title: RPE_donor4_3_deep_group144

data_processing:
Reads from FASTQ files were mapped using YAP (Yet Another Pipeline) software (cemba-data v1.6.9), as previously described (PMID:38092918). First, FASTQ files were demultiplexed for each cell barcode. Next, reads were assessed for quality, then two-pass mapping was performed with bismark (v0.20, with bowtie2 v2.3). BAM file processing and QC was performed using samtools (v1.9) and Picard (v3.0.0). Chromatin contacts were called and methylome profiles were generated using Allcools (v1.0.23). All reads were mapped to the hg38 genome assembly. We filtered for high quality cells based on DNA methylation by requiring mapping rate for both reads R1 and R2 > 0.5; total final reads > 100,000; overall mCCC level < 0.03; overall mCH level < 0.2; and overall mCG level > 0.5. Single-cell DNA methylome profiles were stored in the 'all cytosine' (ALLC) format, a tab-separated table compressed and indexed by bgzip/tabix. We used the generate-dataset command in the ALLCools package to generate a methylome cell-by-feature tensor dataset (MCDS) in Zarr format.
Assembly: hg38
Supplementary files format and content: YAP output files: 3C.contact.tsv.gz
Supplementary files format and content: YAP output files: mC.allc.tsv.gz, mC.allc.tsv.gz.tbi
Supplementary files format and content: bigWig files: Cell type-specific pseudo bulk DNA methylation signal track
Supplementary files format and content: hic files: Hi-C contact matrix
Supplementary files format and content: GSE277361_DMR_Retina.tsv: Cell type-specific DMRs of retina/macula tissues
Supplementary files format and content: GGSE277361_DMR_RPE.tsv: Cell type-specific DMRs of RPE tissue
Library strategy: snm3c-seq

source_name: RPE

extract_protocol:
The eye globes were collected from three unrelated human donors (aged 21, 27 and 43 years) with the collaboration of San Diego Eye Bank. The sample processing time starting from eye enucleation to dissection was restricted within 2 hours to identify the macula, a visible yellowish/whiteish indent that is slightly lateral to the optic disk inside the globe. Prior dissection, the eye globe with proper label was collected in a sterile moist chamber. At first the cornea was removed by cutting 2-3 mm around the limbus using a surgical blade. Pull off the iris and lens. This will leave you with the eye cup, which contains the posterior pole. After removing enough vitreous and aqueous fluid the temporal region can be located by two main blood vessels that curl around the macula. Using a sterile 3mm skin biopsy punch, the macula was collected. Sections were made for the other regions, temporal, nasal, superior and inferior. For all these regions, the retina and RPE tissue were separated and stored immediately in Liquid Nitrogen. Retina tissues were pulverized using a dounce tissue chamber and two pestles ("Loose" and "Tight") on ice for each sample. Pulverized retina tissues were resuspended in 1ml of chilled NIM-DP-L buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl pH 7.5, 1 mM DTT, 1x protease inhibitor (Pierce), 1U μl-1 recombinant RNase inhibitor (Promega, PAN2515) and 0.1% Triton X-100). Tissue was dounce homogenized with a loose pestle (5-10 strokes) followed by a tight pestle (15-25 strokes) or until the solution was uniform. Nuclei were filtered using a 30 μm CellTrics filter (Sysmex, 04-0042-2316) into a LoBind tube (Eppendorf, 22431021). Nuclei were pelleted (at 1000 rcf, for 10 min at 4°C; Eppendorf, 5920R) then resuspended in 1mL PBS with 2% formaldehyde for crosslinking for 10 minutes at room temperature on a rotator. 2.5M glycine was added to achieve 0.22M glycine and incubated for 5 minutes at room temperature on a rotator. Nuclei were pelleted 1000 rcf, for 10 min at 4°Cpelleted (at 1000 rcf, for 10 min at 4°C; Eppendorf, 5920R) then resuspended in 1mL of cold PBS. Nuclei were then pelleted at 2,500 x g for 5 min at 4°C. All supernatant was removed and pellets were snap frozen in liquid nitrogen and stored at -80°C.
In situ 3C treatment was performed during the nucleus preparation, enabling the capture of the chromatin conformation modality as described previously (PMID: 31501549). These steps were performed using the Arima-3C BETA Kit (Arima Genomics). The nuclei were isolated and sorted into 384-well plates using previous described methods (PMID: 34616061). Briefly, nuclei were stained with DRAQ7 (ThermoFisher Scientific, D15106) and then processed for fluorescence-activated nucleus sorting (FANS) using the Sony SH800 cell sorter with single-cell (1 drop single) mode. The snm3C-seq samples were prepared according to a previously described library preparation protocol (PMID: 31501549,34616061). This protocol has been automated using the Beckman Biomek i7 instrument to facilitate large-scale applications. The snm3C-seq libraries were shallow sequenced on the Illumina NovaSeq 2000 to 2-10M reads to check quality. Successful libraries were deeply sequenced on the NovaSeq X Plus instrument, using one lane of 25B flow cell per 4 384-well plates and using 150 bp paired-end mode.

value: RPE

tag: Sex

value: Male


title: RPE_donor4_2_deep_group138

status: Public on Jan 15 2025

type: SRA

city: La Jolla

first: Ying

last: Yuan

address: Palmilla Dr.

