to>Cas9 and to>perCRISPR

value: CT0 and CT12


title: PA1-Dicer mutation S1344L homozygous

description: Library name: PA1-S1344L-HOM

value: ovary

value: S1344L homozygous


title: Grp1, mCherry quartile sort, upper quartile

description: Library name: Grp1, eeBxb1, mCherry bin 4


title: Glomeruli, 5dpf, DMSO-treated (Ctrl), 3

description: Library name: G02_22Jul4

value: DMSO


title: Sublibrary1 pool of all samples

description: Library name: Sublibrary1


title: PA1-wild type

description: Library name: PA1-WT

value: teratocarcinoma of the ovary

value: PA1-wild type


title: Grp1, mCherry quartile sort, upper middle quartile

description: Library name: Grp1, eeBxb1, mCherry bin 3


title: Glomeruli, 5dpf, DMSO-treated (Ctrl), 2

description: Library name: G01_22Jul4

value: Glomeruli

value: Isolated Glomeruli


title: PEC, day 5 T. gondii infection, ADT

city: Indianapolis

first: Americo

middle: H.

last: Lopez-Yglesias

state: IN

address: 635 Barnhill Drive, MS 268

department: Microbiology and Immunology

institute: Indiana University School of Medicine

description: Library name: AL3_ADT

library_source: other

data_processing:
Raw FASTQ files were mapped to the GRCm39 reference genome using 10x Genomics Cell Ranger 9.0.0 to generate gene expression (RNA) and antibody-derived tag counts per cell.
Quality control was performed in Seurat v5: cells with <500 or >7500 detected genes, <1000 or >50000 UMIs, or >20% mitochondrial content were removed.
RNA counts were normalized using SCTransform. PCA was used for dimensionality reduction (top 30 principal components, resolution 0.5) for clustering, visualized by t-SNE.
Antibody-derived tag counts were normalized using the centered log ratio (CLR) method.
Assembly: GRCm39
Supplementary files format and content: Cell Ranger filtered feature-barcode matrix in Matrix Market (MEX) format: AL3_barcodes.tsv.gz (cell barcodes), AL3_features.tsv.gz (gene IDs and antibody features with feature type), AL3_matrix.mtx.gz (combined RNA + ADT counts).
Library strategy: CITE-seq

molecule: protein

source_name: peritoneal cavity

extract_protocol:
Peritoneal exudate cells were collected by peritoneal lavage, stained with the BioLegend TotalSeq-B Mouse Universal Cocktail v1.0, and loaded onto the 10x Genomics Chromium platform.
Single-cell gene expression and cell-surface protein (CITE-seq) libraries were prepared using the 10x Genomics Chromium Single Cell 3' kit with Feature Barcoding for cell-surface protein, per manufacturer instructions.

value: peritoneal cavity

value: peritoneal exudate cells

value: wild-type

value: Toxoplasma gondii, 20 cysts i.p., day 5 post-infection


title: Cetuximab-treated HN5 cells_biol rep3

city: Woluwé

first: Jérôme

last: Ambroise

address: Avenue Hippocrate 54

department: Institut de Recherche Expérimentale et Clinique

country: Belgium

institute: Université catholique de Louvain

description: Library name: HN5.ctx.3

data_processing:
Reads were mapped using  salmon:1.10.3 to the transcriptome index which was built using all Ensembl cDNA sequences obtained in the Homo_sapiens.GRCh38.cdna.all.fa.file
Then, estimated transcript abundances from Salmon were imported into R using the tximport_1.36.1 package
Assembly: Homo_sapiens.GRCh38.cdna.all.fa.file
Supplementary files format and content: excel file with the matrix of reads counts for each gene and each sample

source_name: Oral cavity (tongue)

growth_protocol: All cells were cultured in Dulbeccos’s modified Eagle’s medium (DMEM) containing 25 mM glucose and 4 mM GlutaMAX (#61965026, Thermo Fisher Scientific), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin (Thermo Fisher Scientific) and maintained in exponential growth in 5% CO2/95% air in a humidified incubator at 37°C.

extract_protocol:
Total RNA was extracted from cancer cells using the Monarch Total RNA Miniprep Kit (New England Biolabs). Ribosomal RNA (rRNA) was depleted using the RiboCop rRNA Depletion Kit (Lexogen).
The rRNA-depleted RNA was then used for library preparation with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs), following the manufacturer's instructions. Briefly the key stages of this protocol are successively, the purification of PolyA containing mRNA molecules using poly-T oligo attached magnetic beads from 1μg total RNA (with the Magnetic mRNA Isolation Kit from NEB), a fragmentation using divalent cations under elevated temperature to obtain approximately 300bp pieces, double strand cDNA synthesis and finally Illumina adapters ligation and cDNA library amplification by PCR for sequencing. Sequencing is then carried out on Paired End 100b reads of Illumina NovaSeq.Image analysis and base calling is performed using Illumina Real Time Analysis (3.4.4) with default parameters.

treatment_protocol: HNSCC cells were treated with 1 µg/mL cetuximab (or vehicle) for 3 days.

value: Oral cavity (tongue)

value: Cetuximab


title: Grp1, mCherry quartile sort, lower middle quartile

description: Library name: Grp1, eeBxb1, mCherry bin 2

value: Cultured Cells

value: Recombination into


title: Glomeruli, 5dpf, NFP-treated, 1

description: Library name: F03_22Jul4

value: NFP


title: Cetuximab-treated HN5 cells_biol rep2

description: Library name: HN5.ctx.2


title: Grp1, mCherry quartile sort, lower quartile

description: Library name: Grp1, eeBxb1, mCherry bin 1


title: PEC, day 5 T. gondii infection, GEX

description: Library name: AL3_GEX


title: Larvae, 6dpf, NFP-treated, 2

description: Library name: F02_22Jul4

value: Larvae

value: Whole Larvae


title: Mouse Visium HD spatial gene expression (Tg88_A1)

city: Aurora

first: Eric

last: Prince

state: CO

address: 12800 E 19th AAve

department: Neurosurgery

institute: University of Colorado Anschutz Medical Campus

description:
Library name: TgNotch3R169C_Brain_HD_2
Visium HD CytAssist, Space Ranger 3.1.1

data_processing:
2023 batch: bcl2fastq used for demultiplexing; Space Ranger 2.0.1 count against mm10-2020-A with Visium Mouse Transcriptome Probe Set v1.0; cerebral vessel samples aligned manually via Loupe alignment JSON (v2).
2024 batch: demultiplexed to FASTQ; Space Ranger 3.1.1 count (Visium HD) vs mm10-2020-A; outputs binned at 2 um, 8 um and 16 um (8 um bin submitted).
Assembly: mm10 (refdata-gex-mm10-2020-A)
Supplementary files format and content: 2023 processed files (per sample): filtered/raw_feature_bc_matrix.h5, metrics_summary.csv, cloupe.cloupe, spatial/ (tissue_positions.csv, scalefactors_json.json, tissue_hires/lowres_image.png, aligned_fiducials.jpg, detected_tissue_image.jpg, cytassist_image.tiff).
Supplementary files format and content: 2024 HD processed files (per sample): metrics_summary.csv, top-level spatial images, and the 8 um bin (square_008um) filtered/raw_feature_bc_matrix.h5, cloupe.cloupe, scalefactors_json.json, tissue_positions.parquet. (2 um and 16 um bins and feature_slice.h5 available on request.)
Library strategy: Spatial Transcriptomics

source_name: Mouse tissue, FFPE section

extract_protocol:
FFPE tissue sections placed on 10x Visium CytAssist slides; RNA captured via probe hybridization (Mouse Transcriptome Probe Set v1.0, mm10-2020-A) following the standard 10x Genomics Visium CytAssist for FFPE protocol.
2023 batch: 10x Genomics Visium CytAssist Spatial Gene Expression for FFPE. 2024 batch: 10x Genomics Visium HD Spatial Gene Expression for FFPE (CytAssist). Libraries prepared per the manufacturers standard protocol.

