title: HIT-scISOseq 3d_s1

status: Public on Nov 01 2025

city: guangzhou

first: ming

last: zhou

address: jinsui road 7

institute: Sun Yat-sen University,

description:
HIT-scISOseq
NextSeq High Output Kits

data_processing:
After acquiring the HIT-scISOseq data, we used the accompanying scISA-Tools software for data preprocessing, RNA isoform identification (only the annotated genes were retained as FSM: full splice match), ISM: incomplete splice match, NIC: novel in the catalog, and NNC: novel not in catalog isoforms), gene and isoform matrix generation.
Assembly: Ensembl Macaca_fascicularis_5.0.99
Supplementary files format and content: Read count matrices for each sample were generated by CellRanger counting.

organism: Macaca fascicularis

source_name: cornea

extract_protocol:
The cells were filtered through a 30-µm cell strainer and were re-suspended in 60 µL PBS containing 0.04% BSA to achieve 1,000 cells µl-1 for capture on the 10× Genomics Chromium controller.
We obtained single-cell full-length cDNAs using the 10× Genomics platform, and then used the HIT-scISOseq method for library construction and sequencing.

value: cornea

value: corneal injury

tag: time point

value: 3-day healing stage


title: CN,SMC1_ETP_ChIP_2

type: SRA

city: Dallas

first: Ram

last: Madabhushi

state: TX

address: 5323 Harry Hines Blvd

country: USA

institute: UT Southwestern Medical Center

library_source: genomic

data_processing:
Raw fastq.gz sequencing reads were analyzed for quality control using fastq.gzC, trimmed using Trimmomatic, and aligned to the mm10 reference genome (GRCm38.p6 assembly) using BWA-MEM.
BAM files were marked and filtered for duplicate reads using Sambamba and Samtools, respectively.
Peaks were called using MACS2 from biological duplicates and pseudo-replicates where each ChIP sample was normalized to input DNA.
MACS2-generated bedGraph files were converted to bigWig files using Kent Utils for individual replicates and pooled replicates
Assembly: mm10
Supplementary files format and content: BigWig

molecule: genomic DNA

organism: Mus musculus

source_name: Primary Cortical Neuron

growth_protocol: Dissociated cortical neurons from E16 Swiss-Webster mice (Charles River) were plated at a density of 5 million cells/plate in 10 cm plates. The plates were pre-coated with poly-D-lysine (0.5 mg/ml) overnight at 37°C, followed by washing twice with dH2O. Neurons were maintained in neurobasal media (GIBCO) supplemented with L-glutamine, penicillin/streptomycin, and B27. Etoposide (ETP, 50µM, Sigma E1383) was dissolved in DMSO. Cells were treated by adding drug directly to the culture media 30 minutes prior to collection

extract_protocol:
ChIP was performed for two biological replicates using a ChIP assay kit (MilliporeSigma) as described by the manufacturer. Briefly, DIV13 primary cortical neurons (~15M cells per replicate) were crosslinked with 1% formaldehyde for 10 minutes at 37°C, washed twice with ice-cold PBS, scraped, and resuspended in SDS lysis buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS, protease and phosphatase inhibitor cocktail). Cells were sonicated to shear DNA to 200-500 bp using a bioruptor (Diagenode; high power, 30 seconds on/30 seconds off for 40 cycles). Samples were then centrifuged at 13,000 RPM for 10 minutes at 4°C to remove cellular debris, and the supernatant was diluted up to 2 mL in ChIP dilution buffer (16.7 mM Tris-HCl pH 8, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS). Prior to immunoprecipitation, 1% of each sample transferred to a clean tube to be used for input normalization. Samples were then incubated with appropriate antibody overnight at 4°C followed by incubation with ChIP-grade protein A/G magnetic beads (Thermo) for 3 hours at 4°C. Antibody-conjugated beads were washed with low salt buffer (20 mM Tris-HCl pH 8, 1% Triton-X-100, 2 mM EDTA, 0.1% SDS, 150 mM NaCl), high salt buffer (20 mM Tris-HCl pH 8, 1% Triton-X-100, 2 mM EDTA, 0.1% SDS, 500 mM NaCl), LiCl buffer (10 mM Tris-HCl pH 8, 1% IGEPAL-CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, 0.25 M LiCl), and twice with TE buffer (50 mM Tris-HCl pH 8, 1 mM EDTA) for 5 minutes each with agitation at room temperature. Immunoprecipitated protein-DNA complexes were eluted from the magnetic beads in 500 µl ChIP elution buffer (1% SDS, 0.1 M NaHCO3). DNA-protein crosslinks for immunoprecipitated samples and input DNA were reversed via incubation with an additional 20 mM NaCl for 4 hours at 37°C and residual protein was digested with Proteinase K (NEB) for 1 hour at 45°C. DNA was precipitated using phenol/chloroform isolation and ethanol precipitation, washed with 70% ethanol, and resuspended in 50 µl TE buffer.
Samples were prepared for sequencing using a paired-end library preparation kit compatible with Illumina sequencing instruments (KAPA biosciences) according to the manufacturer’s instructions. Following library amplification, DNA fragment size was assessed via Tapestation (Agilent) to ensure a final library size of 350-450 bp. DNA concentration was quantified by fluorometry (dsDNA high sensitivity Qubit assay, Thermo) to ensure equal DNA concentration for each replicate before replicates were pooled and sequenced on an Illumina NextSeq 500 instrument using V2.5 reagents to an average depth of 25-35 million reads per replicate.

treatment_protocol: DIV13 primary cortical neurons were bath stimulated with 10 micromolar etoposide (ETP) for 30 minutes prior to collection or with 50 micromolar NMDA for 10 minutes followed by washout and recovery in drug-free media for 50 minutes prior to collection

tag: tissue

value: Primary Cortical Neuron

tag: chip antibody

value: SMC1 (Bethyl, A300-055A)

tag: genotype

value: WT

tag: treatment

value: ETP


title: HIT-scISOseq 1d_s2

country: China

description:
HIT-scISOseq
NextSeq High Output Kits

library_source: transcriptomic single cell

molecule: total RNA

value: 1-day healing stage


title: CN,SMC1_NMDA_ChIP_2

value: NMDA


title: CN,SMC1_NMDA_ChIP_1


title: CN,SMC1_ETP_ChIP_1


title: CN,SMC1_CON_ChIP_2


title: CN,SMC1_CON_ChIP_1


title: CN,CTCF_NMDA_ChIP_1


title: CN, CTCF_ETP_ChIP_1


title: S7_J019_T1 [S7_S7_L001]

status: Public on Nov 07 2025

city: Canyon

first: Matthew

last: Scott

address: 3201 Russell Long Blvd

institute: Texas A&M University

library_source: transcriptomic

data_processing:
Illumina RTA v3 software was used for basecalling.
Read quality was assessed in FastQC v0.11.9
Read trimming was performed with Trimmomatic v0.39 with parameters for a 4-base pair sliding window and removing read segments below a minimum base quality score of 20, read length of less than 28 bases, leading and trailing basepairs below a quality score of 3, and removal of all Illumina Universal adaptors.
Read mapping and indexing was performed with HISAT2 v2.1.1, using the bovine whole genome assembly ARS-UCD1.3; parameters -p 14 --dta -x GCF_002263795.2_ARS-UCD1.3
Read assembly and quantification was performed with StringTie v2.2.0 with parameters -p 14 -G GCF_002263795.2_ARS-UCD1.3_genomic.gff, followed by stringtie --merge -p 14 -G GCF_002263795.2_ARS-UCD1.3_genomic.gff -o mergefile.gtf, and stringtie -e -p 14 -G mergefile.gtf -o. A read count matrix was generated with prepDE3.py with parameters python prepDE3.py  -l 150 -i mergelist.txt. ComBat-Seq within sva was used to account for lane bias. Differential expression analysis was performed with edgeR v3.36.0 and glmmSeq v0.2.0.
Assembly: GCF_002263795.2
Supplementary files format and content: Comma-delimited text file of raw gene counts for each sample

organism: Bos taurus

source_name: Whole blood

extract_protocol:
Total RNA was isolated via Tempus Spin RNA Isolation Kits (Applied Biosystems), per product instruction.
RNA libraries were prepared for sequencing using Illumina Stranded mRNA library preparation kits, using standard Illumina protocols.

treatment_protocol: All samples were collected into Tempus RNA Blood tubes (Applied Biosystems) and stored per product recommendation the same day of collection.

value: Whole blood

tag: ID

tag: timepoint

tag: vaccinated

value: no

tag: marketing

value: direct

tag: treated for_brd

value: yes

tag: sequencing lane


title: CN, CTCF_ETP_ChIP_2


title: CN,CTCF_Input


title: CN,CTCF_NMDA_ChIP_2


title: CN,SMC1_Input


title: HIT-scISOseq 3d_s2

description:
HIT-scISOseq
NextSeq High Output Kits


title: CN,CTCF_CON_ChIP_2


title: CN, RNAseq_NMDA_3

data_processing:
Raw fastq.gz sequencing reads were trimmed using Trimmomatic and aligned to the mm10 reference genome (GRCm38.p6 assembly) using TopHat. The aligned reads were processed by the HTSeq-Count pipeline and relative transcript abundance was measured by transcripts per million mapped fragments (TPM).
Assembly: mm10
Supplementary files format and content: raw counts (.txt)

extract_protocol:
Total RNA was extracted from two biological replicates of DIV13 primary cortical neurons (~5 million cells per replicate) using the Qiagen RNeasy Plus Universal Kit (Qiagen) according to the manufacturer’s instructions. RNA quality was assessed using an Agilent Tapestation 4200, and only RNA with RIN score of 8 or higher was used for library preparation. Prior to library preparation, samples were treated with DNase to remove any residual DNA, and RNA concentration was measured by fluorometry (RNA high sensitivity Qubit assay, Thermo).
RNA libraries were prepared for sequencing using a TruSeq stranded mRNA library prep kit with single-end adapters (Illumina) according to the manufacturer’s instructions. Prior to sequencing, library quality was assessed using an Agilent Tapestation 4200, and sample concentration was measured by fluorometry (dsDNA high sensitivity Qubit assay, Thermo). Samples were pooled and sequenced on an Illumina NextSeq 500 instrument with V2.5 reagents to an average depth of 35-45 million reads per replicate.

treatment_protocol: DIV13 primary cortical neurons were bath stimulated with 50 micromolar NMDA for 10 minutes followed by washout and recovery in drug-free media for 50 minutes prior to collection


title: CN, RNAseq_ NMDA_2


title: CN,CTCF_CON_ChIP_1


title: CN, RNAseq_NMDA_1


title: CN, RNAseq_CON_2


title: CN, RNAseq_CON_1


title: CN, RNAseq_CON_3


title: mESC-RBBP5-FKBP, auxin 8hr, biological replicate 1, input, tech rep 2

status: Public on Oct 29 2025

city: La Jolla

first: Alon

last: Goren

state: CA

address: 9375 Gilman Dr

department: Department of Medicine

institute: University of California San Diego

description: Library name: mESC-RBBP5-FKBP_dTAG13_8hr_1_input_2

data_processing:
Samples were sequenced on Illumina NextSeq 550 or Nextseq 2000, samples were demultiplexed with Illumina bcl2fastq.
Adapters were removed with Trimmomatic, reads with Phred score <26 over a 4bp window were truncated.
Reads were aligned to a concatenated genome containing hg38 + sacCer3 + dm6, alignment files were separated by species. Duplicate reads were removed with samtools markdup.
HOMER tag directories were made; by default read counts for each species were filtered for primary alignments with MAPQ > 10, reads were extended to model approximate ChIP-seq fragment size using the option -fragLength 150. Samples were converted to bed files for normalization. Details of spike-in normalization are described in Supplement, normalized data are in bedGraph format.
Read normalized bigwig files were created with HOMER makeBigWig.pl from tag directories.
Assembly: hg38, dm6, sacCer3
Supplementary files format and content: Read-normalized BigWig files for hg38 genome

growth_protocol: HeLa-S3 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1x penicillin-streptomycin, L-glutamine, and 110mg/L sodium pyruvate. Cells were incubated at 37°C and 5% CO2. HCT116-EF1a-OsTIR1-Rpb1 cells were cultured in McCoy’s 5A media (Thermo Scientific 16600082) with 10% FBS and 1x antibiotic-antimycotic. Cells were incubated at 37°C and 5% CO2.