department: MATS

country: USA

institute: UCSD

library_source: genomic

molecule: genomic DNA

organism: Homo sapiens

tag: tissue

tag: age

tag: genotype

value: WT


title: RPE_donor4_3_deep_group143


title: RPE_donor4_2_deep_group139


title: RPE_donor3_3_deep_group132


title: RPE_donor4_3_deep_group141


title: RPE_donor4_3_deep_group142


title: RPE_donor3_3_deep_group130


title: RPE_donor4_2_deep_group137


title: RPE_donor4_1_deep_group135


title: RPE_donor3_3_deep_group131


title: NSCLC_PA070_tcr

status: Public on Jan 17 2025

city: New York

first: Somnath

last: Tagore

state: NY

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

institute: Columbia University

description: Single-nuclei RNA sequencing

library_source: transcriptomic

data_processing:
library strategy: single-cell RNAseq (TCR-seq)
Fastq files were analyzed using vdj in Cell Ranger (v6.1.1, 10x Genomics). Transcripts were aligned to the human GRCh38 reference genome.
Assembly: GRCh38
Supplementary files format and content: Cell Ranger vdj output files (csv)

molecule: total RNA

source_name: NSCLC brain metastasis

extract_protocol:
Single-nuclei suspension from frozen NSCLC metastases were generated using methods published in Slyper et al. (Nature Medicine, 2020) with modifications. The tissue was first cut in 20 µm sections using a cryostat and stored on dry ice until the next step. Then the tissue was put on wet ice and washed once with 5 ml PBS to remove OCT. After this step nuclei were extracted by vigorous pipetting in salt-tris buffer with Tween-20 (TST)  followed by a high-volume wash in salt-tris buffer (ST). Nuclei were counted and 15,000 nuclei were separated for droplet-based RNA capture and reverse transcription using 10x Genomics  5' v1.1 or 5' V2.0  reagents and a Chromium controller.
Chromium Single Cell V(D)J Enrichment Kit for human T cells Reagent Kit, v 1.1 or Single Cell Human TCR Amplification Kit were utilized for snTCR library generation (10X genomics) depending on whether 5' V1 or 5' V2 kits were utilized for cDNA library generation, respectively. Final sequencing libraries were prepared using Single Index Kit T Set A or Library construction kit (catalog no. 1000190) and Dual Index Kit TT set A (catalog no. 1000215).

tag: metastatic site

value: BM

tag: patient id

tag: stk11 mutation status

tag: kras mutation status

tag: sequencing technique

value: snRNA-seq

tag: tcr-seq

value: yes


title: RPE_donor4_1_deep_group133


title: RPE_donor3_2_deep_group128


title: RPE_donor4_1_deep_group136


title: RPE_donor4_1_deep_group134


title: RPE_donor3_3_deep_group129


title: D0_F03_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

molecule: polyA RNA

organism: Mus musculus

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

tag: cell type

value: embryonic stem cell


title: RPE_donor3_1_deep_group124


title: RPE_donor2_3_deep_group120


title: D0_E10_RNAseq


title: RPE_donor2_2_deep_group116


title: RPE_donor3_1_deep_group122


title: RPE_donor3_2_deep_group127


title: RPE_donor2_3_deep_group118


title: RPE_donor3_2_deep_group126


title: NSCLC_STK_18_tcr

description: Single-nuclei RNA sequencing

value: STK11 Q305X

value: KRAS Q61H


title: UOK342-TEAD1-rep2_S19

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-rep2_S19

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

tag: cell line

value: patient-derived cell line

tag: antibody


title: RPE_donor3_2_deep_group125


title: RPE_donor2_2_deep_group113


title: RPE_donor2_1_deep_group112


title: RPE_donor2_3_deep_group117


title: RPE_donor2_2_deep_group114


title: RPE_donor3_1_deep_group123


title: RPE_donor2_3_deep_group119


title: RPE_donor2_1_deep_group111


title: RPE_donor2_1_deep_group110


title: RPE_donor2_2_deep_group115


title: RPE_donor1_3_deep_group108


title: RPE_donor1_2_deep_group104


title: RPE_donor2_1_deep_group109


title: RPE_Donor1_1_deep_group100


title: RPE_donor1_3_deep_group105


title: RPE_donor1_2_deep_group103


title: RPE_Donor1_1_deep_group99


title: RPE_donor1_3_deep_group106


title: RPE_Donor1_1_deep_group97


title: resistant_rep3

first: Richard

middle: A

last: Young

state: MA

address: 9 Cambridge Center

institute: Whitehead Institute for Biomedical Research

description: rRNA depleted total RNA

data_processing:
Reads were mapped to the human genome GRCh38 using STAR aligner (v2.7.1a), allowing up to 100 multiple alignments and up to 200 loci anchors (--outFilterMultimapNmax 100 --winAnchorMultimapNmax 200).
Differential expression analysis of genes and transposable elements comparing triplicates of samples treated with normal or pathogenic concentrations of insulin was performed using TEtranscripts (v2.2.3).
Assembly: hg38
Supplementary files format and content: comma-separated values file contains differential expression data for annotated transcripts
Supplementary files format and content: cntTable.txt: raw counts
Supplementary files format and content: Rlog_annotated.csv: rlog-transformed counts

source_name: Liver

extract_protocol:
Cells were washed with EMEM and RNA was then purified using TRIzolTMreagent following manufacturer’s instructions.
RNA-seq libraries were prepared using a KAPA RNA HyperPrep kit with RiboErase. Spike-ins were included but were not used for analysis.

treatment_protocol: HepG2 cells were treated with 0.1nM (normal) or 3nM (pathogenic) insulin concentrations in EMEM 0.125% BSA for three days.

value: Liver

value: Hepatocellular carcinoma

tag: treatment

value: 3 nM insulin


title: Retina_donor4_3_deep_group96

source_name: Retina

value: Retina


title: Retina_donor4_3_deep_group95


title: RPE_Donor1_1_deep_group98


title: NSCLC_STK_20_slideseq

description: Single-nuclei RNA sequencing, SlideSeqV2

data_processing:
library strategy: single-nuclei RNAseq; SlideSeqV2
We used the Slide-seq tools pipeline (Stickels et al., 2021) to process our Slide-seq data, and loaded the data into Seurat.
Assembly: GRCh38
Supplementary files format and content: Counts data (*.counts.csv.gz) and Spatial coordinates data (*.coords.csv.gz)

source_name: NSCLC primary tumor

extract_protocol:
RNA from 10 uM frozen tumor sections was extracted and spatially indexed as described in Stickels et al. (Nature Biotechnology, 2021). Briefly, sections were cut and then mounted on a "puck". RNA was hybridized for 15 min in 6x SCC and then reveres transcribed using Maxima RT.  Tissue was then digested and cleared using clearing buffer and proteinase K and unbound capture oligos were removed using EXO-I treatment. 2nd strand synthesis was then performed using sn-SMRT oligos and Klenow Enzyme and the libraries were amplified using PCR with Truseq and SMART PCR primers.
Libraries after PCR amplification were tagmented and indexed using Nextera XT kit and Nextera N70x oligos. After SPRI cleanup libraries were quantified using D5000 tapestation and prepared for sequencing.