value: brain

value: transgenic, TgNotch3^R169C (line Tg88)

value: 2024 Visium HD

tag: capture area


title: Cetuximab-treated HN5 cells_biol rep1

description: Library name: HN5.ctx.1


title: input, arp8swr1htz1, 6h

city: Dortmund

first: Anja

last: Deiser

address: Otto-Hahn-Str. 6

department: DOLCE

institute: TU-Dortmund

description: Library name: IN_arp8swr1htz1_6h_L2

data_processing:
Sequencing reads were mapped to the Saccharomyces cerevisiae genome using Bowtie2 (v2.4.2) with the parameters --no-discordant --no-mixed --local SAMtools (v1.9) was used to filter our multiple mapping reads and to select reads with alignment quality higher than 30 (MAPQ > 30).
Forward and reverse reads were seperated using SAMtools.
Bedtools (v2.30.0) was used to generate coverage files (bedgraph format).
Assembly: S.cerevisiae R64 (SacCer3)
Supplementary files format and content: Bedgraph files containing reads coverage

organism: Saccharomyces cerevisiae

growth_protocol: Cells were grown in YP-media supplemented with 2 % raffinose at 30 °C. Exponentially growing cells (OD600 0.5 - 0.7) were synchronized in M phase with 5 µg/mL nocodazole. Cell cycle arrest was ensured by microscopy and the DNA content measured by flow cytometry.

extract_protocol:
Mechanic cell lysis was performed and chromatin was sheared by sonication to fragments of 200-500 bp. 1 % of the chromatin extract was treated with RNase A and proteinase K and crosslinks reversed. DNA was purified using the QIAquick PCR purification kit 250 (QIAGEN 28106).
For strand-specific library preparation the Accel-NGS 1S Plus Library Kit was used accordingly to the manufacturer’s instructions. 2 ng of DNA were used as starting material and libraries amplified in 14 cycles. For clean-up SPRIselect beads were used.

treatment_protocol: Galactose (2 %) was added to the cultures to induce DSBs via the pGAL-AsiSI system. Cells were crosslinked 0, 2, 4 and 6 h after DSB induction with 1 % formaldehyde for 16 min at room temperature and the reaction quenched with glycine (final concentration 400 mM). Cells were harvested (centrifugation at 3500x g at 4 °C), washed with cold PBS and flash frozen in liquid nitrogen.

value: pGAL-AsiSI arp8d swr1dhtz1d

value: galactose


title: Larvae, 6dpf, NFP-treated, 1

description: Library name: F01_22Jul4


title: Vehicle-treated HN5 cells_biol rep3

description: Library name: HN5.ctl.3

value: Vehicle


title: input, arp8swr1htz1, 4h

description: Library name: IN_arp8swr1htz1_4h_L2


title: Mouse Visium HD spatial gene expression (Tg88_148_A1)

description:
Library name: TgNotch3R169C_Brain_HD_1
Visium HD CytAssist, Space Ranger 3.1.1

value: transgenic, TgNotch3^R169C (line Tg88/148)


title: Vehicle-treated HN5 cells_biol rep2

description: Library name: HN5.ctl.2


title: Mouse Visium HD spatial gene expression (Tg129_213_D1)

description:
Library name: TgNotch3WT_Brain_HD_2
Visium HD CytAssist, Space Ranger 3.1.1

value: transgenic, TgNotch3^WT (line Tg129/213)


title: Glomeruli, 5dpf, DMSO-treated (Ctrl), 1

description: Library name: E03_22Jul4

value: Glomeruli

value: Isolated Glomeruli

value: DMSO


title: input, arp8swr1htz1, 2h

description: Library name: IN_arp8swr1htz1_2h_L2


title: Larvae, 6dpf, DMSO-treated (Ctrl), 2

description: Library name: E02_22Jul4


title: Vehicle-treated HN5 cells_biol rep1

description: Library name: HN5.ctl.1

value: Oral cavity (tongue)


title: Larvae, 6dpf, DMSO-treated (Ctrl), 1

description: Library name: E01_22Jul4


title: input, arp8swr1htz1, 0h

description: Library name: IN_arp8swr1htz1_0h_L2


title: Mouse Visium HD spatial gene expression (Ctrl_D1)

description:
Library name: TgNotch3WT_Brain_HD_1
Visium HD CytAssist, Space Ranger 3.1.1

value: transgenic, TgNotch3^WT


title: BMDM_RNY1-/- ProteinFx_4

city: Nashville

first: Cherie

middle: Evelyn

last: Saffold

state: Tennessee

address: 1211 21st Ave S

department: Pathology, Microbiology, and Immunology

institute: Vanderbilt University Medical Center

description:
Library name: 3Q3G6X_20
Processed data file contains both raw gene-level counts and CPM-normalized expression values.

data_processing:
Quality of the fastq files was assessed using FastQC v0.12.1. Reads were then quality filtered using fastp v0.24.0 with poly-X tail trimming, 3' quality-based  tail trimming, a minimum Phred quality score of 15, and a minimum length requirement of 50 bp. Quality-filtered reads were aligned to the reference genome using STAR aligner v2.7.11 with non-canonical splice junction removal and output of unmapped reads, followed by coordinate sorting using samtools v1.22.1. PCR and optical duplicates were removed using UMI-based deduplication with UMIcollapse v1.1.0. Alignment quality metrics, strand specificity, and read distribution across genomic features were assessed using RSeQC v5.0.4 and Qualimap v2.3, with results aggregated into a comprehensive quality control report using MultiQC v1.32. Gene-level expression quantification was performed using featureCounts (subread package v2.1.1) with strand-specific counting, multi-mapping read fractional assignment, exons and three prime UTR as the feature identifiers,  and grouped by gene_id. Final gene counts were annotated with gene biotype and  other metadata extracted from the reference GTF file.  Sample-sample correlations  for sample-sample heatmap and PCA were calculated on normalized counts  (TMM, trimmed mean of M-values) using Pearson correlation. Differential expression  was done with edgeR v4.0.16 using standard practice including filtering for  low-expressed genes with edgeR::filterByExprwith default values. Functional  enrichment, when available, is performed using gene set enrichment analysis with  gseapy v0.12 using the MSigDB Hallmark gene set.
Assembly: GRCm39 (Ensembl release 114)
Supplementary files format and content: Gene-level raw counts and CPM-normalized expression matrix for all samples

source_name: bone marrow

growth_protocol: All mice used for bone marrow derived macrophage (BMDM) culture were derived from female mice. Bone marrow cells were isolated from C57BL/6J murine femurs and tibias. Bone marrow cells were cultured for 5 days in 80% FBS-containing RPMI media + 20% L929 fibroblast conditioned media. On day 6, BMDMs were seeded in 96-well plates at 200,000 cells/well and switched to 100% FBS-containing RPMI media. BMDMs were used for experiments on day 7 of culture.

extract_protocol:
RNA was extracted by Plasmidsaurus according to their protocol used for their 3' end RNA sequencing sample preparation. Briefly, RNA was extracted using a bead-based extraction approach from cells preserved in Zymo DNA/RNA Shield.
Library preparation was performed by Plasmidsaurus according to their protocol for Illumina RNA sequencing. Briefly, polyadenylated mRNA transcripts were enriched and reverse transcribed into cDNA using oligo(dT) primers containing sample barcodes, unique molecular identifiers (UMIs), and Illumina Read 1 adapter sequences. Double-stranded cDNA was subsequently generated by second-strand synthesis. cDNA libraries were fragmented and tagged by tagmentation, which simultaneously incorporated Illumina Read 2 adapter sequences. Final sequencing libraries were amplified by PCR to incorporate unique dual indices (i5 and i7) and Illumina P5/P7 flow cell adapter sequences.

treatment_protocol: EV and Protein-enriched BALF fractions were concentrated to 160 uL and stored at 4°C for 5 days. Before treatment, BMDMs were washed 3x with PBS and resuspended in Opti-mem media without FBS. EV-enriched fractions from RNY1+/+ and RNY1-/- BALF were added to BMDM cultures at equal particle concentrations (3.5x10^7 particles/ul). Protein-enriched fractions from RNY1+/+ and RNY1-/- BALF were added to BMDM cultures at equal protein concentrations (0.75 ug/ul).

value: bone marrow

value: Bone marrow-derived macrophages

value: Wildtype BMDMs

value: Protein-enriched BALF from RNY1 -/- mouse with OVA-induced airway inflammation


title: input, swr1htz1, 6h

description: Library name: IN_swr1htz1_6h_L2

value: pGAL-AsiSI swr1dhtz1d


title: BMDM_RNY1-/- ProteinFx_3

description:
Library name: 3Q3G6X_19
Processed data file contains both raw gene-level counts and CPM-normalized expression values.