extract_protocol:
HCT116 or HeLa-S3 cells were double crosslinked on plates with 2 mM DSG for 30 minutes (Fisher Scientific NC1736108) and 1% formaldehyde (Pierce ThermoFisher PI28906) for 10 minutes. Crosslinking was quenched with 1/20th volume of 2.625 M glycine (EMD Millipore 357002) and 0.5% BSA (NEB B9000S). Cells were washed by resuspending 3 times with 0.5% BSA/PBS + Protease inhibitor (PIC, Roche 11697498001), spinning down at 1200g for 5 minutes at 4°C and removing the supernatant. Washed pellets were snap frozen in dry ice then stored in -80°C. Cells were lysed with LB3 lysis buffer + PIC, then chromatin was sheared on the PIXUL sonicator (Active Motif 53130) for 108 minutes. S2 cells were crosslinked in 1% formaldehyde for 10 minutes at room temperature, then quenched with glycine and 10% BSA to a final concentration of 0.5%, lysed in LB3 buffer, and sonicated on the PIXUL for 60 minutes. S. cerevisiae cultures in log phase were crosslinked with 0.86% formaldehyde for 30 minutes on a room temperature rotating shaker. The cell wall was digested with Zymolase at 37°C for 60 minutes, cells were washed by pelleting 3x at 5000g and washing in PBS + Protease inhibitor, then resuspended in NP-S buffer + PI. Sheared S. cerevisiae chromatin was sonicated on the PIXUL for 108 minutes. All PIXUL sonication was done with settings Pulse (N) = 50, PRF (kHz) = 1.00, Burst Rate (Hz) = 20, only Process Time was varied. Sheared chromatin from each species were clarified by centrifugation at 15000g, for 10 minutes at 4°C, then combined and inputs were taken before immunoprecipitation for each condition, to assess the relative ratios of chromatin. To HeLa-S3 cells, approximately 100k S2 and 500k S. cerevisiae cells were added per immunoprecipitation reaction. To HCT116 cells, approximately 500k S2 and 100k S. cerevisiae cells were added to each immunoprecipitation reaction. To each IP, 10uL of Protein A beads (Thermo Fisher Scientific 10002D), 1% Triton X-100 and 1x PIC were added. 1 million HCT116 cells or HeLa-S3 cells were used per immunoprecipitation reaction.
To each IP, 10uL of Protein A beads (Thermo Fisher Scientific 10002D), 1% Triton X-100 and 1x PIC were added. 1 million HCT116 cells or HeLa-S3 cells were used per immunoprecipitation reaction. Below are the volumes per IP of each antibody used: CST H3K27ac (D5E4): 1uL CST H3K4me3 (C42D8): 1uL CST H3K4me1 (D1A9): 1uL CST H3K36me3 (D5A7): 1uL CST Rpb1 NTD (D8L4Y): 4uL CST H3K9ac (C5B11): 4uL Samples were incubated overnight on a rotator at 4°C. IP Washes were performed: 3x with 180uL of WBI, 3x with 180uL of WBIII, and 2x with 185uL TET, then eluted in 25uL of cold TT Buffer. Input chromatin containing target and spike-in species’ chromatin from each biological condition were diluted 1:62.5x in TT buffer to a final volume of 25uL. Libraries were prepared with the NEB Ultra II Library Prep Kit (NEB E7645L). NEXTflex Dual-Barcode ChIP-seq Adapters (NOVA-514151) were added at 0.25uM or 0.5uM or 0.625uM. Libraries were then de-crosslinked at 55°C for 1hr, then 65°C overnight and cleaned with Speedbeads, 8.6% PEG and 0.8M NaCl and eluted in TT buffer. Next libraries were PCR amplified with 1uM Solexa 1GA and 1GB primers and NEB Ultra II Q5 Mastermix (PCR protocol: 98C for 30’’, then 12 or 13 cycles of 98C for 10’’, 60C for 15’’, 72C for 30’’, then hold at 72C for 2’). A final cleanup was done with Speedbeads, 8.5% PEG and 1M NaCl, washed twice with 80% EtOH, then the final libraries were eluted in 20uL TT. Library size was estimated by running 6uL of eluted library on a 1.8% agarose gel and concentration was quantified with Qubit HS dsDNA kit (Invitrogen Q32854). Libraries were pooled and sequenced on Illumina NextSeq 550 or NextSeq 2000.

treatment_protocol: HeLa-S3 cells were synchronized in G1/S by adding 2 mM thymidine for 16 hr, washed with PBS, released for 3 hr in fresh medium, and pro-metaphase arrested with 10uM S-trityl-L-cysteine (STC) for 12 hr then harvested. HeLa-S3 cells and HCT116 cells were treated with 500nM Triptolide for 1 or 4 hours. HCT116 cells were treated with 500uM auxin for 1 or 4 hours.

tag: spike-in species

value: D. melanogaster, S. cerevisiae

tag: cell line

tag: cell type

value: mouse embryonic stem cell

value: Auxin 8hr

tag: batch


title: mESC-RBBP5-FKBP, auxin 8hr, biological replicate 1, H3K4me3, tech rep 4

description: Library name: mESC-RBBP5-FKBP_dTAG13_8hr_1_H3K4me3_2

value: CST H3K4me3 (C42D8)


title: mESC-RBBP5-FKBP, auxin 2hr, biological replicate 1, input, tech rep 2

description: Library name: mESC-RBBP5-FKBP_dTAG13_2hr_1_input_2

value: Auxin 2hr


title: mESC-RBBP5-FKBP, auxin 8hr, biological replicate 1, Rpb1, tech rep 2

description: Library name: mESC-RBBP5-FKBP_dTAG13_8hr_1_Rpb1_2

value: CST Rpb1 NTD (D8L4Y)


title: mESC-RBBP5-FKBP, auxin 24hr, biological replicate 1, input, tech rep 2

description: Library name: mESC-RBBP5-FKBP_dTAG13_24hr_1_input_2

value: Auxin 24hr


title: mESC-DPY30-mAID, auxin 8hr, biological replicate 1, input, tech rep 2

description: Library name: mESC-DPY30-mAID_IAA_8hr_1_input_2


title: mESC-RBBP5-FKBP, auxin 0hr, biological replicate 1, H3K4me3, tech rep 4

description: Library name: mESC-RBBP5-FKBP_dTAG13_0hr_1_H3K4me3_4

value: Auxin 0hr


title: mESC-RBBP5-FKBP, auxin 2hr, biological replicate 1, Rpb1, tech rep 2

description: Library name: mESC-RBBP5-FKBP_dTAG13_2hr_1_Rpb1_2


title: mESC-RBBP5-FKBP, auxin 24hr, biological replicate 1, H3K4me3, tech rep 4

description: Library name: mESC-RBBP5-FKBP_dTAG13_24hr_1_H3K4me3_4


title: mESC-DPY30-mAID, auxin 8hr, biological replicate 1, Rpb1, tech rep 2

description: Library name: mESC-DPY30-mAID_IAA_8hr_1_Rpb1_2


title: mESC-RBBP5-FKBP, auxin 24hr, biological replicate 1, Rpb1, tech rep 2

description: Library name: mESC-RBBP5-FKBP_dTAG13_24hr_1_Rpb1_2


title: mESC-RBBP5-FKBP, auxin 2hr, biological replicate 1, H3K4me3, tech rep 4

description: Library name: mESC-RBBP5-FKBP_dTAG13_2hr_1_H3K4me3_2


title: mESC-RBBP5-FKBP, auxin 0hr, biological replicate 1, input, tech rep 2

description: Library name: mESC-RBBP5-FKBP_dTAG13_0hr_1_input_2


title: mESC-DPY30-mAID, auxin 2hr, biological replicate 1, Rpb1, tech rep 2

description: Library name: mESC-DPY30-mAID_IAA_2hr_1_Rpb1_2


title: mESC-RBBP5-FKBP, auxin 0hr, biological replicate 1, Rpb1, tech rep 2

description: Library name: mESC-RBBP5-FKBP_dTAG13_0hr_1_Rpb1_2


title: mESC-DPY30-mAID, auxin 24hr, biological replicate 1, Rpb1, tech rep 2

description: Library name: mESC-DPY30-mAID_IAA_24hr_1_Rpb1_2


title: mESC-DPY30-mAID, auxin 8hr, biological replicate 1, H3K4me3, tech rep 4

description: Library name: mESC-DPY30-mAID_IAA_8hr_1_H3K4me3_2


title: mESC-DPY30-mAID, auxin 24hr, biological replicate 1, input, tech rep 2

description: Library name: mESC-DPY30-mAID_IAA_24hr_1_input_2


title: mESC-DPY30-mAID, auxin 24hr, biological replicate 1, H3K4me3, tech rep 4

description: Library name: mESC-DPY30-mAID_IAA_24hr_1_H3K4me3_4


title: mESC-DPY30-mAID, auxin 0hr, biological replicate 1, Rpb1, tech rep 2

description: Library name: mESC-DPY30-mAID_IAA_0hr_1_Rpb1_2


title: MP0427_T1025T

status: Public on Oct 21 2025

city: New York

first: Somnath

last: Tagore

state: NY

address: William Black Building, 17th Floor, Lab 1708E

institute: Columbia University

data_processing:
We used Picard for demultiplexing, followed by Kallisto for abundance calculation and counts generation.
Assembly: GRCh38
Supplementary files format and content: Abundance data (*.tsv) and Counts data (*.csv)

organism: Homo sapiens

extract_protocol:
Briefly, pHAGE constructs of PIK3CA variants and wild-type genes were expanded from the sequence-validated glycerol stocks for plasmid DNA extraction. The backbone pHAGE constructs, together with the packaging plasmids (pCMVR8.74 and pMD2.G), were co-transfected into the LentiX-293T cells. The virus-containing medium was harvested at 36h, 48h, 60h and 72h post-transfection and then filtered with 0.45 μM filter. 3 x 10^5 cells were plated in each well of a 6-well plate 24h before transduction. The cell culture medium were replaced with 3 mL viral medium and underwent spinoculation at 1000g for 3h in the presence of 8 ug/mL polybrene. Cells were washed with PBS 16h after transduction and recovered in culture medium for another 48h. The transduced cells were then selected with puromycin for 96h and recovered for at least 48h before getting used in subsequent culture and assays.
As described in Bush et al., 2017 (PMID: 28740083).
PLATE-seq

value: Epithelial cell

tag: amino acid change

value: Antibiotics selection (Puromycin)

tag: time


title: mESC-DPY30-mAID, auxin 0hr, biological replicate 1, H3K4me3, tech rep 4

description: Library name: mESC-DPY30-mAID_IAA_0hr_1_H3K4me3_4


title: MP0680_E542E


title: MP1254_V105_R108del


title: MP1257_F614I


title: mESC-DPY30-mAID, auxin 0hr, biological replicate 1, input, tech rep 2

description: Library name: mESC-DPY30-mAID_IAA_0hr_1_input_2


title: mESC-DPY30-mAID, auxin 2hr, biological replicate 1, input, tech rep 2

description: Library name: mESC-DPY30-mAID_IAA_2hr_1_input_2


title: MP0016_M1004I


title: MP0681_F70F


title: MP0683_E109_I112delinsD


title: MP0018_M1043L


title: MP1241_E542K


title: mESC-DPY30-mAID, auxin 2hr, biological replicate 1, H3K4me3, tech rep 4

description: Library name: mESC-DPY30-mAID_IAA_2hr_1_H3K4me3_2


title: MP1240_E545K


title: MP1246_E542V


title: MP1248_Q546R


title: MP1244_C420R


title: MP1242_G1007R


title: MP1249_H701R


title: MP0895_R38C


title: MP1253_H1047L


title: MP1239_G11D


title: MP0710_M1043I


title: MP0011_I910V


title: MP0405_R115L


title: MP044I_C604R


title: MP1050_H1047R


title: MP0010_E545D


title: MP1252_M1043V


title: MP1247_E545A


title: MP1243_L456Afs*13


title: MP1245_E453K


title: MP1255_R398H


title: MP0689_E1037K


title: MP0406_R108H


title: MP0020_E542A


title: MP0024_V344M


title: MP0685_E469insDK


title: MP0079_E39K


title: SCA6, cerebellum, 19 months, sample 2

status: Public on Sep 24 2025

city: Los Angeles

first: Genevieve

last: Konopka

address: 650 Charles Young Drive South, 73-214 CHS

department: Neurobiology

institute: UCLA David Geffen School of Medicine

data_processing:
FastQC for raw read quality
Trimmomatic for trimming reads and adapters
STAR for read alignment to MM10 genome
DESeq2 analysis for DEGs
GO analysis using ToppFunn and SynGO
Assembly: MM10
Supplementary files format and content: TXT, Count table

molecule: polyA RNA

source_name: cerebellum

extract_protocol:
RNA extraction
150 bp paired end reads

value: cerebellum

value: HET

value: 19 months


title: wild-type, cerebellum, 19 months, sample 5

value: WT


title: MP0439_P471L


title: SCA6, cerebellum, 19 months, sample 4


title: SCA6, cerebellum, 19 months, sample 5


title: wild-type, cerebellum, 19 months, sample 1


title: SCA6, cerebellum, 19 months, sample 1


title: SCA6, cerebellum, 12 months, sample 3

value: 12 months


title: wild-type, cerebellum, 19 months, sample 4


title: wild-type, cerebellum, 12 months, sample 5


title: MP1250_A1020V


title: wild-type, cerebellum, 19 months, sample 2


title: wild-type, cerebellum, 12 months, sample 3


title: SCA6, cerebellum, 12 months, sample 5


title: SCA6, cerebellum, 12 months, sample 1


title: wild-type, cerebellum, 12 months, sample 4


title: wild-type, cerebellum, 12 months, sample 1


title: SCA6, cerebellum, 12 months, sample 2


title: SCA6, cerebellum, 6 months, sample 5


title: wild-type, cerebellum, 12 months, sample 2


title: SCA6, cerebellum, 12 months, sample 4


title: SCA6, cerebellum, 6 months, sample 4


title: SCA6, cerebellum, 19 months, sample 3


title: wild-type, cerebellum, 19 months, sample 3


title: SCA6, cerebellum, 6 months, sample 3


title: wild-type, cerebellum, 6 months, sample 5


title: SCA6, cerebellum, 6 months, sample 1


title: SCA6, cerebellum, 6 months, sample 2


title: wild-type, cerebellum, 6 months, sample 3


title: wild-type, cerebellum, 6 months, sample 1


title: wild-type, cerebellum, 6 months, sample 2


title: SCA6, cerebellum, 3 months, sample 5

value: 3 months


title: wild-type, cerebellum, 3 months, sample 3


title: SCA6, cerebellum, 3 months, sample 3


title: SCA6, cerebellum, 3 months, sample 2


title: SCA6, cerebellum, 3 months, sample 1


title: Trim66_KO_homo_HiC_rep_2

status: Public on Sep 17 2025

city: Shanghai

first: Yalei

last: Kong

address: No. 280, Chongqing South Road

institute: Shanghai Jiao Tong University

description:
Library name: sample 13
HiC_homo_2

data_processing:
The In situ Hi-C data were processed following the 4DN Hi-C data processing pipeline (https://data.4dnucleome.org/resources/data-analysis/hi_c-processing-pipeline). Briefly, sequencing reads were aligned to the mm10 reference genome with BWA (version 0.7.17). Valid Hi-C contacts were extracted by removing PCR duplicates and short cis contacts within 1 kb using pairtools (version 1.0.1) . Hi-C contact matrices were then generated by binning the genome at multiple resolutions and normalized using matrix balancing implemented in cooler (version 0.9.2). To account for variations in sequencing depth and alignment efficiency, contact frequencies were normalized by dividing the binned interaction counts by the total number of Hi-C contacts at the specified resolution. Contact matrices were visualized using Python libraries including pandas, seaborn, and matplotlib. Aggregate pileup analyses were conducted using the pileup function from the coolpuppy package (version 1.1.0). A/B compartment profiles were derived from ICE-normalized intrachromosomal contact maps at 500 kb resolution by converting them into observed-over-expected (O/E) matrices. Principal component analysis (PCA) was performed, and PC1 values were interpreted based on GC content. Regions with positive PC1 scores, typically gene-rich, were assigned as compartment A, while regions with negative PC1 scores were assigned as compartment B. Compartment strength was assessed and visualized by saddle plots in cooltools (version 0.5.4). TADs were identified by calculating insulation scores via the diamond insulation algorithm implemented in cooltools, with a window size of 125 kb and a bin size of 25 kb.
Assembly: mm10
Supplementary files format and content: Hi-C: mcool files