value: Primary Tumor

value: KRAS WT

value: snRNA-seq, SlideSeqV2

value: no


title: RPE_donor1_2_deep_group101

value: RPE

value: Male

value: WT


title: RPE_donor1_2_deep_group102


title: Retina_donor4_3_deep_group94


title: Retina_donor4_3_deep_group93


title: RPE_donor3_1_deep_group121


title: Retina_donor4_1_deep_group88


title: Retina_donor4_2_deep_group90


title: Retina_donor4_2_deep_group89


title: Retina_donor4_2_deep_group91


title: Retina_donor4_1_deep_group87


title: Retina_donor4_2_deep_group92


title: RPE_donor4_2_deep_group140


title: RPE_donor1_3_deep_group107


title: Retina_donor3_3_deep_group84


title: Retina_donor4_1_deep_group85


title: Retina_donor4_1_deep_group86


title: Retina_donor3_3_deep_group83


title: Retina_donor3_2_deep_group80


title: Retina_donor3_3_deep_group81


title: Retina_donor3_3_deep_group82


title: Retina_donor3_2_deep_group78


title: Retina_donor3_1_deep_group76


title: Retina_donor3_2_deep_group79


title: Retina_donor3_1_deep_group75


title: Retina_Donor2_3_deep_group64


title: RetinaDonor2_deep_group71


title: RetinaDonor2_deep_group72


title: Retina_donor3_2_deep_group77


title: RetinaDonor1_deep_group60


title: RetinaDonor2_deep_group67


title: Retina_donor3_1_deep_group74


title: Retina_donor3_1_deep_group73


title: RetinaDonor1_deep_group59


title: RetinaDonor1_deep_group58


title: RetinaDonor2_deep_group65


title: RetinaDonor2_deep_group68


title: RetinaDonor2_deep_group69


title: RetinaDonor2_deep_group70


title: Retina_Donor2_3_deep_group63


title: Retina_Donor2_3_deep_group62


title: RetinaDonor2_deep_group66


title: Retina_Donor1_3_deep_group52


title: RetinaDonor1_deep_group56


title: D0_C08

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: Retina_Donor2_3_deep_group61


title: D0_A10


title: D0_A06


title: RetinaDonor1_deep_group57


title: RetinaDonor1_deep_group55


title: RetinaDonor1_deep_group54


title: Macula_donor4_3_deep_group48

source_name: Macula

value: Macula


title: Macula_donor4_3_deep_group46


title: RetinaDonor1_deep_group53


title: Macula_donor4_2_deep_group44


title: Macula_donor4_3_deep_group47


title: Retina_Donor1_3_deep_group51


title: Retina_Donor1_3_deep_group49


title: Macula_donor4_3_deep_group45


title: Macula_donor3_3_deep_group36


title: Macula_donor4_2_deep_group42


title: Macula_donor4_2_deep_group43


title: Macula_donor4_1_deep_group38


title: Macula_donor4_1_deep_group40


title: Macula_donor4_2_deep_group41


title: Macula_donor3_2_deep_group32


title: Macula_donor4_1_deep_group39


title: Macula_donor3_2_deep_group30


title: Macula_donor3_3_deep_group35


title: Macula_donor4_1_deep_group37


title: Macula_donor3_3_deep_group34


title: Macula_donor3_1_deep_group28


title: Macula_donor3_1_deep_group26


title: Macula_donor2_3_deep_group24


title: Macula_donor3_3_deep_group33


title: Macula_donor3_2_deep_group31


title: Macula_donor3_2_deep_group29


title: Macula_donor3_1_deep_group25


title: Macula_donor3_1_deep_group27


title: Retina, donor 3, multiome ATAC

data_processing:
CellRanger
Assembly: hg38
Supplementary files format and content: CellRanger output files: RNA.barcodes.tsv.gz, RNA.features.tsv.gz, RNA.matrix.mtx.gz
Supplementary files format and content: CellRanger output files: atac_fragments.tsv.gz, atac_fragments.tsv.gz.tbi
Supplementary files format and content: bigWig files: Cell type-specific pseudo bulk ATAC-seq signal track
Supplementary files format and content: bed files: Peak calling of the specific cell type from pseudo bulk ATAC-seq in bed format
Library strategy: 10x multiome