title: Larvae, 5dpf, NFP-treated, 3

description: Library name: D03_22Jul4

value: Larvae

value: Whole Larvae

value: NFP


title: Cetuximab-treated FaDu cells_biol rep3

description: Library name: FaDu.ctx.3

source_name: Hypopharynx

value: Hypopharynx

value: FaDu

value: Cetuximab


title: Cetuximab-treated FaDu cells_biol rep2

description: Library name: FaDu.ctx.2


title: input, swr1htz1, 4h

description: Library name: IN_swr1htz1_4h_L2

value: galactose


title: Glomeruli, 6dpf, NFP-treated, 3

description: Library name: D02_22Jul4

value: Glomeruli


title: BMDM_RNY1-/- ProteinFx_2

description:
Library name: 3Q3G6X_18
Processed data file contains both raw gene-level counts and CPM-normalized expression values.


title: Cetuximab-treated FaDu cells_biol rep1

description: Library name: FaDu.ctx.1


title: Glomeruli, 6dpf, DMSO-treated (Ctrl), 3

description: Library name: D01_22Jul4

value: Isolated Glomeruli


title: input, swr1htz1, 2h

description: Library name: IN_swr1htz1_2h_L2


title: BMDM_RNY1-/- ProteinFx_1

description:
Library name: 3Q3G6X_17
Processed data file contains both raw gene-level counts and CPM-normalized expression values.


title: Vehicle-treated FaDu cells_biol rep3

description: Library name: FaDu.ctl.3

value: Vehicle


title: input, swr1htz1, 0h

description: Library name: IN_swr1htz1_0h_L2


title: Larvae, 5dpf, DMSO-treated (Ctrl), 3

description: Library name: C03_22Jul4

value: DMSO


title: BMDM_RNY1+/+ ProteinFx_4

description:
Library name: 3Q3G6X_16
Processed data file contains both raw gene-level counts and CPM-normalized expression values.

value: Protein-enriched BALF from RNY1 +/+ mouse with OVA-induced airway inflammation


title: Vehicle-treated FaDu cells_biol rep2

description: Library name: FaDu.ctl.2


title: input, arp8, 6h

description: Library name: IN_arp8_6h_L2

value: pGAL-AsiSI arp8d


title: Glomeruli, 6dpf, DMSO-treated (Ctrl), 2

description: Library name: C02_22Jul4


title: BMDM_RNY1+/+ ProteinFx_3

description:
Library name: 3Q3G6X_15
Processed data file contains both raw gene-level counts and CPM-normalized expression values.

value: bone marrow

value: Bone marrow-derived macrophages


title: Vehicle-treated FaDu cells_biol rep1

description: Library name: FaDu.ctl.1


title: input, arp8, 4h

description: Library name: IN_arp8_4h_L2


title: Glomeruli, 6dpf, NFP-treated, 2

description: Library name: C01_22Jul4


title: BMDM_RNY1+/+ ProteinFx_2

description:
Library name: 3Q3G6X_14
Processed data file contains both raw gene-level counts and CPM-normalized expression values.

value: Wildtype BMDMs


title: Glomeruli, 6dpf, DMSO-treated (Ctrl), 1

description: Library name: B03_22Jul4


title: NF population from HT17B patient_biol rep3

description: Library name: NF_17B_3

source_name: Oral cavity (non-tumor)

growth_protocol: CAFs were cultured in DMEM-GlutaMAX supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin (Thermo Fisher Scientific) and 0.5 nM human recombinant basic fibroblast growth factor (b-FGF, Cat#78003, STEMCELL Technologies) and then maintained in exponential growth in 5% CO2/95% air in a humidified incubator at 37°C. NFs were grown in human fibroblast expansion basal medium (Cat#M106500, Gibco) supplemented with 2% low serum growth supplement (LSGS, Cat#S00310, Thermo Fisher Scientific) and 1% penicillin/streptomycin.

extract_protocol:
Total RNA was extracted from cancer cells using the Monarch Total RNA Miniprep Kit (New England Biolabs). Ribosomal RNA (rRNA) was depleted using the RiboCop rRNA Depletion Kit (Lexogen).
The rRNA-depleted RNA was then used for library preparation with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs), following the manufacturer's instructions. Briefly the key stages of this protocol are successively, the purification of PolyA containing mRNA molecules using poly-T oligo attached magnetic beads from 1μg total RNA (with the Magnetic mRNA Isolation Kit from NEB), a fragmentation using divalent cations under elevated temperature to obtain approximately 300bp pieces, double strand cDNA synthesis and finally Illumina adapters ligation and cDNA library amplification by PCR for sequencing. RNA sequencing was performed on an AVITI platform (Element Biosciences). The RNA-seq libraries were sequenced using 76 bp paired-end reads, and samples were multiplexed to achieve 20 million reads per sample.

treatment_protocol: No treatment

value: Oral cavity (non-tumor)

value: no treatment


title: input, arp8, 2h

description: Library name: IN_arp8_2h_L2


title: BMDM_RNY1+/+ ProteinFx_1

description:
Library name: 3Q3G6X_13
Processed data file contains both raw gene-level counts and CPM-normalized expression values.


title: NF population from HT17B patient_biol rep2

description: Library name: NF_17B_2


title: input, arp8, 0h

description: Library name: IN_arp8_0h_L2


title: Larvae, 5dpf, DMSO-treated (Ctrl), 2

description: Library name: B02_22Jul4

value: Larvae

value: Whole Larvae


title: NF population from HT17B patient_biol rep1

description: Library name: NF_17B_1


title: BMDM_RNY1-/- EVFx_4

description:
Library name: 3Q3G6X_12
Processed data file contains both raw gene-level counts and CPM-normalized expression values.

value: EV-enriched BALF from RNY1 -/- mouse with OVA-induced airway inflammation


title: Larvae, 5dpf, DMSO-treated (Ctrl), 1

description: Library name: B01_22Jul4


title: input, control, 6h

description: Library name: IN_control_6h_L2

value: pGAL-AsiSI


title: BMDM_RNY1-/- EVFx_3

description:
Library name: 3Q3G6X_11
Processed data file contains both raw gene-level counts and CPM-normalized expression values.


title: input, control, 4h

description: Library name: IN_control_4h_L2


title: NF population from HT17A patient_biol rep3

description: Library name: NF_17A_3


title: NF population from HT17A patient_biol rep2

description: Library name: NF_17A_2


title: Glomeruli, 6dpf, NFP-treated, 1

description: Library name: A03_22Jul4

value: NFP


title: BMDM_RNY1-/- EVFx_2

description:
Library name: 3Q3G6X_10
Processed data file contains both raw gene-level counts and CPM-normalized expression values.


title: Larvae, 5dpf, NFP-treated, 2

description: Library name: A02_22Jul4


title: input, control, 2h

description: Library name: IN_control_2h_L2

value: galactose


title: BMDM_RNY1-/- EVFx_1

description:
Library name: 3Q3G6X_9
Processed data file contains both raw gene-level counts and CPM-normalized expression values.


title: NF population from HT17A patient_biol rep1

description: Library name: NF_17A_1


title: input, control, 0h

description: Library name: IN_control_0h_L2


title: Larvae, 5dpf, NFP-treated, 1

description: Library name: A01_22Jul4


title: NF population from HT16 patient_biol rep3

description: Library name: NF_16_3


title: BMDM_RNY1+/+ EVFx_4

description:
Library name: 3Q3G6X_8
Processed data file contains both raw gene-level counts and CPM-normalized expression values.

value: EV-enriched BALF from RNY1 +/+ mouse with OVA-induced airway inflammation


title: ABR484 - ACP7 catalytically-dead OE - Rep5

city: Stanford

last: Richard

state: CA

address: 290 Jane Stanford Way

department: Genetics

institute: Stanford University

description:
Library name: D20
PE150 – 20M Paired Reads/6Gb

data_processing:
Raw FASTQ files were processed with TrimGalore, then aligned to the C. elegans reference genome with STAR using featureCounts
STAR output was processed with PyDESeq2, differential-expression analyses performed
Assembly: WBcel235
Supplementary files format and content: STAR featureCounts output matrix

source_name: Intestine ACP7-7A overexpression

extract_protocol:
RNA was isolated from ~1000 worms per replicate using QIAzol lysis reagent. After RNA isolation, Turbo DNase was used to remove DNA.  Finally, a second purification step was performed using Acid-Phenol:Chloroform.
Total RNA was provided to Novogene, who then prepared the libraries. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.