source_name: Main Olfactory Epithelium (MOE)

extract_protocol: The live cells were fixed with 1% formaldehyde for 10 min and quenched with 0.2 M glycine for 5 min at room temperature. The cells were then washed twice with 100 uL HBSS containing 0.1% BSA. The pelleted cells were resuspended in 100 uL of ice-cold Hi-C lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630, and protease inhibitors) and incubated on ice for 30 min. After centrifugation at 3,000 g for 5 min at 4 °C, the pelleted nuclei were washed once with 100 uL of ice-cold 1x NEB Buffer 3.1. Chromatin was solubilized in 20 uL of 0.3% SDS and incubated at 65 °C for 10 min, followed by quenching with 5 uL of 10% Triton X-100 and incubation at 37 °C for 10 min. The nuclei were digested with 50 U DpnII in a total volume of 50 uL at 37 °C for 3 h in a Thermomixer with interval shaking. DpnII was then inactivated by incubation at 65 °C for 20 min. For biotin labeling, 3 uL of 1 mM biotin-14-dATP, 0.3 uL of 10 mM dGTP, 0.3 uL of 10 mM dTTP, 0.3 uL of 10 mM dCTP, and 10 U Klenow were added. The reaction was performed at 37 °C for 1.5 h in a Thermomixer. Subsequently, 60 uL of ligation mixture (33.6 uL water, 12 uL 10x NEB T4 DNA ligase buffer, 12 uL 10% Triton X-100, 1.2 uL 10 mg/mL BSA, and 1.2 uL 2,000 U/uL T4 DNA ligase) was added, and the reaction was incubated at 25 °C for 4 h in a Thermomixer. Crosslinks were reversed and DNA was purified. The purified DNA was sheared to 300-500 bp using a Covaris M220. End repair, dA-tailing, and adaptor ligation were then performed using the Rapid Plus DNA Lib Prep Kit V2 (ABclonal). Biotin-labeled DNA fragments were enriched using 50 uL of Dynabeads MyOne Streptavidin C1 (Invitrogen). Sequencing libraries were prepared on beads, followed by 6-8 cycles of PCR amplification. Fragments ranging from 300 bp to 1,000 bp were size-selected. All libraries were sequenced on a NovaSeq X Plus platform following the manufacturer's instructions.

value: Main Olfactory Epithelium (MOE)


title: Trim66_KO_het_HiC_rep_2

description:
Library name: sample 10
HiC_het_2


title: wild-type, cerebellum, 3 months, sample 5


title: wild-type, cerebellum, 3 months, sample 2


title: Trim66_KO_het_HiC_rep_1

description:
Library name: sample 9
HiC_het_1


title: wild-type, cerebellum, 3 months, sample 1


title: wild-type, cerebellum, 3 months, sample 4


title: Trim66_KO_homo_HiC_rep_1

description:
Library name: sample 12
HiC_homo_1


title: Trim66_KO_het_Olfr1507_OSN_H3K9me3_CUT&Tag_rep_2

description:
Library name: sample 6
H3K9me3_het_2
Trim66_KO_het_Olfr1507_OSN_H3K9me3_CUT&Tag_merge_RPM.bw

data_processing:
Reads were mapped to the mouse reference genome (mm10) using Bowtie 2 (version 2.5.1) with the “--local --very-sensitive --no-mixed --no-discordant --phred33 -I 10 -X 700” parameters. The olfactory enhancer peaks were visualized in a multi-region track using the UCSC genome browser and deeptools (version 3.5.5).
Assembly: mm10
Supplementary files format and content: CUT&Tag: bigWig files
Library strategy: CUT&Tag

extract_protocol: The MOE from Olfr1507-ires-tdTomato; Trim66 -/- or Olfr1507-ires-tdTomato; Trim66 +/- mice was dissected and dissociated. Then more than 10,000 tdTomato-positive cells were sorted by flow cytometry. Libraries were prepared with the Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme, TD903).


title: Trim66_KO_homo_HiC_rep_3

description:
Library name: sample 14
HiC_homo_3


title: Trim66_KO_het_Olfr1507_OSN_H3K4me1_CUT&Tag_rep_1

description:
Library name: sample 1
H3K4me1_het_1
Trim66_KO_het_Olfr1507_OSN_H3K4me1_CUT&Tag_merge_RPM.bw


title: Trim66_KO_het_Olfr1507_OSN_H3K9me3_CUT&Tag_rep_1

description:
Library name: sample 5
H3K9me3_het_1
Trim66_KO_het_Olfr1507_OSN_H3K9me3_CUT&Tag_merge_RPM.bw


title: Trim66_KO_homo_ATAC_seq_rep_2

description: merge bw file of Homo 1 and 2

data_processing:
Reads were mapped to the mouse reference genome (mm10) using Bowtie 2 (version 2.5.1) with the “--local --very-sensitive --no-mixed --no-discordant --phred33 -I 10 -X 700” parameters. The olfactory enhancer peaks were visualized in a multi-region track using the UCSC genome browser and deeptools (version 3.5.5).
Assembly: mm10
Supplementary files format and content: bigWig files

extract_protocol:
The MOE from Trim66 -/- or Trim66 +/- mice was dissected and dissociated. Then 80,000 cells were sorted by flow cytometry.
Libraries were prepared with the TruePrep® DNA Library Prep Kit V2 for Illumina (Vazyme, TD501) and applied the index from the TruePrep® Index Kit V2 for Illumina (Vazyme, TD202).


title: EZH2_WT_ChIP_seq

data_processing:
ChIP-seq reads were mapped to the mouse reference genome GRCm38.p4 (downloaded from the GENCODE M10 release) with bwa 0.7.15 (mem with default parameters), samtools 1.3.1, and sambamba 0.6.3. RNA-seq reads were mapped to the mouse reference genome mm10 (downloaded from Illumina iGenomes) with tophat 2.0.11. ChIP- seq peaks were called with macs2 2.1.0.20140616 with "-B" to generate read pileups. Overlaps between peaks were identified with bedtools 2.26.0. DNA motifs were identified (findMotifsGenome.pl: "-size 200 -mask") and visualized (motif2Logo.pl: "-pdf -ns 1000 -bit") with Homer 4.8.3. GO analysis was conducted with ClusterProfiler 4.8.1 (after removing peaks that are not on reference chromosomes).
Assembly: mm10
Supplementary files format and content: bigWig files

extract_protocol: Dissected MOE was dissociated by the Papain Dissociation System (Worthington) at 37 °C for 15 min and trituration for 5?15 times with a cut P1000 pipette tip. Cells were filtered by a 40- ?m strainer (Falcon). After spinning at 400×g for 2 min, cells were resuspended in DMEM (Gibco). ChIP-seq was performed on dis- sociated MOE cells with a ChIP-IT High Sensitivity Kit (Active Motif) and an S2 focused-ultrasonicator (Covaris) (settings: frequency sweeping, intensity = 3, duty cycle = 2%, cycles per burst = 200, 15 cycles of 1 min on and 1 min off). The final DNA product was eluted into ~60 uL. Libraries were prepared with an NEBNext Ultra DNA Library Prep Kit (New England Biolabs) and 11?18 cycles of PCR amplification, and then size-selected with PCRClean DX magnetic beads (Aline Biosciences) (0.55× to remove long fragments, and 0.9× to remove short fragments). Libraries were quantified by a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and a High Sensitivity DNA Analysis Kit on a Bioanalyzer 2100 (Agilent), and then sequenced on a MiSeq or a HiSeq 2500 (Illumina).


title: Trim66_KO_het_HiC_rep_3

description:
Library name: sample 11
HiC_het_3


title: Trim66_KO_homo_Olfr1507_OSN_H3K4me1_CUT&Tag_rep_2

description:
Library name: sample 4
H3K4me1_homo_2
Trim66_KO_homo_Olfr1507_OSN_H3K4me1_CUT&Tag_merge_RPM.bw


title: Trim66_KO_homo_ATAC_seq_rep_1


title: wild-type, cerebellum, 6 months, sample 4


title: Trim66_KO_homo_Olfr1507_OSN_H3K9me3_CUT&Tag_rep_1

description:
Library name: sample 7
H3K9me3_homo_1
Trim66_KO_homo_Olfr1507_OSN_H3K9me3_CUT&Tag_merge_RPM.bw


title: EZH2_WT_Input


title: Trim66_KO_het_Olfr1507_OSN_H3K4me1_CUT&Tag_rep_2

description:
Library name: sample 2
H3K4me1_het_1
Trim66_KO_het_Olfr1507_OSN_H3K4me1_CUT&Tag_merge_RPM.bw


title: Trim66_KO_het_ATAC_seq_rep_1


title: Trim66_KO_homo_Olfr1507_OSN_H3K4me1_CUT&Tag_rep_1

description:
Library name: sample 3
H3K4me1_homo_1
Trim66_KO_homo_Olfr1507_OSN_H3K4me1_CUT&Tag_merge_RPM.bw


title: SCA6, cerebellum, 3 months, sample 4


title: Trim66_KO_homo_Olfr1507_OSN_H3K9me3_CUT&Tag_rep_2

description:
Library name: sample 8
H3K9me3_homo_2
Trim66_KO_homo_Olfr1507_OSN_H3K9me3_CUT&Tag_merge_RPM.bw


title: Trim66_KO_het_ATAC_seq_rep_2

description: merge bw file of Het 1 and 2


title: YY1_WT_ChIP_seq


title: TRIM66_WT_ChIP_seq_rep_2


title: ATF5_WT_Input_rep_4


title: TRIM66_WT_ChIP_seq_rep_1


title: ATF5_WT_Input_rep_3


title: ATF5_WT_Input_rep_1


title: ATF5_WT_ChIP_seq_rep_4


title: ATF5_WT_ChIP_seq_rep_3


title: ATF5_WT_Input_rep_2


title: Trim66_KO_het_10X_scRNA_seq

data_processing:
Reads were processed using the Cell Ranger (version 6.1.2) pipeline with default parameters. FASTQs generated from Illumina sequencing output were aligned to the mouse genome (GRCm38, mm10) using the STAR algorithm. Next, Gene-Barcode matrices were generated for each individual sample by counting UMIs and filtering non-cell-associated barcodes. Finally, we generate a gene-barcode matrix containing the barcoded cells and gene expression counts. This output was then imported into the Seurat R toolkit (version 4.3.0) for downstream analysis. The different filtering thresholds are suitable for Trim66 knockout data. For Trim66 knockout data, cells with less than 700 transcripts, or less than 200 genes, or more than 15000 transcripts, or more than 10% of mitochondrial gene expression were filtered out as low-quality cells; genes expressed in less than three cells were excluded. The normalized data (NormalizeData function in Seurat package) after merging the different datasets was performed for extracting a subset of variable genes. Next, we performed principal component analysis (PCA) and reduced the data to the top 30 PCA components after scaling the data with the vars.to.regress parameter to remove the mitochondrial variables. Then we used the Harmony package to integrate the different data. And the integrated data was displayed on a 2D map produced with uniform manifold approximation and projection (UMAP) using harmony embeddings (1-20).
The clusters were obtained using the FindNeighbors and FindClusters functions with the resolution set to 0.9 for the Trim66 knockout, resulting in 25 clusters. The cell types were annotated by overlapping the cluster markers found using the FindAllMarkers function with the known cell-type marker genes. The following cell types were annotated with selected marker genes listed below: HBCs (Krt5, Krt14, Trp63, Cxcl14, Sox2, Meg3); GBCs (Ascl1, Sox2, Lgr5, Hes6, Cxcr4, Ezh2, Neurog1, Neurod1, Tmprss4, Kit, Mki67, Top2a); INPs (Neurog1, Neurod1, Neurod2, Top2a, Mki67, Lhx2, Ebf1); immature OSNs (Ablim1, Drd4, Dbn1, Dpysl5, Crmp1, Ppp2cb, Marcksl1, Atf5, Gnas, Hdac2, Gng8, Dpysl3, Gap43, Stmn1, Stmn2, Ebf2, Lhx2, Cbx8, Trib3); mature OSNs (Cngb1, Adcy3, Slc17a6, Cnga4, Cnga2, Gnal, Omp, Gng13, Stoml3, Ebf2, Cbx8, Rtp1); sustentacular cells (Cyp2g1, Cyp1a2, Notch2, Notch3, Hes1, Hes5, Gpx6, Ermn, Sox2, Hey1, Sult1c1); olfactory ensheathing glia (S100b, Plp1, Mpz, Alx3); olfactory microvillar cells (Coch, Ascl3, Cftr, Hepacam2, Sox9, Trpm5); GC-D cells (Ms4a4c, Ms4a6c, Ms4a7, Pde2a, Car2, Cnga3); immune cells (Cd3d, Cd3e, Cd8a, Cd4, Il7r, Fgfbp2, Fcgr3a, Cx3cr1, Cd19, Cd79a, Ms4a4a); blood cells (Ptprc, Hba-a1, Mzb1, Sdc1, Cd14, S100a12, Clec10a, C1qa, C1qb, C1q).
Assembly: mm10
Supplementary files format and content: Cell Ranger software output files (barcodes.tsv, features.tsv, matrix.mtx)