source_name: retina

extract_protocol: The eye globes were collected from three unrelated human donors (aged 21, 27 and 43 years) with the collaboration of San Diego Eye Bank. The sample processing time starting from eye enucleation to dissection was restricted within 2 hours to identify the macula, a visible yellowish/whiteish indent that is slightly lateral to the optic disk inside the globe. Prior dissection, the eye globe with proper label was collected in a sterile moist chamber. At first the cornea was removed by cutting 2-3 mm around the limbus using a surgical blade. Pull off the iris and lens. This will leave you with the eye cup, which contains the posterior pole. After removing enough vitreous and aqueous fluid the temporal region can be located by two main blood vessels that curl around the macula. Using a sterile 3mm skin biopsy punch, the macula was collected. Sections were made for the other regions, temporal, nasal, superior and inferior. For all these regions, the retina and RPE tissue were separated and stored immediately in Liquid Nitrogen. Retina tissues were pulverized using a dounce tissue chamber and two pestles (“Loose” and “Tight”) on ice for each sample. Pulverized retina tissues were resuspended in 1ml of chilled NIM-DP-L buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl pH 7.5, 1 mM DTT, 1x protease inhibitor (Pierce), 1U μl-1 recombinant RNase inhibitor (Promega, PAN2515) and 0.1% Triton X-100). Tissue was dounce homogenized with a loose pestle (5-10 strokes) followed by a tight pestle (15-25 strokes) or until the solution was uniform. Nuclei were filtered using a 30 μm CellTrics filter (Sysmex, 04-0042-2316) into a LoBind tube (Eppendorf, 22431021). Nuclei were centrifuged 1000 rcf, for 10 min at 4°C then the pellet was resuspended in 1ml NIM-DP buffer (0.25 M sucrose, 25 mM KCl, 5mM MgCl2, 10 mM Tris-HCl pH 7.5, 1mM DTT, 1x protease inhibitor, 1U μl-1 recombinant RNase inhibitor) and pelleted (1000 rcf, 10 min at 4°C). Pelleted nuclei were resuspended in 400 μl 2 μM 7-AAD (Invitrogen, A1310) in sort buffer (1mM EDTA, 1U μl-1 recombinant RNase inhibitor, 1x protease inhibitor, 1% fatty acid-free BSA in PBS). A total of 150,000 nuclei was sorted (Sony, SH800S) into a LoBind tube containing a collection buffer (5U μl-1 recombinant RNase inhibitor, 1x protease inhibitor, 5% fatty acid-free BSA in PBS). Then, 5x permeabilization buffer (50 mM Tris-HCl pH 7.4, 50 mM NaCl, 15 mM MgCl2, 0.05% Tween-20, 0.05% IGEPAL, 0.005% Digitonin, 5% fatty acid-free BSA in PBS, 5mM DTT, 1U μl-1 recombinant RNase inhibitor, 5x protease inhibitor) was added for a final concentration of 1x. Nuclei were incubated in ice for 1 min, then centrifuged (500 rcf, 5 min at 4°C). The supernatant was discarded and 650 μl of wash buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% fatty acid-free BSA in PBS, 1mM DTT, 1U μl-1 recombinant RNase inhibitor, 1x protease inhibitor) was added without disturbing the pellet followed by centrifuging(500 rcf, 5 min at 4°C). The supernatant was removed, and the pellet was resuspended in 7μl of 1x nucleus buffer (nucleus buffer (10x Genomics), 1mM DTT, 1U μl-1 recombinant RNase inhibitor). Nuclei (1μl) were diluted in 1x nucleus buffer, stained with Trypan Blue (Invitrogen, T10282) and counted, In total, 16000-20000 nuclei were used for the tagmentation reaction and controller loading and libraries were generated according to the manufacturer’s recommended protocol (https://www.10xgenomics.com/support/single-cell-multiome-atac-plus-gene-expression).

value: Healthy unaffcted

value: retina


title: RPE, donor 3, multiome ATAC


title: Macula_donor2_3_deep_group23


title: Macula_donor2_2_deep_group20


title: Macula_donor2_2_deep_group19


title: Macula, donor 3, multiome ATAC

source_name: macula

value: macula


title: RPE, donor 2, multiome ATAC


title: Macula_donor2_3_deep_group21


title: Macula_donor2_1_deep_group16


title: Macula_donor2_3_deep_group22


title: Macula_donor2_2_deep_group17


title: Retina, donor 2, multiome ATAC


title: RPE, donor 1, multiome ATAC


title: Retina, donor 3, multiome RNA

molecule: nuclear RNA


title: Macula_donor2_1_deep_group14


title: Retina, donor 1, multiome ATAC


title: Macula, donor 2, multiome ATAC


title: RPE, donor 3, multiome RNA


title: Macula, donor 3, multiome RNA


title: Macula, donor 1, multiome ATAC


title: Macula_donor2_1_deep_group15


title: Macula_donor1_2_deep_group8


title: Retina, donor 2, multiome RNA


title: Macula, donor 2, multiome RNA


title: RPE, donor 2, multiome RNA


title: Macula, donor 1, multiome RNA


title: Macula_donor1_3_deep_group12


title: FMRP-cTag CLIP, CA1, Control 30 minutes, replicate 4

first: Ruth

last: Singer

address: 1230 York Avenue, TSH 314

institute: The Rockefeller University

description:
Library name: FMRP_CLIP_Control_rep4
FMRP_Control_rep4

data_processing:
Filter for quality of reads using fastq_filter.pl (CTK, fastq_filter.pl -f min:0-15:20,mean:16-39:20)
Trim 3' linker sequence (Cutadapt, -m 20 -a GTGTCAGTCACTTCCAGCGG)
Collapse exact duplicates using fastq2collapse.pl (CTK)
Trim 5' barcode and linker (CTK, stripBarcode.pl -len 9)
Remove reads less than 17 nt prior to mapping (cutadapt -m 17)
Mapped reads with STAR to UCSC mm10 transcriptome
Collapse PCR duplicates (CTK, tag2collapse.pl -EM 30 -random-barcode --seq-error-model em-local --weight-in-name --keep-max-score --keep-tag-name)
Assembly: mm10
Supplementary files format and content: tab-delimited text files contain quantification of the numer of FMRP tags mapped per transcript

source_name: CA1 hippocampus

extract_protocol:
After optogenetic activation and UV-crosslinking, tissue was dissected from acute hippocampal slices in HBSS with 0.1 mg/mL cycloheximide. Tissue was homogenized in lysis buffer (1x PBS, 0.1% SDS, 0.5% NP-40, 0.5% Sodium deoxycholate supplemented, 1X cOmplete Mini EDTA-free Protease Inhibitor (Roche) and 0.1 mg/mL cycloheximide) by mechanical homogenization. FMRP-cTag-CLIP was performed as described previously (see associated paper for details).
RNA tags were isolated and cloned using the BrdU-CLIP method as described in associated paper.