title: BMDM_RNY1+/+ EVFx_3

description:
Library name: 3Q3G6X_7
Processed data file contains both raw gene-level counts and CPM-normalized expression values.


title: PIP-seq of Mcm4-Flag, 30min released,treated_rep2

city: London

first: LMS

last: Bioinformatics Core

address: Hammersmith Hospital Campus, Du Cane Road

department: Bioinformatics Core

institute: MRC London Institute of Medical Sciences

data_processing:
Library strategy: ssDNA PIP-Seq
Primary analysis RTA 2.11.3 and Secondary analysis bcl2fastq2_2.20.0
Sequencing was conducted on an Illumina NextSeq 500 system with 75 base pair single-end reads
Sequencing reads were mapped to sacCer3 genome using Bowtie2 aligner (v. 2.3.5)
Bedgraph files were generated using bedtools v2.27.1
Bigwig files were generated from the bedgraphs using UCSC wigToBigWig tool
Genome_build: sacCer3
Supplementary_files_format_and_content: Strand seperated Bigwig files of coverage data

source_name: crosslinked culture

growth_protocol: Cells were grown in 150ml YPD from overnight cultures to OD600 0.6 at 220 rpm and 30°C followed by 2 hrs α-factor arrest at 25°C, 220 rpm and released in 150 ml warm media.

extract_protocol:
Lysates were prepared using standard procedures, sonicated chromatin clarified and Flag-tagged proteins immunprecipitated.
Immunoprecipitated DNA was treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation. After phosphorylation, the first adapter was ligated using T4 DNA Ligase and T4 Polynucleotidekinase. Gaps were filled with phi29 polymerase and samples subjected to 10mM potassium permanganate. DNA was eluted from resin, de-crosslinked and purified. After cleanup and piperidine cleavage, samples were purified again and second adapter were ligated and DNA purified. After 18 cycles of amplification, were cleaned up using AMPure XP beads (Beckman Coulter) according to the manufacturer’s instructions. Libraries were quantified using Qubit fluorometry (Thermo Fisher, dsDNA HS Assay) and qPCR (KAPA Illumina library quantification) before pooling and Illumina next generation sequencing.

treatment_protocol: media was supplemented with 100 nM rapamycin 0.5hrs before releasing and upon release. Treated samples were subjected to Potassium permanganate, untreated samples with buffer lacking permanganate

tag: strain

tag: antibody

value: anti-Flag F-1804, Sigma

tag: cell cycle status

value: 30min released

value: treated


title: NF population from HT16 patient_biol rep2

description: Library name: NF_16_2


title: BMDM_RNY1+/+ EVFx_2

description:
Library name: 3Q3G6X_6
Processed data file contains both raw gene-level counts and CPM-normalized expression values.


title: PIP-seq of Mcm4-Flag, 30min released,treated_rep1


title: NF population from HT16 patient_biol rep1

description: Library name: NF_16_1


title: ABR484 - ACP7 catalytically-dead OE - Rep3

description:
Library name: D18
PE150 – 20M Paired Reads/6Gb


title: PIP-seq of Mcm4-Flag, 30min released,untreated_rep2

value: untreated


title: BMDM_RNY1+/+ EVFx_1

description:
Library name: 3Q3G6X_5
Processed data file contains both raw gene-level counts and CPM-normalized expression values.


title: ABR484 - ACP7 catalytically-dead OE - Rep4

description:
Library name: D19
PE150 – 20M Paired Reads/6Gb


title: PIP-seq of Mcm4-Flag, 30min released,untreated_rep1


title: NF population from HT15B patient_biol rep3

description: Library name: NF_15B_3


title: BMDM_PBS_4

description:
Library name: 3Q3G6X_4
Processed data file contains both raw gene-level counts and CPM-normalized expression values.

value: PBS


title: NF population from HT15B patient_biol rep2

description: Library name: NF_15B_2


title: ABR484 - ACP7 catalytically-dead OE - Rep1

description:
Library name: D16
PE150 – 20M Paired Reads/6Gb


title: PIP-seq of Mcm4-Flag, 22.5min released,treated_rep2

value: 22.5min released


title: BMDM_PBS_3

description:
Library name: 3Q3G6X_3
Processed data file contains both raw gene-level counts and CPM-normalized expression values.

value: bone marrow

value: Bone marrow-derived macrophages


title: NF population from HT15B patient_biol rep1

description: Library name: NF_15B_1

value: Oral cavity (non-tumor)


title: D283_Day4_control_rep1

city: Memphis

first: Paul

middle: A.

last: Northcott

address: 262 Danny Thomas Place

department: Developmental Neurobiology

institute: St. Jude Children's Research Hospital

data_processing:
Sequence reads were trimmed for adaptor sequence/low-quality sequence, cleaned reads were mapped to hg38 (Gencode GRCh38.p12) using STAR
Assembly: hg38
Supplementary files format and content: Tab-separated files

extract_protocol:
For RNA-seq in Group 4 MB PDX, freshly isolated DMB006 cells from mouse brains were infected in triplicate with non-targeting control or KDM2B gRNA viruses at high MOI to ensure more than 90% of cells were infected. Cells were cultured for 7 days and then collected for RNA-seq. Infection efficiency was determined by flow cytometry analysis of GFP percentage on day 4. Editing efficiency was evaluated by targeted deep sequencing. For RNA-seq in D283, cells were infected in triplicate with non-targeting control or KDM2B gRNA viruses at high MOI to ensure more than 80% of cells were infected. Cells were cultured for 7 days. An aliquot of cells was collected on day 4 or day 7 for RNA-seq. Infection efficiency was determined by flow cytometry analysis of GFP percentage on day 4. Editing efficiency was evaluated by targeted deep sequencing.
RNA extraction was performed using RNeasy Plus kit (Qiagen) according to manufacturer’s instructions. RNA libraries for RNA-seq were prepared following manufacturer's protocols

value: medulloblastoma cell line


title: ABR275 - ACP7 OE - Rep5

description:
Library name: B10
PE150 – 20M Paired Reads/6Gb

source_name: Intestine ACP7 overexpression


title: 3a_3b_mut_rep2_T5 biol rep 2

city: New York

first: Samuel

last: Sternberg

address: 701 W. 168th Street, HHSC 726

institute: Columbia University

description: Library name: ST190_10_D

data_processing:
Raw reads were processed with Cutadapt (v5.0) to filter out adapter sequences, trim low-quality bases, and remove reads shorter than 15 bp. Cutadapt was run using a tiling approach across the 3′ adapter as previously described, with parameters -e 0.2, -O 10, -q 30, and -m 45 (error rate, minimum overlap, quality cutoff, and minimum length, respectively). Filtered reads were aligned with bwa-mem2 (v2.2.1) under default parameters to a composite reference comprising the E. coli MG1655 genome (NCBI: NC_000913.3), the relevant plasmid sequence, and either the λ-vir (NCBI: NC_001416.1) or T5 (NCBI: NC_005859.1) phage genome. Alignments were sorted and indexed using SAMtools (v1.17). Strand-specific coverage tracks were generated with bamCoverage (v3.5.6), normalized to sequencing depth based on the total number of reads passing trimming and length filtering. Reads that failed to align with bwa-mem2 were extracted using SAMtools fasta, and the fraction of unmapped reads was calculated using SAMtools flagstat. Genome build: E. coli MG1655 (NC_000913.3); λ-vir (NC_001416.1); T5 (NC_005859.1).
Assembly: ASM584v2
Supplementary files format and content: .fasta — Unmapped reads extracted from BAM alignments using SAMtools fasta. Each file contains all reads from a given sample that failed to align to the composite reference, used as input for downstream MEME motif enrichment analysis and motif counts per sample.