extract_protocol:
The MOE tissues were dissected and transported in sterile culture dish with 10 ml 1´Dulbecco's Phosphate-Buffered Saline (DPBS; Thermo Fisher, 14190144) on ice to remove the residual tissue storage solution, then minced on ice. We used the dissociation enzyme, 0.25% Trypsin (Thermo Fisher, 25200-072) and 10 μg/mL DNase I (Sigma, 11284932001) dissolved in PBS with 5% Fetal Bovine Serum (FBS; Thermo Fisher, SV30087.02) to digest the tissues. The MOE tissues were dissociated at 37 °C with a shaking speed of 50 rpm for about 40 min. We repeatedly collected the dissociated cells at interval of 20 min to increase cell yield and viability. Cell suspensions were filtered using a 40 μm nylon cell strainer and red blood cells were removed by 1´Red Blood Cell Lysis Solution (Thermo Fisher, 00-4333-57). Dissociated cells were washed with 1´DPBS containing 2% FBS. Cells were stained with 0.4% Trypan blue (Thermo Fisher, 14190144) to check the viability on Countess® II Automated Cell Counter (Thermo Fisher). Each of the solutions above contained transcriptional inhibitors (5 μg/mL Actinomycin D., 10 μg/mL Anisomycin, and 10 μM Triptolide, Sigma)
Beads with unique molecular identifier (UMI) and cell barcodes were loaded close to saturation, so that each cell was paired with a bead in a Gel Beads-in-emulsion (GEM). After exposure to cell lysis buffer, polyadenylated RNA molecules hybridized to the beads. Beads were retrieved into a single tube for reverse transcription. On cDNA synthesis, each cDNA molecule was tagged on the 5’end (the 3’end of a messenger RNA transcript) with UMI and cell label indicating its cell of origin. Briefly, 10´beads that were then subject to second-strand cDNA synthesis, adaptor ligation, and universal amplification. Sequencing libraries were prepared using randomly interrupted whole-transcriptome amplification products to enrich the 3’ end of the transcripts linked with the cell barcode and UMI. All the remaining procedures including the library construction were performed according to the standard manufacturer’s protocol (CG000206 Rev D). Sequencing libraries were quantified using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 and the Qubit High Sensitivity DNA Assay (Thermo Fisher Scientific). The libraries were sequenced on NovaSeq6000 (Illumina) using 2´150 chemistry.


title: TRIM66_WT_Input


title: Trim66_KO_homo_10X_scRNA_seq


title: ATF5_WT_ChIP_seq_rep_1


title: 14 days post injury (dpi) prior to pulldown

status: Public on Aug 26 2025

city: Madison

first: Jianhong

last: Ou

state: Wisconsin

address: 330 N Orchard Street

institute: Morgridge Institute For Research

data_processing:
The data were analyzed by following the description of CTK pipeline (https://doi.org/10.1093/bioinformatics/btw653)
3' adapter were trimmed by cutadapt (v1.18) by two steps to remove the adapters (NNNNNNNNAGATCGGAAGAGCGTCGTG, NNNNNNNNAGATCGGAAGAGCGTCGTGT, AGATCGGAAGAGCGTCGTG, and AGATCGGAAGAGCGTCGTGT) with parameters(--times 1 -e 0.1 -O 1 --quality-cutoff 5 -m 20)
The trimmed reads were mapped to danRer10 by STAR (2.7.4a)
Reads were counted after PCR duplicates were collapsed.
Assembly: danRer10
Supplementary files format and content: csv file with reads count for given exons

organism: Danio rerio

source_name: zebrafish hearts

extract_protocol: Van Nostrand, Eric L., et al. "Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP)."Nature methods13.6 (2016): 508-514.

value: zebrafish hearts

tag: group

value: 14 days post injury

tag: fraction

value: input

value: Z-CAT transgenic fish

value: Tamoxifen treatment


title: DEN30w

status: Public on Jul 30 2025

city: Yokohama

first: Xian-Yang

last: Qin

state: Kanagawa

address: 1-7-22 Suehiro, Tsurumi

country: Japan

institute: RIKEN Center for Integrative Medical Sciences

description:
Library name: Sample4
Fresh frozen Spatial trascriptome sequencing

data_processing:
Sequencing data were processed using the Space Ranger software (v1.3.1, 10x Genomics) with the spaceranger count pipeline and STAR aligner to map reads to the mouse reference genome (mm10-2020-A).
Assembly: mm10
Supplementary files format and content: barcodes.tsv.gz: list of spatial varcodes
Supplementary files format and content: features.tsv.gz: list of gene IDs
Supplementary files format and content: matrix.mtx.gz: gene expression count data in Matrix Market Exchange Format
Supplementary files format and content: tissue_positions_list.csv, tissue_hires_image.png, tissue_lowres_image.png, detected_tissue_image.jpg, aligned_fiducials.jpg, scalefactors_json.json: These files correspond to the spatial transcriptomics data generated using the 10x Genomics Visium platform.
Library strategy: Spatial Transcriptomics

source_name: liver

extract_protocol:
Fresh frozen tissue were OCT embedded, and 10-μm-thick frozen sections were cut with cryostat
After H&E staining to correlate morphological features with gene expression, tissue sections were permeabilized, and spatially barcoded cDNA synthesis was carried out in situ. Libraries were prepared using Visium Spatial Gene Expression Reagent Kits (10x Genomics).

tag: tissue

value: liver


title: 14 dpi sample 1

value: IP

tag: genotype


title: inC-RNA-seq of K562 single-cell derived colonies cultured in the presence of epigenetic drugs (CAGE_Colonies)

status: Public on Sep 11 2025

city: Boston

first: Ignas

last: Mazelis

state: MA

address: 210 Longwood Avenue

department: System Biology

institute: Harvard Medical School

description: Library name: K562_Clone_seq

data_processing:
BCL sequencing files received from Illumina BaseSpace portal were converted to raw FASTQ files for each of the 4 (R1, R2, R3, R4) sequencing reads without read trimming or masking by using bcl2fastq software (Illumina, v2.20.0.422). Raw FASTQ files were processed using a custom pipeline (available at https://github.com/AllonKleinLab/paper-data/tree/master/Mazelis2025_CAGEs). In brief, sequencing reads were demultiplexed based on Read1 index sequence (stored as read3) (index used: "Control_A_1":"ATAGAGAG", "Control_A_2":"AGAGGATA", "Control_A_3":"CTCCTTAC", "Control_A_4":"TATGCAGT", "Control_A_1":"TACTCCTT", "Control_A_2":"AGGCTTAG", "Control_A_3":"ATTAGACG", "Control_A_4":"CGGAGAGA", "Vorinostat1_1":"CTAGTCGA",  "Vorinostat1_2":"AGCTAGAA","Vorinostat1_3":"ACTCTAGG","Vorinostat1 4":"TCTTACGC","Vorinostat2_1":"CTTAATAG","Vorinostat2_2":"ATAGCCTT","Vorinostat2_3":"TAAGGCTC","Vorinostat2_4":"TCGCATAA","Decitabine1_1":"GTAACGTT","Decitabine1_2":"ACTGAGTT","Decitabine1_3":"ATACTCTT","Decitabine1_4":"GACAACTT","Decitabine2_1":"GAACGATT","Decitabine2_2":"AAGATTGT","Decitabine2_3":"AATCGGGT","Decitabine2_4":"TTGAGGGT","Azacytidine1_1":"AAGCTTCT", "Azacytidine1_2":"TATTGCCT","Azacytidine1_3":"CAGTCCCT","Azacytidine1_4":"ATGCGGAT","Azacytidine2_1":"CAAGTCAT","Azacytidine2_2":"ATATGCAT","Azacytidine2_3":"ATGGAAAT","Azacytidine2_4":"CTGCGTTG","Control_C_1":"AAGCGTAC", "Control_C_2":"TAGTGGAC","Control_C_3":"CGGACAAC","Control_C_4":"ATGGATTA","Control_C_1":"CCACATTA","Control_C_2":"GCAGGGTA","Control_C_3":"ATCCGCTA","Control_C_4":"TTCACATA","Control_B_1"…"TGAAATGA","Control_B_2":"AAACTCGA","Control_B_3":"ACCCACGA","Control_B_4":"CGACGTCA","Control_B_1":"TGTGCTCA","Control_B_2":"CTGGTGCA","Control_B_3":"TACCGGCA","Control_B_4":"ATGAAGCA"), and were filtered based on expected structure (all barcodes matching a barcode whitelist with hamming distance of 1). The demultiplexed reads were then processed using Trimmomatic (1) (with parameters: LEADING 28; SLIDINGWINDOW 4:20; MINLEN 20), followed by Poly-A length trim to 4 nt and selecting reads that are longer than MINLEN. For surviving reads, the fraction of length composed out of same base repeats (only considering > 5) was determined, rejecting reads whose fraction is greater than 0.5. Count matrices were generated using InDrops pipeline.
Count matrices were filtered by selecting barcodes with >2000 UMIs and fraction of mitochondrial counts being below 0.4. Mouse and human cells were filered based on gene expression. Read mapping and cell filtering based on counts and mitochondrial fraction were carried out as described above. Raw counts were then normalized to 10,000 counts per cell (CP10K). Highly variable genes were selected using Scanpy's implementation of the Seurat v3 approach (sc.pp.highly_variable_genes with flavor='seurat_v3'; 4,000 genes for K562 cells and 5,000 genes for L1210 cells; using unnormalized counts for variance calculations). Visualization by UMAP embedding. For visualization only, batch effects from library preparation and cell seeding were corrected using Harmony (Korsunsky et al., 2019) using the scanpy implementation, with the following non-default parameters: theta=4.0, lambda=10.0, and sigma=0.05. Harmony was applied to the 15 NMF program usages per cell. A nearest neighbor graph was constructed using the Harmony-corrected program usages with n_neighbors=10, and scanpy’s UMAP was then applied to the batch-corrected embedding with default parameters. Heatmaps of NMF gene loadings. For heatmaps of gene contributions to NMF programs, the NMF loading matrix (“H” matrix in scikit-learn) was normalized by dividing each gene's loading by its mean non-zero expression level (calculated as mean expression when expressed / fraction of cells expressing). The genes shown in the final heatmaps were filtered to remove mitochondrial genes and non-coding genes to improve interpretability, and ordered by descending normalized loading values. NMF usage means, CVs and Fano Factors. NMF program usage values were first normalized for each cell such that the sum of usages across all programs equaled 1. The mean, CV, and Fano factor of each program's usage across colonies was then calculated at each timepoint. To calculate mock colony CV and Fano factors, we corrected for systematic differences in means over time by regressing the CVs using the power law relationship observed between mean and CV. For comparisons across conditions, each metric was normalized to its value in control colonies at the same timepoint.
Assembly: Mixed Human GRCh38.101 and mouse GRCm.101 genome assembly
Supplementary files format and content: h5ad cell x genes matrices

source_name: Blood cell line

growth_protocol: K562 and L1210 cells were pre-treated with drugs separately by seeding 150,000 (L1210) and 200,000 (K562) cells into 6-well plate wells (Corning). Cells were grown in 2mL of IMDM with 10% FBS, 1X Pen/Strep in the presence of 0.05 μM Dec, 0.7μM Aza, 0.4μM Vor (MedChemExpress) or no drugs (3 repeats) for 48 hours. Next, cells were collected and K562 and L1210 cell-lines were pooled based on the drug condition at a 1:1 ratio. Cell suspensions were encapsulated into CAGEs using a final cell concentration of 1.4e6 cells/mL. Each drug condition was encapsulated for 2 hours, collecting the droplets into four 30 min fractions. One fraction of each condition was sampled instantly by lysing the cells inside the CAGEs and freezing the lysate containing CAGEs at -80ºC in 3X SSCI with 10% DMSO and 0.05U/μL Murine RNAse Inhibitor. Remaining fractions of packed hydrogel CAGEs were seeded into a different 24-well plate well into 1mL of IMDM with 10% FBS and 1x Pen/Strep, 0.1% F127 and 0.1% L31 with appropriate drug. 10,000 K562 and L1210 cells from appropriate drug conditions were seeded outside the CAGEs for the first two days. Every two days, media was exchanged with fresh media with appropriate drugs and one well of each condition was sampled for inC-RNA-seq by lysing the cells and freezing at -80ºC as described above.