value: CA1 hippocampus

value: Camk2a-Cre x Fmr1 cTag

value: AAV-Control

tag: time post activation


title: FMRP-cTag CLIP, CA1, Control 30 minutes, replicate 3

description:
Library name: FMRP_CLIP_Control_rep3
FMRP_Control_rep3


title: Macula_donor1_3_deep_group11


title: FMRP-cTag CLIP, CA1, Control 30 minutes, replicate 2

description:
Library name: FMRP_CLIP_Control_rep2
FMRP_Control_rep2


title: Retina, donor 1, multiome RNA


title: RPE, donor 1, multiome RNA


title: Macula_donor1_3_deep_group10


title: D0_A11

value: embryonic stem cell


title: FMRP-cTag CLIP, CA1, Control 30 minutes, replicate 1

description:
Library name: FMRP_CLIP_Control_rep1
FMRP_Control_rep1


title: FMRP-cTag CLIP, CA1, Opto, 30 minutes, replicate 4

description:
Library name: FMRP_CLIP_Opto_rep4
FMRP_ChR1_rep4


title: Macula_donor2_2_deep_group18


title: Macula_donor1_3_deep_group9


title: FMRP-cTag CLIP, CA1, Opto, 30 minutes, replicate 1

description:
Library name: FMRP_CLIP_Opto_rep1
FMRP_ChR1_rep1


title: FMRP-cTag CLIP, CA1, Opto, 30 minutes, replicate 3

description:
Library name: FMRP_CLIP_Opto_rep3
FMRP_ChR1_rep3


title: FMRP-cTag CLIP, CA1, Opto, 30 minutes, replicate 2

description:
Library name: FMRP_CLIP_Opto_rep2
FMRP_ChR1_rep2


title: hc35_RNA

status: Public on Dec 05 2024

first: Bing

last: Ren

state: CA

address: 9500 Gilman Drive

description: 10x Genomics v3.1 snRNA-seq

library_source: transcriptomic single cell

data_processing:
Raw sequencing was processed using cellranger-arc (10x Genomics), generating snRNA-seq UMI count matrices for intronic and exonic reads mapping in the sense direction of a gene for hg38 GRCh38 human genome assembly and Gencode v32 gene annotations.
We performed unsupervised clustering with RNA UMI counts using Seurat v5 integrative analysis pipeline. First, cell barcodes were filtered for low quality nuclei by requiring ≥ 500 ATAC fragments, > 5 TSS enrichment (GRCh38 GENCODE v46) using SnapATAC2, ≥ 200 genes detected per nuclei.
Assembly: hg38 GRCh38
Supplementary files format and content: The raw_feature_bc_matrix.h5 is a Cell Ranger output that includes feature-barcode matrices containing cellular, non-cellular, and background barcodes.
The atac_fragments.tsv.gz is a Cell Ranger output that contains one line per unique fragment. And the atac_fragments.tsv.gz.tbi is the Tabix index for the fragment file.
hippocampus_RNA_seurat_object_filtered_cells.h5: Seurat object after quality control
hippocampus_RNA_seurat_object_filtered_cells_metadata.tsv: cell metadata for the Seurat object
Bigwig files were generated using deepTools bamCoverage with default settings and --normalizeUsing RPKM. Reads were combined across donors for indicated cell type or cell type by age group.

source_name: hippocampus

extract_protocol:
Frozen brain tissue was pulverized using a mortar and pestle on dry ice and pre-chilled with liquid nitrogen. Pulverized brain tissue was resuspended in 1mL of chilled NIM-DP-L buffer (0.25M sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris-HCl pH 7.5, 1mM DTT, 1X Protease Inhibitor (Pierce), 1U/μL Recombinant RNase inhibitor (Promega, PAN2515), and 0.1% Triton X-100). Tissue was Dounce homogenized with a loose pestle (5-10 strokes) followed by a tight pestle (15-25 strokes) or until solution was uniform. Nuclei were filtered using a 30μm CellTrics filter (Sysmex, 04-0042-2316) into a LoBind tube (Eppendorf, 22431021) and pelleted (1000 rcf, 10 min at 4˚C) (Eppendorf, 5920 R). Pellet was resuspended in 1 mL NIM-DP buffer (0.25M sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris-HCl pH 7.5, 1mM DTT, 1x Protease Inhibitor, 1U/μL Recombinant RNase inhibitor) and pelleted (1000 rcf, 10 min at 4˚C). Pelleted nuclei were resuspended in 400uL 2μM 7-AAD (Invitrogen, A1310) in Sort Buffer (1mM EDTA, 1U/μL Recombinant RNase inhibitor, 1X Protease Inhibitor, 1% fatty acid-free BSA in PBS). 120,000 nuclei were sorted (Sony, SH800S) into a LoBind tube containing Collection Buffer (5U/uL Recombinant RNase inhibitor, 1X Protease Inhibitor, 5% fatty acid-free BSA in PBS). 5X Permeabilization Buffer (50mM Tris-HCl pH 7.4, 50mM NaCl, 15mM MgCl2, 0.05% Tween-20, 0.05% IGEPAL, 0.005% Digitonin, 5% fatty acid-free BSA in PBS, 5mM DTT, 1U/μL Recombinant RNase inhibitor, 5X Protease Inhibitor) was added for a final concentration of 1X. Nuclei were incubated on ice for 1 minute, then centrifuged (500 rcf, 5min at 4C). Supernatant was discarded and 650uL of Wash Buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1%.Tween-20, 1% fatty acid-free BSA in PBS, 1mM DTT, 1U/μL Recombinant RNase inhibitor, 1X Protease Inhibitor) was added without disturbing the pellet followed by centrifuging (500 rcf, 5 min at 4˚C). Supernatant was removed, and the pellet was resuspended in 7uL of 1X Nuclei Buffer (Nuclei Buffer (10x Genomics), 1mM DTT, 1 U/μL Recombinant RNase inhibitor). 1 μL of nuclei was diluted in 1X Nuclei Buffer, stained with Trypan Blue (Invitrogen, T10282) and counted. 16-20k nuclei were used for tagmentation reaction and controller loading.
Libraries were generated following manufacturer’s recommended protocol (https://www.10xgenomics.com/support/single-cell-multiome-atac-plus-gene-expression). 10x multiome ATAC-seq and RNA-seq libraries were paired-end sequenced on NextSeq 500 and NovaSeq 6000 to a depth of ~50,000 reads per cell for each modality.
snATAC-seq
snRNA-seq

value: hippocampus

tag: donor id


title: hc9_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc40_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc212191_RNA

description: 10x Genomics v3.1 snRNA-seq


title: Macula_donor1_2_deep_group7

tag: Sex


title: hc19_RNA

description: 10x Genomics v3.1 snRNA-seq

tag: tissue


title: hc73_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc11_RNA

description: 10x Genomics v3.1 snRNA-seq


title: Macula_Donor1_1_deep_group4

tag: age


title: hc12_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc98_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc69984_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc1216_RNA