molecule: other

organism: Escherichia coli

extract_protocol:
cDIP elution was performed by removing the supernatant and resuspending beads with 80 µL of IP wash buffer containing 5 µg RNase A (Thermo Fisher Scientific). Inputs were resuspended with 70 µL of the same wash buffer. Both inputs and immunoprecipitation samples were incubated at 37 °C for 30 min. SDS was then added to a final concentration of 1%, along with 25 µg of proteinase K (Thermo Fisher Scientific). Supernatants containing eluted DNA were transferred to new tubes and DNA was isolated with the Monarch Spin PCR and DNA Cleanup kit (NEB), following the Oligonucleotide Cleanup protocol.
Eluted DNA samples were heated at 95 °C for 2 min and immediately placed on ice to denature DNA. Adapter ligation and conversion of ssDNA to dsDNA were performed using the xGen ssDNA & Low-Input DNA Library Prep Kit (IDT). Indexing PCR was performed using NEBNext Ultra II Q5 Master Mix (NEB). Libraries were sequenced on the Element AVITI system in single- or paired-end mode with 150 cycles per end.


title: Y666F_rep2 biol rep 2

description: Library name: ST190_18_D


title: Y666F_rep1 biol rep 1

description: Library name: ST190_17_D


title: PIP-seq of Mcm4-Flag, 22.5min released,treated_rep1


title: NF population from HT10 patient_biol rep3

description: Library name: NF_10_3

value: no treatment


title: ABR275 - ACP7 OE - Rep4

description:
Library name: B9
PE150 – 20M Paired Reads/6Gb


title: ABR484 - ACP7 catalytically-dead OE - Rep2

description:
Library name: D17
PE150 – 20M Paired Reads/6Gb


title: BMDM_PBS_2

description:
Library name: 3Q3G6X_2
Processed data file contains both raw gene-level counts and CPM-normalized expression values.

value: Wildtype BMDMs


title: TH205

city: Edmonton

first: Jessica

last: Chang

address: 250 Heritage Medical Research Centre

department: Medicine

institute: University of Alberta

hyb_protocol: (Affymetrix, Santa Clara, CA).  Amplified RNA (10ug) was hybridized for 16 hr at 45C on  GeneChip® PrimeView™ Human Gene Expression Array . GeneChips were washed and stained in the Affymetrix Fluidics Station 450.

scan_protocol: GeneChips were scanned using Gene Array Scanner 3000 7G (Affymetrix) and processed with GeneChip® Command Console® Software (AGCC)(Affymetrix).

data_processing: Microarray data files were preprocessed using robust multi-chip averaging (RMA) implemented in Bioconductor.

label: biotin

source_name: NoABMRM

label_protocol: Total RNA (0.5µg) was labeled using GeneChip 3` IVT Express Labeling kit. Quality of labeled aRNA was assessed on an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA) before hybridization to GeneChip® PrimeView™ Human Gene Expression Array

extract_protocol: Tissue was homogenization in 0.5ml of Trizol reagent (Invitrogen, Carlsbad, CA) and total RNA was extracted and purified using the Rneasy Micro Kit (Quiagen, Ontario, Canada) (average yield 8µg/core, (RNA integrity number >7).

treatment_protocol: All human heart biopsies taken for clinical indication during the period specified above were included. Tissue was immediately placed in RNA later.

value: heart biopsy


title: PIP-seq of Mcm4-Flag, 22.5min released,untreated_rep2


title: BMDM_PBS_1

description:
Library name: 3Q3G6X_1
Processed data file contains both raw gene-level counts and CPM-normalized expression values.


title: NF population from HT10 patient_biol rep2

description: Library name: NF_10_2


title: ABR275 - ACP7 OE - Rep3

description:
Library name: B8
PE150 – 20M Paired Reads/6Gb


title: PIP-seq of Mcm4-Flag, 22.5min released,untreated_rep1


title: ABR275 - ACP7 OE - Rep2

description:
Library name: B7
PE150 – 20M Paired Reads/6Gb


title: NF population from HT10 patient_biol rep1

description: Library name: NF_10_1


title: AVF-Dilution-0428, late

city: Miami

first: Anthony

last: Griswold

state: FL

address: 1501 NW 10th Ave, BRB 318

institute: University of Miami

description:
Library name: AVF-Dilution-0428
10x Genomics

data_processing:
For single cell RNAseq, FASTQ files were generated with Cell Ranger's mkfastq pipeline (version 6.0.2).
The Cell Ranger's count pipeline (version 6.0.2) was used to generate raw gene-barcode matrices from alignment to the 10X Genomics pre-built Cell Ranger mouse reference package (version mm10-2020-A), from GRCm38 Ensembl build 98/GENCODE M23 gene annotations.
Bulk RNAseq was processed through standard pipeline of alignment to the GRCm38 genome with the STAR aligner.
Bulk RNA gene counts were created using the GeneCounts features within STAR against the ENSEMBL M23 gene build
Assembly: GRCm38
Supplementary files format and content: barcodes.tsv.gz is a gzipped text file listing individual cell barcodes included in the sample analysis
Supplementary files format and content: features.tsv.gz is a gzipped tab-delimited file of the gene features included in the gene quantification for each sample
Supplementary files format and content: matrix.mtx.gz is a gzipped tab-delimited file of the counts per feature per cell barcode for each sample
Supplementary files format and content: Final_counts-AVF_timepoints.csv is a comma seperated text file of raw counts per gene

source_name: AVF

extract_protocol:
Contralateral veins and AVFs were harvested at different timepoints. For bulk RNA-seq, tissues were ground to a fine powder in a Spex/Mill 6770 cryogenic grinder, followed by total RNA extraction using Trizol and treatment with DNase I. For single cell RNA-seq, samples were enzymatically digested for 90 minutes at 37°C with shaking using a combination of collagenase type II, soybean trypsin inhibitor, elastase, Dispase, DNAse I, collagenase type XI, hyaluronidase type I, and HEPES in HBSS with Ca2+ and Mg2+. After 90 minutes, an equal volume of Accumax was added to the dissociation mix and incubated for an additional 5 minutes. Single cells were filtered through a 40 μM strainer, washed with HBSS twice, and incubated with red blood cell lysis buffer for 5-10 min. Cells were washed once again with HBSS and resuspended in 0.1% BSA in PBS for counting and RNA sequencing.
Poly(A)-selected bulk mRNA libraries were prepared by Azenta using an ultra-low input RNA sequencing protocol. Libraries were sequenced on the Illumina HiSeq X using a paired-end 150-bp configuration and an output of ~350 million reads per lane. Single cell samples were run using the Chromium Single Cell 3′ Library & Gel Bead Kit v3 (10X Genomics) with a target capture of 10,000 cells. Each sample was processed on an independent Chromium Single Cell A Chip (10X Genomics) and subsequently run on a thermocycler (Eppendorf). Sequencing libraries were evaluated for quality on the Agilent Tape Station (Agilent Technologies, Palo Alto, CA) and quantified using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and qPCR before sequencing on the Illumina NovaSeq 6000.

value: AVF

tag: Sex

value: pooled male and female


title: PIP-seq of Mcm4-Flag, 15min released,treated_rep2

value: 15min released


title: NF population from HT3 patient_biol rep3

description: Library name: NF_3_3


title: ABR275 - ACP7 OE - Rep1

description:
Library name: B6
PE150 – 20M Paired Reads/6Gb


title: H3K9me3 Daxx wt/wt CNT, replicate 2

city: Bonn

first: Giuliano

last: Crispatzu

address: Venusberg-Campus 1, Gebäude 99, Venusberg Campus 1/Gebäude 99

department: Nuclear Function in CNS pathophysiology

institute: DZNE

description: Library name: H3K9me3_16496_WT_S12

data_processing:
CUT&Tag reads were aligned using bowtie2 (v2.2.1; PMID: 22388286) to the GRCm39 / mm39 (or mm10 for Fig5 cluster analysis) reference genome with settings: "--local --very-sensitive-local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700". BAM files are then created and sorted using samtools (v1.11; PMID: 19505943), before removing PCR duplicates using picard (v2.20.4; "Picard Toolkit." 2018. Broad Institute, GitHub Repository. http://broadinstitute.github.io/picard/; Broad Institute) subroutine MarkDuplicates.
Bigwig files were created using deeptools (v3.5.3) with 1x-normalization (RPGC).
Assembly: GRCm39 / mm39
Supplementary files format and content: bigwig
Library strategy: CUT&Tag

source_name: brain

growth_protocol: Mice were maintained on a 12-hour light/dark cycle with ad libitum access to regular chow food (ssniff Spezialdiäten GmbH). All experimental procedures were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen (LANUV).