extract_protocol:
After all the time points were collected, CAGEs were thawed, washed, and treated with Turbo DNAse I and Proteinase K. Finally, CAGEs were washed three times with 3X SSCI buffer and three times with HI10. Resulting CAGEs were subjected to the inC-RNA-seq protocol.  Reverse-transcription reaction was set on ice and performed in PCR strip tubes. A standard 100μL reaction was composed out of 60.5μL of packed capsules, 2.5μL of 10μM RT poly(T) primer housing a 10nt UMI and 6nt barcode (Table S3, RT), 3μL of 10% Igepal CA-630 (Millipore Sigma, I8896-50ML), 20μL of 5X SMARTseq3 RT buffer (100mM Tris pH 8.3, 150mM NaCl, 12.5mM MgCl2, 5mM GTP, 40mM DTT), 0.5μL of 10mM dNTPs (NEB, N0447L), 2.5μL of 40U/μL Recombinant RNase Inhibitor (Takara Bio, 2313A), 1.5μL of 50μM TSO, 5μL of Maxima H- reverse transcriptase (ThermoFisher, EP0753). Different timepoints were pooled and were kept separate from other timepoints. Reaction volumes were scaled according to the amount of capsules used and number of reactions performed. To set up the reaction, capsules, RT primer and Igepal CA-630 (Millipore Sigma, I8896-50ML) were mixed on ice in a PCR tube, with each housing a different RT barcode. Capsules were incubated on ice for 10 min to allow the RT primer to diffuse in, followed by heating at 70ºC for 5 min and cooling down on ice. Next, RT master mix, composed out of the remaining reagents was added to each tube, ensuring a thought mixing of capsules with pipetting. Tubes were incubated on ice for 5 min and the reaction was carried out at 42ºC for 90 min, followed by cycling between 50ºC and 42ºC incubation of 2 mins for 15 cycles. Reactions were left at room temperature overnight and processed the next day. After the RT reaction, capsules were washed three times with TI100 (pH 8.0), twice with TIbS (pH 8.0), three times with ice-cold TI100 (pH 8.0) with 2 min incubations on ice. Finally capsules were washed twice with TI10 (pH 8.0). Next, Capsule dU PCR was performed to carry out cDNA amplification. After the cDNA amplification, capsules were washed three times with TI100 (pH 8.0), twice with TIbS (pH 8.0), three times with TI100 (pH 8.0), twice with TI10 (pH 8.0). Washed capsules were subjected to tree rounds of split-and-pool barcoding. Following split-and-pool barcoding, capsules were split into 8 tubes and were dissolved  using 10M NaOH. DNA was purified using MinElute PCR purification kit and barcoded cDNA was eluted into 20μL of the elution buffer. Barcoded and purified DNA was amplified using PCR. After the reaction, amplified barcoded cDNA was purified using 0.8X AMPure XP beads (Beckman Coulter, A63881) and was eluted into a 15μL Elution Buffer.
To construct Illumina sequencing libraries, 50 ng of barcoded and amplified cDNA was fragmented, blunted and dA-tailed by mixing it with 3.5 μL of NEBNext Ultra II FS Reaction buffer (NEB), 1 μL of fully mixed NEBNext Ultra II FS Enzyme mix (NEB) and nuclease free water to a final volume of 17.5 μL. The reaction mix was vortexed for 10 s, spanned down and placed into a preheated PCR machine for incubation at 37°C for 10 min; 65°C for 30 min; hold 4°C (lid at 75ºC). Fragmented DNA was purified using 0.8X AMPure XP beads (Beckman Coulter, A63881) and eluted into 16μL of water. Single-stranded DNA adapter, containing Illumina’s TruSeq read 1 (Adp. TruSeq R1 FW and RV) sequences were annealed together at the final concentration of 1.5 μM by mixing 1.5 μL of forward ligation primer and reverse ligation primer (each at 100 μM) with 97 μL of nuclease-free water and heating the solution to 95°C for 2 min and cooling it at room temperature for 5 min. 16 μL of the purified fragmented DNA was mixed with 1.25 μL of 1.5 μM annealed adapters, 1.5μL of 10X T4 DNA ligase buffer, 15 μL of NEBNext Ultra II Ligation Master Mix (NEB, E7805S kit) and 0.5 μL of NEBNext Ultra II Ligation Master Enhancer (NEB, E7805S kit). The ligation reaction was carried out by incubating in the thermocycler for 15 min at 20°C (lid at 30ºC). After ligation, the reaction mix was diluted to 100 μL and was purified using AMPure XP beads (Beckman Coulter, A63881) with 0.8X volume ratio and eluted into 40 μL nuclease-free water. Indexing PCR was carried out by mixing purified DNA with 25 μL of 2x NEBNext® High-Fidelity 2X PCR Master Mix (NEB, M0543L) and 2.5 μL of 20μM P7 binding primer, 48 sets of 2.5μL of 20μM indexing primer that houses an 8bp library index and water to 50μL. PCR cycling program: 98 °C, 2 min; 3 cycles of 98 °C, 20 s; 55 °C, 30 s; 72 °C, 20 s and 9 cycles 98 °C, 20 s; 65 °C, 30 s; 72 °C, 20 s; with a 1 min final extension at 72 °C, and 4 °C, hold at the end. Following final library indexing, DNA was double-side purified using 0.6x-0.8x SPRIselect (Beckman Coulter, B23318) magnetic beads and eluted into 15μL.

value: Blood cell line

value: Lymphoblast

tag: treatment


title: Uninjured prior to pulldown

value: Uninjured


title: ATF5_WT_ChIP_seq_rep_2


title: Uninjured sample 2


title: 14 dpi sample 2


title: inC-RNA-seq of L1210 single-cell derived colonies cultured in the presence of epigenetic drugs (CAGE_Colonies)

description: Library name: L1210_Clone_seq

value: 0.05 μM Dec, 0.7μM Aza, 0.4μM Vor (MedChemExpress) or no drugs (3 repeats)


title: RNA-seq in CHD4cd (catalytic dead)-ADNPdN (N-terminal mutant) fusion mESC, replicate 3

status: Public on Jul 21 2025

city: Basel

first: Fabio

last: Mohn

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

description: Library name: CHD4cd_ADNPdN_RNAseq_fusion_r3

data_processing:
Demultiplexing and fastq conversion were performed with bcl2fastq2 v1.17.
Sequenced reads were aligned using STAR 2.7.3 and repeat sensitive settings according to Teissandier et al (Mobile DNA, 2019) (--outFilterType BySJout --outFilterMultimapNmax 100000  --outFilterMismatchNmax 3 --winAnchorMultimapNmax 20000 --alignMatesGapMax 350 --seedSearchStartLmax 30 alignTranscriptsPerReadNmax 30000  --alignWindowsPerReadNmax 30000 --alignTranscriptsPerWindowNmax 300 --seedPerReadNmax 3000 --seedPerWindowNmax 300  --seedNoneLociPerWindow 1000--outSAMattributes NH HI NM MD AS nM --outMultimapperOrder Random --outSAMmultNmax --limitBAMsortRAM 10000000000).
Assembly: GRCm39 / mm39
Supplementary files format and content: bigWig files were generated by using samtools and bedtools to calculate per base coverage, then counts were normalized to 1 mio genome mapping reads and bigwigs were generated using bedGraphToBigWig (USCS tools).

growth_protocol: Mouse embryonic stem cells were grown under standard conditions in medium containing serum, LIF and 2i.

extract_protocol:
For RNA-seq, RNA was collected using RNAeasy miniprep kit (Qiagen) according to manufacturer's instructions. For TT-seq, RNA was isolated using Trizol according to manufacturer's instructions.
Libraries were prepared using TruSeq Ilumina total RNA-seq

tag: genetic background

value: hybrid 129-C57BL/6

value: embryonic stem cells (ESC)


title: Uninjured sample 1


title: DEN22w

description:
Library name: Sample3
Fresh frozen Spatial trascriptome sequencing


title: RNA-seq in CHD4cd (catalytic dead)-ADNPdN (N-terminal mutant) fusion mESC, replicate 1

description: Library name: CHD4cd_ADNPdN_RNAseq_fusion_r1


title: STAM16w

description:
Library name: Sample1
Fresh frozen Spatial trascriptome sequencing


title: RNA-seq in CHD4cd (catalytic dead)-ADNPdN (N-terminal mutant) fusion mESC, replicate 2

description: Library name: CHD4cd_ADNPdN_RNAseq_fusion_r2


title: RNA-seq in CHD4cd (catalytic dead)-ADNPwt fusion mESC, replicate 2

description: Library name: CHD4cd_ADNPwt_RNAseq_fusion_r2


title: STAM20w

description:
Library name: Sample2
Fresh frozen Spatial trascriptome sequencing


title: RNA-seq in CHD4cd (catalytic dead)-ADNPwt fusion mESC, replicate 3

description: Library name: CHD4cd_ADNPwt_RNAseq_fusion_r3


title: RNA-seq in CHD4cd (catalytic dead)-ADNPwt fusion mESC, replicate 1

description: Library name: CHD4cd_ADNPwt_RNAseq_fusion_r1


title: RNA-seq in ADNP N-terminal mutant mESC, for fusion exp, replicate 1

description: Library name: ADNPdN_RNAseq_fusion_r1


title: RNA-seq in ADNP N-terminal mutant mESC, replicate 2

description: Library name: ADNPdN_RNAseq_r2


title: RNA-seq in ADNP N-terminal mutant mESC, for fusion exp, replicate 2

description: Library name: ADNPdN_RNAseq_fusion_r2


title: RNA-seq in CHD4wt-ADNPdN (N-terminal muntant) fusion mESC, replicate 3

description: Library name: CHD4wt_ADNPdN_RNAseq_fusion_r3


title: RNA-seq in CHD4wt-ADNPwt fusion mESC, replicate 2

description: Library name: CHD4wt_ADNPwt_RNAseq_fusion_r2


title: RNA-seq in ADNP N-terminal mutant mESC, replicate 1

description: Library name: ADNPdN_RNAseq_r1


title: RNA-seq in CHD4wt-ADNPwt fusion mESC, replicate 1

description: Library name: CHD4wt_ADNPwt_RNAseq_fusion_r1


title: RNA-seq in ADNP PxVxL mutant mESC, replicate 2

description: Library name: ADNPpxvxl_RNAseq_r2


title: RNA-seq in ADNP WT mESC, for fusion exp, replicate 1

description: Library name: ADNPwt_RNAseq_fusion_r1


title: RNA-seq in ADNP N-terminal mutant mESC, replicate 3

description: Library name: ADNPdN_RNAseq_r3


title: RNA-seq in ADNP KO mESC, replicate 2

description: Library name: ADNPko_RNAseq_r2


title: RNA-seq in ADNP KO mESC, replicate 3

description: Library name: ADNPko_RNAseq_r3


title: RNA-seq in ADNP WT mESC, replicate 1

description: Library name: ADNPwt_RNAseq_r1


title: RNA-seq in CHD4wt-ADNPwt fusion mESC, replicate 3

description: Library name: CHD4wt_ADNPwt_RNAseq_fusion_r3


title: RNA-seq in ADNP WT mESC, for fusion exp, replicate 2

description: Library name: ADNPwt_RNAseq_fusion_r2


title: RNA-seq in CHD4wt-ADNPdN (N-terminal muntant) fusion mESC, replicate 1

description: Library name: CHD4wt_ADNPdN_RNAseq_fusion_r1


title: RNA-seq in ADNP WT mESC, for fusion exp, replicate 3

description: Library name: ADNPwt_RNAseq_fusion_r3


title: RNA-seq in CHD4wt-ADNPdN (N-terminal muntant) fusion mESC, replicate 2

description: Library name: CHD4wt_ADNPdN_RNAseq_fusion_r2


title: RNA-seq in ADNP WT mESC, replicate 2

description: Library name: ADNPwt_RNAseq_r2


title: RNA-seq in ADNP KO mESC, replicate 1

description: Library name: ADNPko_RNAseq_r1


title: RNA-seq in ADNP PxVxL mutant mESC, replicate 1

description: Library name: ADNPpxvxl_RNAseq_r1


title: RNA-seq in ADNP N-terminal mutant mESC, for fusion exp, replicate 3

description: Library name: ADNPdN_RNAseq_fusion_r3


title: RNA_EPI.17_DBA_55

status: Public on Jun 21 2025

first: Richard

last: Koche

state: New York

institute: Memorial Sloan Kettering Cancer Center

description: Table_RNAseq_EPI_rawCounts_updateMarch2026.txt

data_processing:
RNA-seq reads were filtered for quality and 3' trimmed for adapter sequences.
Reads were aligned to mouse genome assembly mm10 using STAR (v2.4) with default parameters.
Read count tables were created using HTSeq v0.6.1.
Normalization and differential expression analysis was performed with DESeq2.
Assembly: mm10
Supplementary files format and content: Tab-delimited text file with un-normalized read counts over all genes.

growth_protocol: EpiSCs were cultured on irradiated MEFs and N2B27 containing 12.5 ng/mL hsFGF, 20 ng/mL Activin A, and 175 nM NVP-TNKS6561.  For DE, EpiSCs are plated at 110,000 cells/cm2 on laminin in chemically defined media (CDM) containing 0.7 ug/mL insulin, 1% KOSR, 2 uM thiazovivin, and the above hsFGF, Activin, and NVP. After 5 hours, the cells are washed and media replaced with CDM containing 3 uM CHIR and 40 ng/mL Activin A. After 16 hours, the cells are washed and media replaced with CDM supplemented with 100 ng/mL Activin A and 100 nM LDN-193189 for 24 additional hours1. Definitive endoderm cells were dissociated in Accutase, washed in FACS buffer, counted, and resuspended with rat anti-mouse CD184/CXCR4 Alexa Fluor 647-conjugated antibody at a concentration of 1 µl/106 cells and incubated for 1 h at 4°C. Cells were then washed with FACS buffer, centrifuged (200 g for 3 min at 4°C) and resuspended in a solution composed by 80% FACS buffer and 20% anti-rat IgG MicroBeads (Miltenyi Biotec, 130-048-501). Cells were incubated in the mixture for 15 min at 4°C, then washed and resuspended in 500 µl buffer. The MS column was placed in an OctoMACS separator (Miltenyi Biotec, 130-048-501) on the MACS multistand. The cell suspension was added to the column and rinsed three times with buffer. Finally, the column was removed from the separator and the magnetically labeled cells were collected.

extract_protocol:
Phase separation in cells lysed in TRIzol was induced with 50% isopropanol and 0.5% 2-mercaptoethanol and RNA was extracted from the aqueous phase using the MagMAX mirVana Total RNA Isolation Kit (Thermo Fisher, A27828) on a KingFisher Flex Magnetic Particle Processor according to the manufacturer's protocol with 1-2 million cells input.
EpiSC: 100-500 ng of total RNA with RIN values of 9.5-10 underwent polyA selection and TruSeq library preparation according to instructions provided by Illumina (TruSeq Stranded mRNA LT Kit), with eight cycles of PCR. Samples were barcoded and run on a NovaSeq 6000 in a PE100 run, using the NovaSeq 6000 S4 Reagent Kit (200 Cycles) (Illumina).  DE: 2 ng total RNA with RNA integrity numbers ranging from 7.7 to 10 underwent amplification using the SMART-Seq v4 Ultra Low Input RNA Kit, with 12 cycles of amplification. Subsequently, 10 ng of amplified cDNA was used to prepare libraries with the KAPA Hyper Prep Kit using eight cycles of PCR. Samples were barcoded and run on a NovaSeq 6000 in a PE100 run, using the NovaSeq 6000 S4 Reagent Kit (200 Cycles) (Illumina).