description: 10x Genomics v3.1 snRNA-seq


title: Macula_donor2_1_deep_group13

tag: genotype


title: hc1203_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc46426_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc1271_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc73787_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc1265_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc26_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc8_RNA

description: 10x Genomics v3.1 snRNA-seq


title: Macula_Donor1_1_deep_group1


title: hc13394_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc6021_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc1153_RNA

description: 10x Genomics v3.1 snRNA-seq


title: Macula_donor1_2_deep_group5


title: hc5551_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc81_RNA

description: 10x Genomics v3.1 snRNA-seq


title: Macula_Donor1_1_deep_group2


title: Macula_Donor1_1_deep_group3


title: hc5610_RNA

description: 10x Genomics v3.1 snRNA-seq


title: Retina_Donor1_3_deep_group50

value: Retina


title: hc5087_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc4781_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc5021_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc937_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc935_RNA

description: 10x Genomics v3.1 snRNA-seq


title: Macula_donor1_2_deep_group6


title: hc5614_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc13414_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc1134_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc6052_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc76_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc77_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc1745_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc78_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc5579_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc9_ATAC

description: 10x Genomics snATAC-seq

library_source: genomic single cell


title: hc35_ATAC

description: 10x Genomics snATAC-seq


title: hc40_ATAC

description: 10x Genomics snATAC-seq


title: hc13344_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc212191_ATAC

description: 10x Genomics snATAC-seq


title: hc26_ATAC

description: 10x Genomics snATAC-seq


title: hc12_ATAC

description: 10x Genomics snATAC-seq


title: hc73_ATAC

description: 10x Genomics snATAC-seq


title: hc11_ATAC

description: 10x Genomics snATAC-seq


title: hc29_RNA

description: 10x Genomics v3.1 snRNA-seq


title: hc1265_ATAC

description: 10x Genomics snATAC-seq


title: hc1203_ATAC

description: 10x Genomics snATAC-seq


title: hc73787_ATAC

description: 10x Genomics snATAC-seq


title: hc1271_ATAC

description: 10x Genomics snATAC-seq


title: hc1153_ATAC

description: 10x Genomics snATAC-seq


title: hc69984_ATAC

description: 10x Genomics snATAC-seq


title: hc46426_ATAC

description: 10x Genomics snATAC-seq


title: hc98_ATAC

description: 10x Genomics snATAC-seq


title: hc1216_ATAC

description: 10x Genomics snATAC-seq


title: hc5610_ATAC

description: 10x Genomics snATAC-seq


title: hc6021_ATAC

description: 10x Genomics snATAC-seq


title: hc8_ATAC

description: 10x Genomics snATAC-seq


title: hc5551_ATAC

description: 10x Genomics snATAC-seq


title: hc19_ATAC

description: 10x Genomics snATAC-seq


title: hc4781_ATAC

description: 10x Genomics snATAC-seq


title: hc1134_ATAC

description: 10x Genomics snATAC-seq


title: hc5021_ATAC

description: 10x Genomics snATAC-seq


title: hc13394_ATAC

description: 10x Genomics snATAC-seq


title: hc5087_ATAC

description: 10x Genomics snATAC-seq


title: hc81_ATAC

description: 10x Genomics snATAC-seq


title: hc5614_ATAC

description: 10x Genomics snATAC-seq


title: hc937_ATAC

description: 10x Genomics snATAC-seq


title: hc13344_ATAC

description: 10x Genomics snATAC-seq


title: hc935_ATAC

description: 10x Genomics snATAC-seq


title: hc13414_ATAC

description: 10x Genomics snATAC-seq


title: hc76_ATAC

description: 10x Genomics snATAC-seq


title: hc5579_ATAC

description: 10x Genomics snATAC-seq


title: hc1745_ATAC

description: 10x Genomics snATAC-seq


title: E12.5_mouse_proximal_forelimb_WT_SUZ12_ChIPseq

status: Public on Oct 21 2024

city: Montreal

first: marie

last: Kmita

state: Quebec

address: 110 Pine avenue west

country: Canada

institute: IRCM

data_processing:
ChIP-seq reads were aligned to the mm10 genome using bowtie v.2.3.1 with the following settings: bowtie2 -p 8 --fr --no-mixed --no-unal –x. Sam files were converted into bam files using Samtools v1.4.1. Duplicates were removed using Picard MarkDuplicates v.2.9.0. ChIP-seq data were visualized on the IGV software using BigWig files generated using bamCoverage tools using the following parameters : --normalizeUsing CPM -bs 1.
Mouse (MM10) and human (HG38) genome FASTA files were downloaded from the UCSC Genome Browser Website on September 15th 2022. Adapter sequences were removed from reads using robtools (https://github.com/francoisrobertlab/robtools) with the command “robtools trimmomatic --trimmers "ILLUMINACLIP:adapters.fa:2:30:10: TRAILING:3 MINLEN:25"” which requires Trimmomatic . Robtools version 2.0 and Trimmomatic version 0.36 was used. Reads were aligned to the genome using robtools with the commands “robtools bowtie2 -x mm10.fa.index -is -paired" and “robtools bowtie2 -x hg38.fa.index -is -paired" which require Bowtie2 (https://github.com/BenLangmead/bowtie2). Bowtie2 version 2.3.4.3 was used. Unmapped reads, low quality reads and duplicated reads were removed using robtools with the command “robtools filterbam” which requires samtools (https://github.com/samtools/samtools). Samtools version 1.9 was used. BAM files of replicates were merged together using robtools with the command “robtools mergebam --suffix -dedup” which requires samtools. Fragments were converted to BED format using robtools with the command “robtools bam2bed” which requires bedtools. Bedtools version 2.27.1 was used. Genome coverage files were generated using robtools with the command “robtools genomecov -g mm10.chrom.sizes --spike-suffix -human” which requires bedtools.
From the BED files containing the fragments, we counted the number of fragments per transcript for each category obtained from the SUZ12 and RING1B ChIP-seq data using a custom script called “TssFragmentCount.py” which requires bedtools. The number of fragments were scaled using a custom script called “TssScaleFragmentCount.py”. These custom scripts are available at https://github.com/francoisrobertlab/kmita-202210.
Peak calling was done using a custom script called macs2.py that requires MACS2. MACS2 version 2.2.7.1 was used. For each peak, TSSs from “knownGene.txt.gz” that are within a window size of 500bp around peak were keep for further analysis using a custom script called “TssWithPeaks.py”. Scaled number of fragments for each TSS were obtained using custom scripts “TssFragmentCount.py” and “TssScaleFragmentCount.py”. TSSs for which the EED mutant fragment count are between half and twice the WT counts are considered as maintained genes in the EED mutant. TSSs for which the EED mutant fragment count are above twice the WT counts are considered as gain genes in the EED mutant.
Scaled number of fragments for each TSS from “knownGene.txt.gz” were obtained using custom scripts “TssFragmentCount.py” and “TssScaleFragmentCount.py”. TSSs for which the WT fragment count is twice above the input fragment count were kept for further analysis. From these TSSs, only TSSs for which the WT fragment count is twice above the median of non-zero input fragment count of all TSSs were kept for further analysis. TSSs for which the EED mutant fragment count are between half and twice the WT counts were considered as additional maintained genes in the EED mutant.
Comparison of fragment counts were done using Perseus  version 1.6.14.0.
Assembly: mm10