extract_protocol:
Microglia from mice brains were isolated using the Adult Brain Dissociation Kit from Miltenyi Biotech (Bergisch Gladbach, Germany) according to the manufacturer’s guidelines. In brief, after euthanasia, mice brains were mechanically and enzymatically digested at 37°C. Fully digested tissue suspensions were filtered with a 70 µm cell strainer followed by subsequent removal of myelin, cell debris and erythrocytes. The cell suspension was then incubated with CD11b MicroBeads (Miltenyi Biotech) for 15 minutes at 4°C followed by a separation in MS columns (Miltenyi Biotech) using a magnetic field on a MACS MultiStand and QuadroMACS (Miltenyi Biotech).
H3.3 CUT&Tag libraries were prepared using Active Motif CUT&Tag-IT Assay Kit. Briefly, around 100,000-200,000 microglia per reaction were bound to activated Concanavalin A beads. After successive incubations with primary (overnight at 4°C) and secondary (60 min) antibodies in Dig-wash buffer, the beads were washed and resuspended in pA-Tn5 transposomes (1:100 dilution) in Dig-300 buffer for 60 min at room temperature. Then, samples were incubated in Tagmentation buffer for 60 min at 37°C. After tagmentation, DNA fragments were further extracted and purified using a column. Subsequently, PCR amplification was performed using i7 and i5 primers for 14 cycles, followed by clean-up with 65 µl of SPRIselect beads (Beckman Coulter). Finally, CUT&Tag-seq libraries were  quantified using D1000 assay kit on a Tapestation 4200 (Agilent).                                           For H3K9me3 CUT&Tag experiment, we used the protocol described by Henikoff et al (DOI: dx.doi.org/10.17504/protocols.io.bqwvmxe6). Briefly, around 50,000 nuclei per reaction were bound to activated Concanavalin A beads. After incubation with primary H3K9me3 (Active Motif, 1:20) overnight at 4°C, followed by the incubation with secondary at room temperature for 60 min. Then, beads were washed and resuspended in pAG-Tn5 in 300-wash buffer (1:20 dilution) for 60 min at room temperature. After tagmentation, beads were washed 3x in 50 µl TAPS buffer and then resuspended in 5 µl SDS release buffer for 60 min at 58°C. The SDS was neutralized with 15 µl of 0.67% Triton X-100, and 4 µl of dual-indexed primers from the ‘IDT for Illumina Nextera DNA UD Indexes Set A’ (Illumina) as well as 25 µl of NEBNext high-fidelity 2×PCR master mix (NEB) were added. Gap filling and 14 cycles of PCR were performed, followed by clean-up with 65 µl of SPRIselect beads (Beckman Coulter).

treatment_protocol: To induce Cre activation, Cx3cr1-CreERT2 positive mice at 12-week-old age were administrated with 80 mg/kg/day tamoxifen (T5648, Sigma-Aldrich) via oral gavage for five consecutive days.

value: brain

value: microglia

value: Cx3cr1CreERT,Daxx wt/wt

value: Tamoxifen


title: H3K9me3 Daxx wt/wt CNT, replicate 1

description: Library name: H3K9me3_16494_WT_S11


title: PIP-seq of Mcm4-Flag, 15min released,treated_rep1

value: anti-Flag F-1804, Sigma

value: treated


title: Control N2 strain - rep 5

description:
Library name: A5
PE150 – 20M Paired Reads/6Gb

source_name: Wild Type

value: Wild Type


title: NF population from HT3 patient_biol rep2

description: Library name: NF_3_2


title: PIP-seq of Mcm4-Flag, 15min released,untreated_rep2

value: untreated


title: H3K9me3 Daxx ff/ff CNT, replicate 3

description: Library name: H3K9me3_16456_FF_S21

value: Cx3cr1CreERT,Daxx flox/flox


title: NF population from HT3 patient_biol rep1

description: Library name: NF_3_1

value: Oral cavity (non-tumor)


title: PIP-seq of Mcm4-Flag, 15min released,untreated_rep1


title: Control N2 strain - rep 4

description:
Library name: A4
PE150 – 20M Paired Reads/6Gb


title: H3K9me3 Daxx ff/ff CNT, replicate 2

description: Library name: H3K9me3_16453_FF_S20


title: CAF population from HT17B patient_biol rep3

description: Library name: CAF_17B_3

source_name: Larynx (tumor)

value: Larynx (tumor)


title: Control N2 strain - rep 3

description:
Library name: A3
PE150 – 20M Paired Reads/6Gb


title: H3K9me3 Daxx ff/ff CNT, replicate 1

description: Library name: H3K9me3_16451_FF_S19


title: CAF population from HT17B patient_biol rep2

description: Library name: CAF_17B_2

value: no treatment


title: PIP-seq of Mcm4-Flag, G1 arrested,treated_rep1

value: G1 arrested


title: PIP-seq of Mcm4-Flag, G1 arrested,treated_rep2


title: H3.3 Daxx wt/wt CNT, replicate 3

description: Library name: H3-3_16496_WT_S8


title: Control N2 strain - rep 1

description:
Library name: A1
PE150 – 20M Paired Reads/6Gb


title: PIP-seq of Mcm4-Flag, G1 arrested,untreated_rep2


title: CAF population from HT17B patient_biol rep1

description: Library name: CAF_17B_1


title: Control N2 strain - rep 2

description:
Library name: A2
PE150 – 20M Paired Reads/6Gb


title: CAF population from HT17A patient_biol rep3

description: Library name: CAF_17A_3

source_name: Oral cavity (tumor)

value: Oral cavity (tumor)


title: Rep #3 eor-1(cs28); tra-4(bc250)

city: Cambridge

first: Dongyeop

last: Lee

state: MA

address: 31 Ames St

institute: HHMI/MIT

description: Library name: 3_eor-1; tra-4

data_processing:
The raw files were aligned to C. elegans genome/WBcel235 using STAR-2.5.3.
Sequences located at transcripts were quantified by using RSEM-1.3.1.
Differentially expressed genes (DEG) analysis was performed using DESeq2-1.24.0.
Assembly: C. elegans genome/WBcel235
Supplementary files format and content: Tab-delimited text files containing read counts estimated by using RSEM

source_name: Embryo

extract_protocol:
Total RNA extraction using the Qiagen RNeasy mini RNA extraction kit
NEB Ultra II RNAseq

value: Embryo


title: H3.3 Daxx ff/ff CNT, replicate 2

description: Library name: H3-3_16451_FF_S9


title: H3.3 Daxx ff/ff CNT, replicate 1

description: Library name: H3-3_16289_FF_S7


title: PIP-seq of Mcm4-Flag, G1 arrested,untreated_rep1


title: Rep #3 eor-1(cs28)

description: Library name: 3_eor-1


title: CAF population from HT17A patient_biol rep2

description: Library name: CAF_17A_2


title: Male_midgut_control_3

data_processing:
After demultiplexing, raw RNAseq reads were aligned against the Ensembl Drosophila genome reference sequence assembly (dm6) and transcript annotations obtained from Illumina iGenomes using TopHat2 (v2.0.11; Kim et al., 2013).
Gene-based read counts were then obtained using the featureCounts function from the Rsubread Bioconductor package (v.1.24.2; Liao et al., 2013, Liao et al., 2014)
Assembly: dm6
Supplementary files format and content: tab-delimited csv file includes raw counts for each sample

source_name: Midgut

growth_protocol: Virgin males and females for each genotype were collected and aged for 6 days at 18C. Subsequently, the animals were switched to 29C for 4 days and dissected for RNAseq.

extract_protocol:
Total RNA was extracted using the Rneasy Mini kit (Qiagen). 200ng were used for the construction of the sequencing libraries.
PolyA enrichment was performed using NEBNext® Poly(A) mRNA Magnetic Isolation module, and NGS RNASeq libraries made using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® according to manufacturer’s instructions
For each sample, a minimum of 40 million Paired End 100bp reads were generated on an Illumina HiSeq2500 with single 6bp indexing.