value: epiblast stem cells


title: RNA_EPI.9_C57B6_44


title: RNA_EPI.4_NOD_AC576

value: NOD/ShiLtJ


title: RNA_EPI.8_WSB_AC635

value: WSB/EiJ


title: RNA_EPI.19_C57B6_1


title: RNA_EPI.7_NZO_AC671

value: NZO/HiLtJ


title: RNA_EPI.6_PWK_AC698

value: PWK/PhJ


title: RNA_EPI.3_C57B6_5.1


title: RNA_EPI.5_NOD_AC595


title: RNA_EPI.18_C57B6_4


title: RNA_DE.O_WSB_AC627

description: Table_RNAseq_DE_rawCounts_updateMarch2026.txt

value: definitive endoderm


title: RNA_EPI.13_NZO_AC653


title: RNA_EPI.16_PWK_ALT


title: RNA_EPI.10_CAST_WM080

value: CAST/EiJ


title: RNA-seq in ADNP WT mESC, replicate 3

description: Library name: ADNPwt_RNAseq_r3


title: RNA_DE.R_DBA_55


title: RNA_DE.L_C57B6_5.1


title: RNA_DE.I_PWK_AC698


title: RNA_EPI.15_WSB_AC627


title: RNA_EPI.14_PWK_AC695


title: RNA_EPI.12_C57B6_2


title: RNA_EPI.11_CAST_WM078


title: RNA_DE.Q_C57B6_1


title: RNA_DE.N_PWK_ALT


title: RNA_DE.M_C57B6_4


title: RNA_DE.X_NZO_AC653


title: RNA_DE.K_WSB_AC635


title: RNA_DE.J_NOD_AC576


title: RNA_DE.E_CAST_WM078


title: iCell-RPE, AgeD, experiment 4

status: Public on Jun 20 2025

city: Aachen

first: Jacopo

last: Di Russo

address: Pauwelsstraße 30

country: Germany

institute: Universitätsklinikum Aachen, AöR

description:
Library name: AgeD-iRPE 4
Sample name in salmon.merged.gene_counts.csv: DE55NGSUKBR144392

data_processing:
The raw data was processed using the nf-core/rnaseq v3.12.0 (https://doi.org/10.5281/zenodo.1400710)
Sequence reads were quality checked using FastQC before and after adapter trimming.
Adapter sequences and low-quality bases were trimmed using Cutadapt (3' adapter: AAAAAAAA).
Trimmed reads were aligned to the GENCODE GRCh38 v44 genome using STAR (key parameters: max intron size 1,000,000; max mismatches 10%; max multimaps 20).
Alignment quality was assessed using Samtools, RSeQC, and QualiMap.
Gene-level quantification was performed using featureCounts (grouped by gene_type).
Assembly: GRCh38 (GENCODE v44)
Supplementary files format and content: .csv file contains raw gene counts for all samples

source_name: iCell-RPE (Fujifilm 01279)

growth_protocol: HiPSCs-derived RPE cells (Fujifilm Cellular Dynamics, R1102) were seeded on 4 kPa polyacrylamide hydrogels with a basement membrane-like coating and were grown for 10 days in a medium recommended by the manufacturer.

extract_protocol:
RNA isolation was performed using the RNeasy Micro RNA isolation kit (Qiagen, 74004) with DNase treatment. 100 ng RNA was used for the library preparation.
RNA libraries for RNAseq were prepared using the QuantSeq 3’-mRNA Library Prep/ Lexogen enrichment.

treatment_protocol: On day 10 after seeding, AAV5 were used at 7x106 infectious units per cell for 24 h to transduce the cells with FKBP-casp8 containing plasmid (VectorBuilder, VB230127-1043eaq) in addition to a non-transduced control. On day 15, FKBP-casp8 was dimerised by addition of 100 nM AP20187 in medium (Sigma Aldrich, SML2838) for 24 h. Paired non-transduced cells  were also subjected to the AP20187 treatment. The RNA isolation was performed on day 20, or four days after apoptosis treatment finalisation.

value: iCell-RPE (Fujifilm 01279)

value: retinal pigment epithelial cells

value: apoptosis via 100 nM AP20187


title: RNA_DE.H_PWK_AC695


title: RNA_DE.C_PWK_AC699


title: VPSP-ASD-BA9

status: Public on Jun 17 2025

first: Nasser

last: Elhajjaoui

address: 695 Charles E Young Dr S

department: Human Genetics

institute: University of California, Los Angeles

description: Library name: Sample 49

data_processing:
We aligned the snmCT-seq data to the GENCODE v40 reference (GRCh38.p13 genome and genic annotation files), building upon the settings described extensively in the flagship manuscript for snmCT-seq1. Core changes are the implementation of paired-end alignment and quantification methods, increasing mapping rate and coverage and ensuring that read 1 and read 2 features are not quantified twice when insert size is low. Additionally, we add read deduplication to the RNA reads. Detailed descriptions are available on the pipeline git repository (github.com/chooliu/snmCTseq_Pipeline, release v1.1.0) and summarized below.
We used cell barcodes specific to each reaction well (of 384-well plate) to demultiplex sequence reads into individual nuclei with custom scripts, then trimmed Phred score base calls, Illumina adapter sequences and components specific to snmCT-seq library (the well-barcode, adaptase tail) using fastp2 v0.23.2 (-f 17 -t 10 -F 15 -T 10 -l 30 --cut_right -q 20 -u 50 -y -Y 15 -x --adapter_fasta).
Methylome reads for each nuclei are then mapped using Bismark3 v0.24.0 in a two-stage process in order to increase mapping efficiency for PBAT-based reactions and other single-cell methylome preparations4: (i) first, reads are mapped in paired-end mode, then (ii) reads unable to map in stage i and singleton reads are mapped in single-end mode. We define singletons as reads without its pair passing trimming quality control. The --maxins 2000 and --pbat (for paired-end and read 1) flags are used, with default settings otherwise. PCR and sequencer optical duplicates (pixel distance 2500) are then removed using Picard MarkDuplicates5. Reads with mapping score ≤ 10 were excluded (samtools view -h -q 10; samtools6 v1.16.1). A core innovation of the snmCT-seq bench workflow is its simultaneous profiling of RNA and DNA in one reaction well without requiring their physical separation; instead, we perform in silico separation of fully methylated RNA libraries introduced by methylated dNTPs (resulting in full methylation, including at CpH- contexts) from the low mCH/CH levels present in libraries originating from genomic DNA. Using the “XM:Z” alignment flag, we then therefore classified the Bismark alignments containing at least three CpH-context cytosines and mCH/CH ≤ 0.50 as DNA reads. Finally, the final resultant DNA reads were used to infer cytosine-level methylation states: for each cytosine c, we tabulated the number of alignments suggesting that the cytosine is methylated (mc; C>T bisulfite conversion) versus unmethylated (uc; no conversion) using allcools7 v1.0.8 (“.allc” file, github.com/lhqing/ALLCools).
In parallel, RNA-originating reads were mapped with STAR8 v2.7.10a, also using the two-stage (paired-end, followed by single-end) mapping procedure for RNA quantification. Non-default mapping settings included --alignEndsType EndToEnd --sjdbOverhang 149 --outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 –outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.04 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 as established previously. Alignments that were duplicates identified by Picard or had mapping score ≤ 10 were excluded. To retain RNA reads, each alignment with at least three CpH- context cytosines and mCH/CH ≥0.90 based on the “MD:Z” flag were retained. Finally, RNA counts of each gene were then quantified using featureCounts9.
We re-validated the performance of the in silico separation of RNA and DNA in this paired-end workflow by using three datasets generated with the related, DNA methylation-only array10 (snmC-seq2). Using existing data of this DNA-only assay profiling similar nuclei (BA10 cortex from 25 year old male; Sequence Read Archive SRR6911760, SRR6911772, SRR6911776), we again found high in silico discrimination between RNA and DNA based on alignment mCH/CH. When the RNA pipeline (STAR 🡪 mCH/CH filtering) was applied to these DNA-only reads, the majority (98.9%) of these bisulfite-converted DNA-only reads failed to align to the genome, and only 0.09% of reads were ultimately incorrectly classified as RNA reads and 0.0089% were quantified as transcript counts using featureCounts. With the DNA pipeline (Bismark 🡪 mCH/CH filtering), 0.075% of reads were incorrectly classified as RNA reads and discarded.
Assembly: hg38
Supplementary files format and content: tab delimited text files of methylcytosine calls (CGN)
Supplementary files format and content: tab delimited text files of methylcytosine calls (CAN)
Supplementary files format and content: tab delimited pseudo-bulk count matrix of genes (row names) by cell-type (column names)
Supplementary files format and content: tab delimited text file of age-related differentially-methylated regions

source_name: brodmann area 9

extract_protocol:
Nuclei were isolated from postmortem human cortical tissue (Brodmann area 9) stored at −80°C. Tissue was pulverized in liquid nitrogen using a pre-chilled mortar and pestle, and resuspended in NIBT buffer (250 mM sucrose, 10 mM Tris-Cl pH 8.0, 25 mM KCl, 5 mM MgCl₂, 0.1% Triton X-100, 1 mM DTT, protease inhibitors, and RNase inhibitors). Lysates were homogenized in a Dounce homogenizer (40 strokes loose and tight pestles), mixed with 20% iodixanol, and overlaid on a 25% iodixanol cushion. Nuclei were pelleted by centrifugation at 10,000×g at 4°C for 20 minutes. Nuclei were resuspended in DPBS supplemented with RNase inhibitors, stained with Hoechst 33342 (2 μM), blocked with 5 mg/mL BSA, and labeled with anti-NeuN AlexaFluor-488 antibody (clone A60, MilliporeSigma MAB377XMI). Nuclei were sorted into 384-well plates containing reverse transcription reagents and stored on ice until library preparation.
Single-nucleus libraries were prepared using the snmCT-seq and snm3C-seq3 protocols (PMID: 35419551, protocols.io [https://www.protocols.io/view/snmcat-v2-x54v9jby1g3e/v2] and [https://www.protocols.io/view/snm3c-seq3-kqdg3x6ezg25/v1]). Reverse transcription included Superscript II RT, 5mM DTT, 0.1% Triton X-100, dNTPs, a biotinylated oligo-dT primer (dT30VN_4_5bio), random primer (N6_2), and template-switching oligo (TSO_4_5bio), with RNase inhibitors. Following RT, cDNA was amplified with KAPA2G polymerase for 14–16 cycles depending on neuronal identity. Unincorporated oligos were digested using uracil DNA glycosylase. Bisulfite conversion was performed with Zymo EZ DNA Methylation-Direct kit reagents, followed by multiple rounds of clean-up with M-Binding and M-Wash Buffers. Libraries were prepared using random priming, exonuclease digestion, SPRI bead clean-up, Adaptase-mediated end repair and ligation, and final indexing with KAPA HiFi HotStart ReadyMix and custom indexing primers. Libraries were purified and quantified using the Qubit dsDNA BR Assay Kit. Sequencing was performed using the Illumina NovaSeq 6000 with S4 flowcells in 100 bp paired-end mode.

value: brodmann area 9

tag: donor

value: VPSP

tag: donor alt

tag: Sex

value: male

tag: genome build

tag: diagnosis

value: ASD

tag: age

tag: age category

value: Mid

tag: age detailed

tag: brainbank

value: ABN

tag: pmi

tag: rin


title: iCell-RPE, Ctrl, experiment 1

description:
Library name: iRPE 1
Sample name in salmon.merged.gene_counts.csv: DE61NGSUKBR144381

tag: cell line

tag: cell type

value: WT


title: RNA_DE.F_C57B6_2


title: iCell-RPE, Ctrl, experiment 4

description:
Library name: iRPE 4
Sample name in salmon.merged.gene_counts.csv: DE12NGSUKBR144390


title: iCell-RPE, AgeD, experiment 1

description:
Library name: AgeD-iRPE 1
Sample name in salmon.merged.gene_counts.csv: DE07NGSUKBR144383


title: RNA_DE.G_C57B6_44


title: RNA_DE.B_NZO_AC671


title: iCell-RPE, AgeD, experiment 3

description:
Library name: AgeD-iRPE 3
Sample name in salmon.merged.gene_counts.csv: DE39NGSUKBR144389


title: PH2A-CTL-BA9

description: Library name: Sample 47

tag: tissue

value: CTL


title: RNA_DE.D_NOD_AC595

tag: genotype

value: NOD/ShiLtJ


title: iCell-RPE, AgeD, experiment 2

description:
Library name: AgeD-iRPE 2
Sample name in salmon.merged.gene_counts.csv: DE23NGSUKBR144386

tag: treatment


title: PZ65-CTL-BA9

description: Library name: Sample 48


title: HQ4T-CTL-BA9

description: Library name: Sample 42


title: NEPX-ASD-BA9

description: Library name: Sample 45

value: NEPX

value: Young


title: EUFW-CTL-BA9

description: Library name: Sample 39

value: EUFW


title: iCell-RPE, Ctrl, experiment 2

description:
Library name: iRPE 2
Sample name in salmon.merged.gene_counts.csv: DE77NGSUKBR144384


title: KMC3-ASD-BA9

description: Library name: Sample 44

value: Old


title: iCell-RPE, Ctrl, experiment 3

description:
Library name: iRPE 3
Sample name in salmon.merged.gene_counts.csv: DE93NGSUKBR144387