source_name: proximal forelimb

extract_protocol:
ChIP experiments were performed in dissected proximal and distal forelimb buds of CD1 (wild type) and Prrx1Cre; EedDel/Flox (Eedc/-) mice at E12.5 following the same conditions as previously described (Berlivet et al., 2013). Chromatin was cross-linked for 13 minutes using formaldehyde and sonicated using Fisher Scientific, Model 100 sonic dismembrator to obtain fragments between 100-600 bp. Protein A and Protein G Dynabeads (Invitrogen) were incubated for 6 hours at 4℃ with 5ug of antibody. The chromatin was coupled to the beads overnight at 4℃. The immunoprecipitated samples were then washed with different wash buffers depending on the antibody present. Lastly, the DNA was purified on QIAquick columns (Qiagen).
Library and flow cells were prepared by the IRCM Molecular Biology Core Facility according to Illumina’s recommendations and sequenced on Illumina Hiseq 2500 in a 50 cycles paired-end configuration.

value: proximal forelimb

value: SUz12 cell signaling :3737


title: E12.5_mouse_distal_forelimb_EED_RYBP_ChIPseq

source_name: distal forelimb

value: distal forelimb

value: RYBP millipore Sigma :ab3637


title: E12.5_mouse_distal_forelimb_WT_SUZ12_ChIPseq


title: E12.5_mouse_INPUT

source_name: forlimb

value: forlimb

value: none (input)


title: E12.5_mouse_distal_forelimb_WT_RING1B_ChIPseq

value: WT

value: RING1B active motif :39664


title: E12.5_mouse_distal_forelimb_WT_H3K27me3_ChIPseq

value: H3K27me3 millipore :07-449


title: E12.5_mouse_distal_forelimb_WT_RYBP_ChIPseq


title: E12.5_mouse_distal_forelimb_EED_H3K27me3_ChIPseq


title: E12.5_mouse_proximal_forelimb_WT_H3K27ac_ChIPseq

value: H3K27ac abcam :ab4729


title: E12.5_mouse_INPUT_Rx

source_name: forelimb

value: forelimb


title: E12.5_mouse_proximal_forelimb_WT_RING1B_ChIPseq


title: E12.5_mouse_distal_forelimb_WT_H3K27ac_ChIPseq


title: E12.5_mouse_distal_forelimb_WT_H2AK119ub_ChIPseq

value: H2AK119ub Cell Signaling :8240


title: E12.5_mouse_distal_forelimb_WT_CBX2_ChIPseq

value: CBX2 abcam :ab80044


title: hc77_ATAC

description: 10x Genomics snATAC-seq


title: hc78_ATAC

description: 10x Genomics snATAC-seq


title: E12.5_mouse_distal_forelimb_EED_H2AK119ub_ChIPseq


title: RNF2_EED_PROX_E12_5_REP1

data_processing:
Mouse (GRCm38.p6) genome FASTA and GTF files were downloaded from the Ensembl Website (http://useast.ensembl.org/info/about/publications.html) on October 4th 2022. Adapter sequences were removed from reads using robtools (https://github.com/francoisrobertlab/robtools) with the command “robtools trimmomatic --trimmers "ILLUMINACLIP:adapters.fa:2:30:10 HEADCROP:10 SLIDINGWINDOW:4:15 TRAILING:20 MINLEN:40"” which requires Trimmomatic. Reads were aligned to the genome using STAR (https://github.com/alexdobin/STAR ) using the command “STAR --readFilesCommand zcat --readFilesIn "$sample"-paired_R1.fastq.gz "$sample"-paired_R2.fastq.gz --genomeDir mouse-star-index --genomeLoad NoSharedMemory --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --sjdbScore 1 --quantMode TranscriptomeSAM --outSAMtype BAM Unsorted --outSAMheaderHD @HD VN:1.4 SO:unsorted --outSAMunmapped Within --outSAMattributes NH HI AS NM MD --outFilterType BySJout --outFilterMultimapNmax 20 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.04 --outFileNamePrefix "$sample".star/"$sample"_“ where “$sample” is replaced by the sample name. STAR version 2.7.9a was used. The number of fragments per transcript were counted using RSEM (http://deweylab.github.io/RSEM/ ) with the command “rsem-calculate-expression --alignments --paired-end "$sample".star/"$sample"_Aligned.toTranscriptome.out.bam rsem-index/Mus_musculus "$sample".rsem/"$sample"” where “$sample” is replaced by the sample name. RSEM version 1.3.3 was used.
Assembly: mm10

extract_protocol:
RNA was extracted from dissected proximal and distal limb buds from 2 independent pools of wild type limb buds (n=2) and from two independent pools of mutant limb buds (n=2). The extraction was performed using the RNAeasy Plus mini kit. Next, mRNA enrichment, library preparation and flow-cell preparation for sequencing were performed by the IRCM Molecular Biology Core Facility according to Illumina’s recommendations. Sequencing was done on a Nextseq500 instrument with a paired-end 50 cycles protocol.
Library and flow cells were prepared by the IRCM Molecular Biology Core Facility according to Illumina’s recommendations and sequenced on Illumina in a 50 cycles paired-end configuration.