treatment_protocol: Adults were switch from 18C to 29C to induce the expression of the dsxRNAi.

value: Midgut

value: Male

tag: developmental stage

value: Adult 10 days after hatching

value: y[1] v[1] / w[*] ; mex1-Gal4 / + ; UAS-GFP / tub-Gal80ts

value: Temperature switch 18C (6 days) to 29C (4 days)


title: H3.3 Daxx wt/wt CNT, replicate 2

description: Library name: H3-3_16239_WT_S6

value: brain

value: microglia

value: Cx3cr1CreERT,Daxx wt/wt

value: Tamoxifen


title: Rep #3 tra-4(bc250) coh-1(n6618)

description: Library name: 3_tra-4 coh-1


title: Male_midgut_control_2


title: CAF population from HT17A patient_biol rep1

description: Library name: CAF_17A_1


title: H3.3 Daxx wt/wt CNT, replicate 1

description: Library name: H3-3_16237_WT_S5


title: CAF population from HT16 patient_biol rep3

description: Library name: CAF_16_3

source_name: Oropharynx (tumor)

value: Oropharynx (tumor)


title: Rep #3 coh-1(n6618)

description: Library name: 3_coh-1


title: RNA-seq KO Flu 3

city: Seoul

first: Jun Young

last: Hong

address: 50, Yonsei-ro, Seodaemun-gu

department: Systems Biology

country: South Korea

institute: Yonsei University

description:
Library name: Sample 12
Column name in processed_counts.csv: KO_Flu_3

data_processing:
Paired-end sequencing was performed with NovaSeqX with 101 bp reads from each end. Reads were aligned to the mouse genome mm39 by STAR v2.7.10a and quantified using featureCounts v2.0.3. Counts per million (CPM) values were calculated using edgeR v4.0.16.
Assembly: mm39
Supplementary files format and content: a tab-delimited csv file including Ensembl ID and CPM (counts per million)

source_name: lung

extract_protocol:
RNA was purified from mouse lung tissues using QIAGEN RNeasy columns (74106) with on-column DNase digestion according to the manufacturer’s instructions.
RNA-seq libraries were constructed following the Illumina TruSeq stranded mRNA Reference Guide #1000000040498 v00.

treatment_protocol: Female Balb/cJ mice or Vanin-deficient mice were anesthetized with a ketamine/xylazine mixture, and the specified number of plaque-forming units (PFU) of the influenza virus (strain A/WSN/33) in 30 μl PBS was administered intranasally dropwise.

value: lung

value: Vanin-/-

value: Flu


title: Male_midgut_control_1


title: Rep #3 Wild type

description: Library name: 3_wt


title: RNA-seq KO Flu 2

description:
Library name: Sample 11
Column name in processed_counts.csv: KO_Flu_2


title: CAF population from HT16 patient_biol rep2

description: Library name: CAF_16_2

value: no treatment


title: CAF population from HT16 patient_biol rep1

description: Library name: CAF_16_1


title: Male_midgut_dsxRNAi_3

value: y[1] v[1] / w[*] ; dsxRNAi / mex1-Gal4 ; tub-Gal80ts / +


title: Male_midgut_dsxRNAi_2


title: CAF population from HT15B patient_biol rep3

description: Library name: CAF_15B_3

value: Larynx (tumor)


title: Rep #2 eor-1(cs28); tra-4(bc250)

description: Library name: 2_eor-1; tra-4

value: Embryo


title: RNA-seq KO Flu 1

description:
Library name: Sample 10
Column name in processed_counts.csv: KO_Flu_1


title: Rep #2 eor-1(cs28)

description: Library name: 2_eor-1


title: Male_midgut_dsxRNAi_1

value: Midgut


title: CAF population from HT15B patient_biol rep2

description: Library name: CAF_15B_2


title: Rep #2 tra-4(bc250) coh-1(n6618)

description: Library name: 2_tra-4 coh-1


title: RNA-seq KO baseline 2

description:
Library name: Sample 8
Column name in processed_counts.csv: KO_baseline_2

value: Control


title: RNA-seq KO baseline 1

description:
Library name: Sample 7
Column name in processed_counts.csv: KO_baseline_1


title: Rep #2 coh-1(n6618)

description: Library name: 2_coh-1


title: Rep #2 Wild type

description: Library name: 2_wt


title: RNA-seq WT Flu 3

description:
Library name: Sample 6
Column name in processed_counts.csv: WT_Flu_3

value: WT


title: Female_midgut_control_1

value: Female

value: Adult 10 days after hatching

value: y[1] v[1] / w[*] ; mex1-Gal4 / + ; UAS-GFP / tub-Gal80ts


title: Rep #1 eor-1(cs28); tra-4(bc250)

description: Library name: 1_eor-1; tra-4


title: CAF population from HT10 patient_biol rep3

description: Library name: CAF_10_3


title: Female_midgut_dsxRNAi_3

value: Temperature switch 18C (6 days) to 29C (4 days)


title: RNA-seq WT Flu 2

description:
Library name: Sample 5
Column name in processed_counts.csv: WT_Flu_2


title: CAF population from HT10 patient_biol rep1

description: Library name: CAF_10_1


title: CAF population from HT10 patient_biol rep2

description: Library name: CAF_10_2


title: Rep #1 eor-1(cs28)

description: Library name: 1_eor-1


title: Female_midgut_dsxRNAi_2


title: Female_midgut_dsxRNAi_1


title: CAF population from HT3 patient_biol rep2

description: Library name: CAF_3_2

value: Oral cavity (tumor)


title: Rep #1 tra-4(bc250) coh-1(n6618)

description: Library name: 1_tra-4 coh-1


title: RNA-seq WT baseline 2

description:
Library name: Sample 2
Column name in processed_counts.csv: WT_baseline_2


title: Rep #1 coh-1(n6618)

description: Library name: 1_coh-1


title: CAF population from HT3 patient_biol rep1

description: Library name: CAF_3_1


title: control_15h_2

city: Boston

first: Carol

middle: A

last: Kumamoto

address: 150 Harrison Ave.

department: Department of Molecular Biology and Microbiology

institute: Tufts University

description:
Library name: Sample 2
10x Genomics

data_processing:
Initial data analysis including demultiplexing, barcode processing and aligning reads to the human genome was performed using Cell Ranger.
Assembly: human GRCh38
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Colonoid line CJ50

growth_protocol: Preparation of colonoid monolayers on transwells. Colonoid line CJ50 was derived from colonic biopsies obtained from a 67 year old Chinese female undergoing routine outpatient colonoscopy for colorectal cancer screening at Tufts Medical Center (IRB #11652, principal investigator: Jatin Roper). The cells were propagated in Matrigel (Corning 356231) as described previously [29]. Medium compo-sition was as described [22] except that L-WRN conditioned medium (L-WRN cells ATCC CRL- 3276 [30]) was used at 65% instead of L-WNT3A, R-Spondin and Noggin conditioned media, Primocin (InvivoGen ant-pm-1) was used instead of pen-strep and 5 uM Y27632 (Rho kinase inhibitor, Sigma Y0503) was added for the first 48 hrs of culturing. For preparation of monolayers on transwells, colonoid spheres were recovered from Matrigel, washed with PBS and digested with TrypLE Express (Gibco 12605-010) for 2.5 min at 37oC. After digestion, cells were diluted with 1 volume advanced DMEM (Invi-trogen 12634-028), 1% Glutamax (Gibco 35050-061), 20% FBS (Fisher 26140079) and filtered through a 40 um cell strainer. Cells were diluted with 0.4% Trypan blue, counted with a Corning CytoSmart counter and diluted to 2.5 x 106 cells/ml with warmed 65% LWRN medium containing Y27632. Matrigel was diluted with CMGF- to 33 or 66 ug protein/mL, 125 ul was applied to each 6.5mm diameter, 0.4 um pore, polycarbonate filter transwell (Costar 3413) and incubated at 37oC for 2 hours. Liquid was removed from the transwell, the basolateral compartment was filled with 0.5 ml 65% medium and 50 ul of medium was added to the apical compartment. Cells (2.5 x 105 cells per 6.5 mm diameter transwell) were added and the seeded transwells were incubated in a tissue culture incubator with 5% CO2 at 37oC. The next day, transwells were given fresh 65% L-WRN medium containing 5 uM Y27632 (Sigma Y0503), replacing the basolateral medium and adding 0.2 ml to the apical compartment. The next day, the medium was changed to 65% without Y27632 (0.4 ml apically and 0.5 ml basolaterally) and was changed again after 2 days when the mono-layer was typically confluent. Confluent monolayers were differentiated by washing with CMGF-, followed by feeding with Differentiation Medium (DM) [22, 31] containing Primocin. Medium was changed the next day with DM containing Primocin. Monolayers were fed 2 days later (day 3) (DM without Primocin) and in the late afternoon of day 4 or in the morning of day 5 (DM without Primocin). Monolayers were inoculated with pre-germinated spores on day 5. For measurement of trans-epithelial electrical resistance (TEER), a Millicell ERS-2 volt/ohm meter was used following manufacturer’s protocol.