title: E4FN-CTL-BA9

description: Library name: Sample 38


title: 9714-ASD-BA9

description: Library name: Sample 36

value: Harvard-ATP


title: Hct002-CTL-BA9

description: Library name: Sample 43

value: UM-BTB

tag: ancestry

value: White


title: FXMW-ASD-BA9

description: Library name: Sample 40

tag: donor

value: FXMW

value: FXMN


title: CQ56-CTL-BA9

description: Library name: Sample 37

value: brodmann area 9

tag: donor alt


title: 6041-ASD-BA9

description: Library name: Sample 32

tag: Sex

value: male


title: PA3P-ASD-BA9

description: Library name: Sample 46

tag: genome build

value: ASD


title: 75QW-CTL-BA9

description: Library name: Sample 34

tag: diagnosis


title: 5969-ASD-BA9

description: Library name: Sample 30

tag: age


title: G7YU-CTL-BA9

description: Library name: Sample 41

tag: age category


title: 6096-CTL-BA9

description: Library name: Sample 33

value: Mid

tag: age detailed


title: 5978-ASD-BA9

description: Library name: Sample 31

tag: brainbank


title: 5917-CTL-BA9

description: Library name: Sample 29

tag: pmi


title: 5841-ASD-BA9

description: Library name: Sample 26

tag: rin


title: 5577-CTL-BA9

description: Library name: Sample 25


title: 5294-ASD-BA9

description: Library name: Sample 16


title: 5842-ASD-BA9

description: Library name: Sample 27


title: 5565-ASD-BA9

description: Library name: Sample 23

value: Black


title: 5408-CTL-BA9

description: Library name: Sample 21


title: 5576-CTL-BA9

description: Library name: Sample 24


title: 8XCF-ASD-BA9

description: Library name: Sample 35

value: ABN


title: 5558-CTL-BA9

description: Library name: Sample 22

value: CTL


title: 5302-ASD-BA9

description: Library name: Sample 17


title: 4670-CTL-BA9

description: Library name: Sample 12


title: 5308-ASD-BA9

description: Library name: Sample 18


title: 4337-CTL-BA9

description: Library name: Sample 11


title: 3HUF-ASD-BA9

description: Library name: Sample 8


title: 5360-CTL-BA9

description: Library name: Sample 19


title: 3152-CTL-BA9

description: Library name: Sample 6


title: 4332-CTL-BA9

description: Library name: Sample 9


title: 5115-ASD-BA9

description: Library name: Sample 13


title: 5144-ASD-BA9

description: Library name: Sample 14


title: 5403-ASD-BA9

description: Library name: Sample 20


title: 5242-CTL-BA9

description: Library name: Sample 15


title: 4334-ASD-BA9

description: Library name: Sample 10


title: 5895-CTL-BA9

description: Library name: Sample 28


title: 2GGP-CTL-BA9

description: Library name: Sample 4


title: 285A-ASD-BA9

description: Library name: Sample 3


title: 13161-ASD-BA9

description: Library name: Sample 2


title: 00493-ASD-BA9

description: Library name: Sample 1


title: D2_G12_RNAseq

status: Public on May 31 2025

city: Cambridge

first: Laura

last: Biggins

address: Babraham Research Campus

department: Bioinformatics

country: United Kingdom

institute: The Babraham Institute

description: 2i_release_NMTSeq_RNAseq_filtered_normalized.txt

data_processing:
All sequencing was carried out on HiSeq instrument. RNA-seq libraries were pooled as 96-plex or 192-plex pools and sequenced using 75-bp paired-end reads.
scRNA-seq alignments were performed using Hisat 2 as described previously (Clark et al. Nature Communications, 2018).
Genome_build: GRCm38
Supplementary_files_format_and_content: Comma-delimited file with Ensembl Gene IDs, quantified values show number of reads per gene per sample

growth_protocol: E14 mESC were cultured in 2i+LIF media consisting out of N2B27 medium, composed out of DMEM/F12 (Life Technologies, 12634010) and Neurobasal (Life Technologies, 21103049) in a volume/volume (v/v) ratio 1:1, 0.1 mM 2-mercaptoethanol, 2 mM L-Glutamine, 1:200 v/v N2 (Life Technologies, 17502048) and 1:100 v/v B27 supplement (Life Technologies, 17504044), supplemented with 3 µM CHIR99021, 1 µM PD0325901 and 20 ng/ml of leukemia inhibitory factor (LIF)  (all Department of Biochemistry, University of Cambridge). mESCs were cultured on tissue culture plastic pre-coated with 0.1 % gelatine in H2O. Cultures were passaged when approaching confluence (every 2 days). All cells were cultured in a humidified incubator at 37 °C in 5 % CO2 and 20 % O2. mESCs were subject to routine mycoplasma testing using the MycoAlert testing kit (Lonza) and tested negative. For induction for 2i-relase experiment, the cell been rinsed with PBS before the culture media was changed to serum + LIF medium consisting of DMEM (Life Technologies, 10566-016), containing 15% Fetal Bovine Serum (FBS) (Gibco, 10270106), 1x non-essential amino acids (NEAA) (Life Technologies, 11140050), 0.1 mM 2-mercaptoethanol (Life Technologies, 31350-010), 2 mM L-Glutamine (Life Technologies, 25030-024), supplemented with 20 ng/ml of LIF (Department of Biochemistry, University of Cambridge).

extract_protocol:
0h, 24h, and 48h after induction of 2i-release  the cells been dissociated into single cells using accutase before flow sorting  (BD Influx) into 96-well plate containing 2.5μl of methylase reaction buffer (1 × M.CviPI Reaction buffer (NEB), 2 U M.CviPI (NEB), 160 μM S-adenosylmethionine (NEB), 1 U μl−1 RNasein (Promega), 0.1% IGEPAL CA-630 (Sigma)). Samples were incubated for 15 min at 37 °C to methylate accessible chromatin before the reaction was stopped with the addition of RLT plus buffer (Qiagen) and samples frozen down and stored at −80 °C before processing.
All downstream library preparation steps been performed as previously described by Clark et al. (Clark et al. Nature Communications, 2018).