extract_protocol:
Monolayers were cultured on 12 mm diameter 0.4 um polycarbonate filters (Millicell PIHP01250) and differentiated as described above. Spores were treated with glycine and sodium cholate, as above, collected by centrifugation (Eppendorf 5415, 13200 rpm, 5 min) resuspended in PBS and 1 x 105 spores in 10 ul or PBS was added to the apical medium. Transwells were incubated as described above. After 4, 15, 20 or 25 hours of incubation, monolayers were washed with 100 ul PBS, EDTA 0.5 mM, pH 8 and incubated with TrypLE Express, 600 ul in the basolateral and 400 ul in the apical compartments at 37oC for 12 min. One hundred ul 50% FBS was added to the apical compartment and the cells were suspended by pipeting up and down several times. Cells were passed through a 40 um cell strainer and recovered in tubes precoated with 0.1% BSA. The transwells were then washed with 400 ul CMGF-, which was passed through the strainer and combined with the previous filtrate. Cells were collected by centrifugation at 300g x 5 min. Most of the supernatant was gently removed. Cells were resuspended in 1 ml of 0.04% BSA, 5 uM Y27632 and collected as above. Using a wide bore tip, the pellet was resuspended in the remaining supernatant by pipetting up and down. Cells were counted with a Corning Cytosmart counter and adjusted to 1 x 106 cells/ml.
The remaining steps were performed following manufacturer’s protocol. Briefly, the cell suspension was loaded along with reagents, barcoded Single Cell 3’ v3.1 Gel Beads and Partitioning Oil onto the Chromium Next GEM Chip G. Cells were partitioned into GEMS using the Chromium Controller. Gel beads were then dissolved, releasing primers, and the cells were lysed. Reverse transcription was performed to generate cDNA tagged with cell specific barcodes and Illumina TruSeq Read 1 sequencing primer. GEMS were broken and first strand cDNA was purified and PCR amplified. After enzymatic fragmentation followed by size selection, the cDNA was PCR amplified, incorporating a sample index and primers P5, P7, and TruSeq Read 2 for Illumina sequencing. Library quality was assessed using an Agilent Bioanalyzer. Paired end sequencing was performed by the Tufts University Genomics Core Facility.

treatment_protocol: Co-culturing C. difficile with colonoid monolayers. Pre-germinated spores (approximately 5 x 107 per ml) were prepared as above and 10ul of the preparation was added gently to the apical medium of the transwell. Control samples were inoculated with 10ul of buffer. The plates were wrapped with Parafilm and incubated in a standard tissue culture incubator (37oC, 5% CO2, room air). At various times, transwells were taken into the anaerobic chamber (2 transwells at a time) to estimate the number of bacteria per transwell. The first 170 ul of apical medium was carefully removed. This medium was not turbid and was discarded. The second approximately 170 ul of medium was collected into the pipet tip and used to wash the monolayer twice, then collected into a pre-weighed tube. For inoculated transwells, this sample was typically turbid. 10 ul of this sample was immediately diluted with 1 ml of pre-reduced PBS to measure CFU. The remainder of the sample was removed from the chamber and weighed to determine the volume recovered from each transwell after correction for the 10 ul removed for CFU. Transwell monolayers were removed from the chamber and fixed with 4% para-formaldehyde for 15 min at room temp, followed by washing 3 times with PBS at room temperature. Transwells were stored at 4oC in PBS. For some experiments, transwells were inoculated with vegetative C. difficile cells. Strains were grown in pre-reduced TY broth and harvested during log phase. A sample of each culture was diluted 1/50 into pre-reduced PBS. DM medium was pre-reduced and 0.4 ml was transferred to a gasketed screw cap tube for each inoculation. 10 ul of bacteria diluted in PBS was added to each tube (or PBS only for mock inoculation). The caps were screwed on tightly and tubes removed from the chamber. The transwells were inoculated by removing their apical medium and replacing it with the bacteria-containing medium. Transwells were inoculated with approximately 5 x 104 CFU.

value: Colonoid line CJ50

tag: infection

value: non-infected


title: control_15h_1

description:
Library name: Sample 1
10x Genomics


title: Rep #1 Wild type

description: Library name: 1_wt


title: RNA-seq WT baseline 1

description:
Library name: Sample 1
Column name in processed_counts.csv: WT_baseline_1

value: lung


title: W10, Rep3, bACE

status: Public on Jun 21 2026

city: Philadelphia

first: Rahul

middle: M.

last: Kohli

address: 3610 Hamilton Walk, 502B Johnson Pavilion

department: Department of Medicine

institute: University of Pennsylvania

data_processing:
Raw reads were trimmed for base quality and adaptor sequences using Trim Galore! (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and reads below a length of 20 bases were discarded.
The trimmed reads were aligned to the reference genomes using Bismark (v0.22.3) with the following parameters -D 15 -R 2 -L 20 -N 0 –score_min L,0,-0.2.
To account for A3A failure due to double-stranded DNA, reads with three or more consecutive non-converted cytosines in the CpH context were removed using the filter_non_conversion script included in the Bismark package.
Assembly: mm10
Supplementary files format and content: Tab-delimited file containing the chromosome, start position, end position, methylation percentage, count methylated, count unmethylated

source_name: Cortex

extract_protocol:
To isolate gDNA, brain was thawed and digested in lysis buffer (50mM Tris, pH8.0, 100mM EDTA, 0.5% SDS) with proteinase K (Sigma-Aldrich) overnight at 55°C. Genomic DNA was isolated using Phenol:Chloroform:Isoamyl Alcohol (Fisher BP17501-400) and ethanol precipitation. DNA samples resuspended in TE were stored at 4°C.
Purified mouse gDNA (200 ng) with 1 ng each of phiX, mCpG pUC19 (Zymo), and 5hmC T4 spike-in controls were sheared to approximately 350 bp using a Covaris instrument. Sheared gDNA was taken through end prep and adaptor ligation using the NEBNext Ultra II DNA Library Prep Kit (NEB). Custom-made pyrrolo-cytosine adaptors (IDT) were substituted for kit-supplied adaptors. Adapted samples were bisulfite treated using the Premium Bisulfite Kit (Diagenode) and eluted in nuclease-free water. Post bisulfite clean-up, each sample was split into two 10 µL aliquots. One aliquot was further purified with a 1.2x left-side SPRI purification (Beckman). The SPRI-purified aliquot was denatured and APOBEC3A-treated (NEB, E1725L). After A3A deamination, the sample was purified via SPRI beads (1.2x, left-sided). Quantitative PCR was used to determine the number of cycles for the indexing PCR. Indexing PCR was performed using the unique dual index primer pairs from NEBNext Multiplex Oligos for Enzymatic-Methyl Seq (NEB). After the indexing PCR, samples were purified with two 0.8x left-sided SPRI purifications. Libraries were quantified with a Qubit HS kit (Invitrogen,Q32851) and assessed for quality with an Agilent Bioanalyzer. The resulting libraries were then pooled and sequenced on an Illumina MiSeq using 150-bp read length on a nano or micro sequencing kit (Illumina).
BS-Seq, bACE-Seq

value: Cortex

value: Bisulfute + A3A (bACE-Seq)

tag: time

value: Week 10


title: W10, Rep3, BS

value: Bisulfute (BS-Seq)


title: W10, Rep2, BS


title: W10, Rep2, bACE


title: W8, Rep3, BS

value: Week 8


title: W8, Rep3, bACE


title: W10, Rep1, BS


title: W8, Rep2, bACE