tag: strain

value: embryonic stem cell


title: D2_H06_RNAseq

tag: cell type


title: D2_H10_RNAseq


title: D2_H03_RNAseq


title: D2_G05_RNAseq


title: D2_G08_RNAseq


title: D2_G10_RNAseq


title: D2_H12_RNAseq


title: D2_H09_RNAseq


title: D2_H11_RNAseq


title: D2_H02_RNAseq


title: D2_G06_RNAseq


title: D2_H07_RNAseq


title: D2_H04_RNAseq


title: D2_G09_RNAseq


title: D2_G07_RNAseq


title: D2_G11_RNAseq


title: D2_H05_RNAseq


title: D2_G01_RNAseq


title: D2_H01_RNAseq


title: D2_G03_RNAseq


title: D2_G04_RNAseq


title: D2_G02_RNAseq


title: D2_F12_RNAseq


title: D2_F11_RNAseq


title: D2_F06_RNAseq


title: D2_F01_RNAseq


title: D2_E10_RNAseq


title: D2_F02_RNAseq


title: D2_E08_RNAseq


title: D2_E06_RNAseq


title: D2_E01_RNAseq


title: D2_F05_RNAseq


title: D2_F10_RNAseq


title: D2_F04_RNAseq


title: D2_F09_RNAseq


title: D2_F07_RNAseq


title: D2_E07_RNAseq


title: D2_E11_RNAseq


title: D2_E04_RNAseq


title: D2_E09_RNAseq


title: D2_D11_RNAseq


title: D2_D12_RNAseq


title: D2_E02_RNAseq


title: D2_E03_RNAseq


title: D2_F08_RNAseq


title: D2_E12_RNAseq


title: D2_F03_RNAseq


title: D2_D10_RNAseq


title: D2_D09_RNAseq


title: D2_D08_RNAseq


title: D2_D02_RNAseq


title: D2_D07_RNAseq


title: D2_D03_RNAseq


title: D2_C12_RNAseq


title: D2_D04_RNAseq


title: D2_C05_RNAseq


title: D2_C07_RNAseq


title: D2_B11_RNAseq


title: D2_C08_RNAseq


title: D2_C02_RNAseq


title: D2_D06_RNAseq


title: D2_C11_RNAseq


title: D2_B12_RNAseq


title: D2_D01_RNAseq


title: D2_C09_RNAseq


title: D2_C06_RNAseq


title: D2_C04_RNAseq


title: D2_C01_RNAseq


title: D2_C10_RNAseq


title: D2_C03_RNAseq


title: D2_D05_RNAseq


title: D2_B09_RNAseq


title: D2_B05_RNAseq


title: D2_B07_RNAseq


title: D2_B04_RNAseq


title: D2_B08_RNAseq


title: D2_B02_RNAseq


title: D2_A03_RNAseq


title: D2_B10_RNAseq


title: D1_H12_RNAseq


title: D2_A07_RNAseq


title: D2_A12_RNAseq


title: D2_A08_RNAseq


title: D2_B01_RNAseq


title: D2_A01_RNAseq


title: D2_B03_RNAseq


title: D2_B06_RNAseq


title: D2_A04_RNAseq


title: D2_A10_RNAseq


title: D1_H11_RNAseq


title: D2_A06_RNAseq


title: D2_A11_RNAseq


title: D2_A05_RNAseq


title: D2_A02_RNAseq


title: D2_A09_RNAseq


title: D1_H03_RNAseq


title: D1_H01_RNAseq


title: D1_H02_RNAseq


title: D1_G08_RNAseq


title: D1_G05_RNAseq


title: D1_H08_RNAseq


title: D1_G04_RNAseq


title: D1_H07_RNAseq


title: D1_H04_RNAseq


title: D1_G12_RNAseq


title: D1_H06_RNAseq


title: D1_G06_RNAseq


title: D1_H09_RNAseq


title: D1_G11_RNAseq


title: D1_G10_RNAseq


title: D1_G03_RNAseq


title: D1_H10_RNAseq


title: D1_G09_RNAseq


title: D1_H05_RNAseq


title: D1_G02_RNAseq


title: D1_G01_RNAseq


title: D1_F12_RNAseq


title: D1_G07_RNAseq


title: D1_F09_RNAseq


title: D1_E12_RNAseq


title: D1_F05_RNAseq


title: D1_F04_RNAseq


title: D1_F01_RNAseq


title: D1_E08_RNAseq


title: D1_F08_RNAseq


title: D1_E09_RNAseq


title: D1_E07_RNAseq


title: D1_F06_RNAseq


title: D1_F03_RNAseq


title: D1_F11_RNAseq


title: D1_E06_RNAseq


title: D1_F02_RNAseq


title: D1_F07_RNAseq


title: D1_E05_RNAseq


title: D1_E04_RNAseq


title: D1_D10_RNAseq


title: D1_E03_RNAseq


title: D1_E01_RNAseq


title: D1_D11_RNAseq


title: D1_C12_RNAseq


title: D1_C11_RNAseq


title: D1_D12_RNAseq


title: D1_D01_RNAseq


title: D1_D03_RNAseq


title: D1_D09_RNAseq


title: D1_D08_RNAseq


title: D1_D06_RNAseq


title: D1_D02_RNAseq


title: D1_D05_RNAseq


title: D1_D04_RNAseq


title: D1_D07_RNAseq


title: D1_C08_RNAseq


title: D1_C06_RNAseq


title: D1_B11_RNAseq


title: D1_B08_RNAseq


title: D1_C09_RNAseq


title: D1_C07_RNAseq


title: D1_B05_RNAseq


title: D1_C03_RNAseq


title: D2_H08_RNAseq


title: D1_C01_RNAseq


title: D1_B12_RNAseq


title: D1_C04_RNAseq


title: D1_C05_RNAseq


title: D1_B10_RNAseq


title: D1_B06_RNAseq


title: D1_C02_RNAseq


title: D1_B04_RNAseq


title: D1_B03_RNAseq


title: D1_B09_RNAseq


title: D1_A09_RNAseq


title: D1_B02_RNAseq


title: D1_A07_RNAseq


title: D1_A11_RNAseq


title: D1_A12_RNAseq


title: D1_B01_RNAseq


title: D1_A10_RNAseq


title: D1_A05_RNAseq


title: D1_A08_RNAseq


title: D1_A06_RNAseq


title: D0_H11_RNAseq


title: D1_A02_RNAseq


title: D1_A04_RNAseq


title: D1_A01_RNAseq


title: D0_H10_RNAseq


title: D0_H12_RNAseq


title: D0_H09_RNAseq


title: D0_H08_RNAseq


title: D1_F10_RNAseq


title: D0_H05_RNAseq


title: D0_H07_RNAseq


title: D0_H04_RNAseq


title: D0_H03_RNAseq


title: D0_H06_RNAseq


title: D0_H01_RNAseq


title: D0_G10_RNAseq


title: D0_G03_RNAseq


title: D0_G01_RNAseq


title: D0_G11_RNAseq


title: D0_G09_RNAseq


title: D0_G12_RNAseq


title: D0_G07_RNAseq


title: D0_G08_RNAseq


title: D0_G05_RNAseq


title: D0_G02_RNAseq


title: D0_H02_RNAseq


title: D0_G06_RNAseq


title: D0_F04_RNAseq


title: D0_F09_RNAseq


title: D0_F10_RNAseq


title: D0_F01_RNAseq


title: D0_F11_RNAseq


title: D0_E09_RNAseq


title: D0_F02_RNAseq


title: D0_E11_RNAseq


title: D0_E12_RNAseq


title: D0_E08_RNAseq


title: D0_G04_RNAseq


title: D0_F06_RNAseq


title: D0_F12_RNAseq


title: D0_E07_RNAseq


title: D0_E02_RNAseq


title: D0_E05_RNAseq


title: D0_E04_RNAseq


title: D0_E03_RNAseq


title: D0_E01_RNAseq


title: D0_E06_RNAseq


title: D0_D09_RNAseq


title: D0_D07_RNAseq


title: D0_D12_RNAseq


title: D0_D10_RNAseq


title: D0_D08_RNAseq


title: D0_D06_RNAseq


title: D0_F05_RNAseq


title: D0_F07_RNAseq


title: D0_F08_RNAseq


title: D0_C09_RNAseq


title: D0_C12_RNAseq


title: D0_D05_RNAseq


title: D0_C10_RNAseq


title: D0_D01_RNAseq


title: D0_D04_RNAseq


title: D0_D03_RNAseq


title: D0_C04_RNAseq


title: D0_D02_RNAseq


title: D0_C11_RNAseq


title: D0_C03_RNAseq


title: D0_C01_RNAseq


title: D0_B12_RNAseq


title: D0_C08_RNAseq


title: D0_C05_RNAseq


title: D0_C07_RNAseq


title: D0_B08_RNAseq


title: D0_C02_RNAseq


title: D1_E10_RNAseq


title: D0_C06_RNAseq


title: D0_B11_RNAseq


title: D0_B10_RNAseq


title: D0_B09_RNAseq


title: D0_B07_RNAseq


title: D0_B04_RNAseq


title: D0_A08_RNAseq


title: D0_B02_RNAseq


title: D0_B01_RNAseq


title: D0_A11_RNAseq


title: D0_B03_RNAseq


title: D0_A09_RNAseq


title: D0_A06_RNAseq


title: D0_A07_RNAseq


title: D0_A12_RNAseq


title: D0_A04_RNAseq


title: D0_A05_RNAseq


title: D0_A10_RNAseq


title: D0_A03_RNAseq


title: D0_A02_RNAseq


title: D0_A01_RNAseq


title: D2_H11

data_processing:
All sequencing was carried out on HiSeq instrument. BS-seq libraries were sequenced in 96-plex pools using 125-bp paired-end reads. RNA-seq libraries were pooled as 96-plex or 192-plex pools and sequenced using 75-bp paired-end reads.
Alignments of the single-cell bisulfite sequencing were performed using Bismark as well as subsequent CpG methylation and GpC accessibility calling.
Genome_build: GRCm38
Supplementary_files_format_and_content: The Bismark CpG coverage report is tab-delimited, uses 1-based genomic coordinates for every covered cytosine position in the experiment and is in the following format: <chromosome> <start position> <end position> <methylation percentage> <count methylated> <count non-methylated>

extract_protocol:
0h, 24h, and 48h after induction of 2i-release  the cells been dissociated into single cells using accutase before flow sorting  (BD Influx) into 96-well plate containing 2.5μl of methylase reaction buffer (1 × M.CviPI Reaction buffer (NEB), 2 U M.CviPI (NEB), 160 μM S-adenosylmethionine (NEB), 1 U μl−1 RNasein (Promega), 0.1% IGEPAL CA-630 (Sigma)). Samples were incubated for 15 min at 37 °C to methylate accessible chromatin before the reaction was stopped with the addition of RLT plus buffer (Qiagen) and samples frozen down and stored at −80 °C before processing.
E14 mESC were cultured in 2i+LIF media. For induction for 2i-relase experiment, the cell been rinsed with PBS before the culture media was changed to serum + LIF medium. 0h, 24h, and 48h after induction of 2i-release single cells were distracted and sc-NMT sequencing was performed.


title: D2_H12


title: D2_H10


title: 35BV-ASD-BA9

description: Library name: Sample 7

tag: tissue


title: 2NA6-ASD-BA9

description: Library name: Sample 5


title: D2_H01


title: D2_H05


title: D2_H06


title: D2_H08


title: D2_H09


title: D2_H04


title: D2_H07


title: D2_H02


title: D2_H03


title: D1_B07_RNAseq


title: D2_G10


title: D2_G09


title: D2_G06


title: D2_F12


title: D2_G08


title: D2_G04


title: D2_G05


title: D2_G07


title: D2_G03


title: D2_F09


title: D2_G12


title: D2_G01


title: D2_F11


title: D2_F08


title: D2_G02


title: D2_F07


title: D2_F03


title: D2_F06


title: D2_F04


title: D2_F05


title: D2_E09


title: D2_E11


title: D2_E08


title: D2_F02


title: D2_E12


title: D2_E06


title: D2_E10


title: D2_E07


title: D2_E05


title: D2_E04


title: D2_E02


title: D2_E03


title: D2_D06


title: D2_D07


title: D2_D04


title: D2_D03


title: D2_D08


title: D2_D05


title: D2_D10


title: D2_D12


title: D2_E01


title: D2_C12


title: D2_C09


title: D2_D02


title: D2_C05


title: D2_C10


title: D2_C11


title: D2_D01


title: D2_C04


title: D2_C08


title: D2_C06


title: D2_C07


title: D2_C03


title: D0_D11_RNAseq


title: D2_C02


title: D2_B09


title: D2_B12


title: D2_C01


title: D2_B08


title: D2_B10


title: D2_B11


title: D2_B07


title: D2_B06


title: D2_B05


title: D2_B04


title: D2_B02


title: D2_B01


title: D2_B03


title: D2_A11


title: D2_A12


title: D2_A10


title: D2_A09


title: D2_A08


title: D2_A07


title: D2_A03


title: D2_A02


title: D2_A05


title: D1_H12


title: D2_A04


title: D1_H10


title: D2_A01


title: D1_H11


title: D1_H09


title: D2_A06


title: D1_H08


title: D1_H06


title: D1_H05


title: D1_E11_RNAseq


title: D1_H04


title: D1_H03


title: D0_B06_RNAseq


title: D1_H02


title: D1_G07


title: D1_H07


title: D1_G10


title: D1_G11


title: D1_G12


title: D1_G05


title: D1_H01


title: D1_G09


title: D0_B05_RNAseq


title: D1_G04


title: D1_G03


title: D1_G08


title: D1_G02


title: D1_G01


title: D1_F12


title: D1_F07


title: D1_F08


title: D1_F02


title: D1_E12


title: D1_F05


title: D1_F09


title: D1_F10


title: D1_F04


title: D1_F01


title: D1_F06


title: D1_E11


title: D1_F03


title: D1_E09


title: D1_E08


title: D1_E04


title: D1_E05


title: D1_E02


title: D1_D10


title: D1_D11


title: D1_E06


title: D1_E01


title: D1_D08


title: D1_D12


title: D1_E07


title: D2_F01


title: D1_E03


title: 3-week retina, miR-9-2 knockout, biol rep 3

status: Public on Apr 01 2026

city: Seattle

first: Timothy

middle: Joel

last: Cherry

state: WA

department: Pediatrics

institute: University of Washington/Seattle Children's Research Institute

description:
Library name: KO3WF1
10X Genomics
wk3.rds

data_processing:
Cell Ranger from 10X Genomics was used align sequencing reads, demultiplex samples, and generate feature-barcode matrices. Raw feature-barcode matrices from Cell Ranger were input into Cellbender to remove ambient RNA and generate filtered feature-barcode matrices. Resulting matrices were used as input into Seurat snRNA-sequencing analysis pipeline.
Assembly: mm10
Supplementary files format and content: .h5; filtered count matrix output from Cellbender processing
Supplementary files format and content: .rds; individual Seurat objects for each timepoint, including RNA/SCT-normalized assays, annotated celltypes, and PCA/UMAP/HarmonyUMAP dimensional reductions.

molecule: nuclear RNA

source_name: Retina

extract_protocol:
Nuclei were isolated from flash frozen retinal tissue by adapting the 10X Protocol for Nuclei Isolation from Mouse Brain Tissue for Single Cell ATAC Sequencing. Briefly, for modifications, tissue was broken up by dounce before transfer to tube, and digitonin was not included in buffers for samples used with the Chromium Next GEM Single Cell 3ʹ v3.1 kit.
Single-nuclei RNA-sequencing libraries were constructed according to 10X Genomics protocols for the  Chromium Next GEM Single Cell 3ʹ v3.1 kit (E16.5, P0, enhancer KO) or the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression (3-weeks), targeting 10,000 cells.

treatment_protocol: miR-9-2 knockout mice were generated by crossing Mir9-2 tm1Mtm/Mmjax mice with ROSA26::FLPe knock in and E2a-Cre mice to create a animals with a constitutive germline knockout of the miR-9-2 hairpin loop. miR-9-2 enhancer KO mice were generated by CRISPR targeting a ~300bp region corresponding to accessibility for the putative miR-9-2 enhancer.

value: Retina

tag: genotype

value: miR-9-2 knockout

tag: developmental stage

value: 3 weeks

value: female

tag: batch


title: D1_D03


title: D1_D06


title: D1_D05


title: D1_D04


title: D1_D09


title: D1_D07


title: D2_D09


title: D1_D02


title: D1_D01


title: D1_C12


title: D1_C07


title: D1_C09


title: D1_C03


title: D1_C08


title: D1_C11


title: D1_C06


title: D1_C05


title: D1_C02


title: D1_B12


title: D1_C01


title: D1_C04


title: D1_B10


title: D1_B11


title: D1_B09


title: D1_C10


title: D1_B08


title: D1_B03


title: D1_B04


title: D1_A09


title: D1_B06


title: D1_B05


title: D1_A12


title: D1_A07


title: D1_A08


title: D1_A10


title: D1_B01


title: D1_B02


title: D1_B07


title: D1_A05


title: D1_A06


title: D1_A11


title: D0_H04


title: D1_A04


title: D1_A03


title: D0_H12


title: D0_H09


title: D0_H06


title: D1_A02


title: D1_A01


title: D0_H11


title: D0_H07


title: D0_H05


title: D0_H10


title: D0_H08


title: HAGLR_8h_rep1_RNAseq

city: Boulder

first: Mingfeng

last: Liu

state: CO

address: 709 Mohawk Dr

institute: University of Colorado

description: Library name: HAGLR_8_1

data_processing:
Quality control, read mapping, and quantification were performed using nf-core/rnaseq v3.14.0 (Ewels et al. 2020).
Assembly: The cleaned reads were aligned to the GRCh38.p13 human reference genome and quantified using GENCODE v38 annotations.
Supplementary files format and content: .csv, raw gene counts and tpm

source_name: hiPSCs

extract_protocol:
RNA was harvested using Directzol RNA Miniprep kit (ZymoResearch). RNA integrity and quantity were assessed using Agilent 2100 Bioanalyzer and NanoDrop.
Ribosomal RNA was depleted from total RNA using a commercial rRNA depletion kit. Strand-specific sequencing libraries were constructed following standard Illumina-compatible protocols. Library quality was assessed, and paired-end sequencing was performed on an Illumina platform. All library construction and sequencing steps were conducted by Novogene Co., Ltd.

tag: cell line

value: hiPSCs


title: D0_G09


title: D0_H01


title: D0_H02


title: D0_H03


title: D0_G11


title: D0_G08


title: D0_G05


title: D0_G06


title: D0_G10


title: D0_G07


title: D0_G04


title: D0_G12


title: D0_G03


title: D0_F11


title: D0_F09


title: D0_G01


title: D0_F05


title: D0_G02


title: D0_F03


title: D0_F12


title: D0_F08


title: D0_F10


title: D0_F04


title: D0_F07


title: D0_F06


title: D0_F01


title: D0_E11


title: D0_E12


title: D0_F02


title: D0_E10


title: D0_E09


title: D0_E08


title: D0_E03


title: D0_E04


title: D0_E07


title: D0_E01


title: D0_E06


title: D0_D08


title: D0_E02


title: D0_D10


title: D0_D11


title: D0_D12


title: D0_E05


title: D0_D07


title: D0_D05


title: D0_D06


title: D0_D09


title: D0_D02


title: D0_D01


title: D0_D04


title: D0_C10


title: D2_E05_RNAseq


title: D0_C11


title: D0_C09


title: D0_C12


title: D0_C05


title: D0_C03


title: D0_C04


title: D0_C02


title: D0_C06


title: D0_B07


title: D0_B09


title: D0_C01


title: D0_B08


title: D0_B10


title: D0_B04


title: D0_B12


title: D0_B06


title: D0_B05


title: D0_B03


title: D0_B11


title: D0_C07


title: D0_A12


title: D0_A08


title: D0_A05


title: D0_A09


title: D0_A02


title: D0_A07


title: D0_A03


title: D0_D03


title: UOK342-TEAD1-rep1_S7

status: Public on May 28 2025

city: Baltimore

first: Danfeng

last: Cai

address: 615 N Wolfe St

institute: Johns Hopkins Bloomberg School of Public Health

description: Library name: UOK342-TEAD1-rep1_S7

data_processing:
ChIP-seq data were processed using the following pipeline. Raw sequencing reads in FASTQ format were quality-checked using FastQC. Reads were aligned to the human reference genome (hg38) using Bowtie2 (v2.3.5.3). The resulting BAM files were used to generate coverage tracks (bigWig format) using bamCoverage from the deepTools suite, without additional normalization.
Assembly: hg38
Supplementary files format and content: .bigwig file

source_name: kidney

extract_protocol:
The cells, washed in ice-cold PBS, were then lysed in cell lysis buffer, with subsequent nutation at 4°C. Cells were treated by dounce homogenization on ice, followed by centrifuge at 500 x g for 10min. The supernatant was discarded, and the pellets was resuspended using nuclear lysis buffer. Nuclear lysates were then sheared by Covaris E220 to achieve average chromatin length of 300-400 bp.
DNA was purified with ChIP DNA Clean & ConcentratorTM (ZYMO research, D5205) and library preparation was performed using NEBNext® Multiplex Oligos for Illumima®

value: kidney

value: patient-derived cell line

tag: antibody


title: D0_A04


title: D0_A01


title: D0_B02


title: UOK122-TEAD1-rep2_S16

description: Library name: UOK122-TEAD1-rep2_S16


title: UOK121-TEAD1-rep2_S27

description: Library name: UOK121-TEAD1-rep2_S27


title: HK2-TEAD1-rep2_S24

description: Library name: HK2-TEAD1-rep2_S24

source_name: kidney; Cortex; Proximal tubule

value: kidney; Cortex; Proximal tubule


title: UOK121-TEAD1-rep1_S5

description: Library name: UOK121-TEAD1-rep1_S5