title: RNF2_EED_PROX_E12_5_REP2


title: E12.5_mouse_distal_forelimb_WT_CBX8_ChIPseq

value: CBX8 abcam :ab259849


title: RNF2_EED_DIST_E12_5_REP2


title: E12.5_mouse_distal_forelimb_EED_RING1B_ChIPseq


title: RNF2_PROX_E12_5_REP2


title: RNF2_EED_DIST_E12_5_REP1


title: E12.5_mouse_distal_forelimb_EED_CBX8_ChIPseq


title: RNF2_PROX_E12_5_REP1


title: RNF2_DIST_E12_5_REP2


title: EED_DIST_E12_5_REP1


title: E12.5_mouse_distal_forelimb_EED_CBX2_ChIPseq


title: EED_PROX_E12_5_REP2


title: EED_DIST_E12_5_REP2


title: WT_PROX_E12_5_REP2


title: WT_DIST_E12_5_REP1


title: WT_PROX_E12_5_REP1


title: EED_PROX_E12_5_REP1


title: 82-199-2017-4-26-2017_S82

status: Public on Sep 18 2024

city: Canyon

first: Matthew

last: Scott

state: TX

address: 3201 Russell Long Blvd

institute: Texas A&M University

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 36 bases, and leading and trailing basepairs below a quality score of 3
Read mapping and indexing was performed with HISAT2 v2.1.1, using the bovine whole genome assembly ARS-UCD1.2; parameters -p 14 --dta -x GCF_002263795.1_ARS-UCD1.2
Read assembly and quantification was performed with StringTie v2.1.7 with parameters -p 14 -G GCF_002263795.1_ARS-UCD1.2_genomic.gff, followed by stringtie --merge -p 14 -G GCF_002263795.1_ARS-UCD1.2_genomic.gff -o mergefile.gtf, and stringtie -e -p 14 -G mergefile.gtf -o. A read count matrix was generated with prepDE.py with parameters python prepDE.py  -l 150 -i mergelist.txt. Differential expression analysis was performed with edgeR v3.32.1.
Genome_build: GCF_002263795.1
Supplementary_files_format_and_content: comma-delimited text file of gene counts for each sample

organism: Bos taurus

source_name: Whole blood

extract_protocol:
Total RNA was isolated from Tempus Blood Tubes with Tempus Spin RNA Isolation Kit
RNA libraries were prepared for sequencing using TruSeq stranded mRNA library preparation kits, using standard Illumina protocols

treatment_protocol: All blood samples were collected in Tempus blood tubes (Applied Biosystem). Samples were stored per product recommendation.

tag: brd severity

value: Healthy

tag: year

tag: sample day

value: Male

tag: animal id

tag: developmental stage

value: Bull


title: 76-169-2017-4-26-2017_S76


title: 77-177-2017-4-26-2017_S77


title: RNF2_DIST_E12_5_REP1


title: 79-185-2017-4-26-2017_S79


title: 75-168-2017-4-26-2017_S75

value: Steer


title: WT_DIST_E12_5_REP2


title: 78-181-2017-4-26-2017_S78


title: 80-187-2017-4-26-2017_S80


title: 71-158-2017-4-26-2017_S71


title: 73-162-2017-4-26-2017_S73


title: 72-160-2017-4-26-2017_S72


title: 69-155-2017-4-26-2017_S69


title: 64-123-2017-4-26-2017_S64


title: 74-167-2017-4-26-2017_S74


title: 63-115-2017-4-26-2017_S63


title: 70-156-2017-4-26-2017_S70


title: 66-130-2017-4-26-2017_S66


title: 60-104-2017-4-26-2017_S60


title: 56-192-2017-3-29-2017_S56


title: 67-133-2017-4-26-2017_S67


title: 55-185-2017-3-29-2017_S55


title: 65-125-2017-4-26-2017_S65


title: 68-139-2017-4-26-2017_S68


title: 54-181-2017-3-29-2017_S54


title: 81-195-2017-4-26-2017_S81


title: 53-177-2017-29-2017_S53


title: 61-105-2017-4-26-2017_S61


title: 58-187-2017-3-29-2017_S58


title: 52-169-2017-3-29-2017_S52


title: 59-199-2017-3-29-2017_S59


title: 62-109-2017-4-26-2017_S62


title: 57-195-2017-3-29-2017_S57


title: 49-166-2017-3-29-2017_S49


title: 48-162-2017-3-29-2017_S48


title: 50-167-2017-3-29-2017_S50


title: 46-158-2017-3-29-2017_S46


title: 41-133-2017-3-29-2017_S41


title: 34-105-2017-3-29-2017_S34


title: 43-147-2017-3-29-2017_S43


title: 40-130-2017-3-29-2017_S40


title: 44-155-2017-3-29-2017_S44


title: 39-125-2017-3-29-2017_S39


title: 29-114-2017-3-3-2017_S29


title: 28-85-2015-5-11-2015_S28


title: 28-123-2017-3-29-2017_S38


title: 35-109-2017-3-29-2017_S35


title: 42-139-2017-3-29-2017_S42


title: 30-147-2017-3-3-2017_S30


title: 47-160-2017-3-29-2017_S47


title: 28-40-2015-3-30-2015_S18


title: 27-75-2015-5-11-2015_S27


title: 37-115-2017-3-29-2017_S37


title: 23-40-2015-5-11-2015_S23


title: 24-60-2015-5-11-2015_S24


title: 26-71-2015-5-11-2015_S26


title: 21-20-2015-5-11-2015_S21


title: 25-68-2015-5-11-2015_S25


title: 22-28-2015-5-11-2015_S22


title: 19-60-2015-3-30-2015_S19


title: 15-20-2015-3-30-2015_S15


title: 20-71-2015-3-30-2015_S20


title: 31-166-2017-3-3-2017_S31


title: 32-192-2017-3-3-2017_S32


title: 17-33-2015-3-30-2015_S17


title: 04-60-2015-3-2-2015_S4


title: 05-68-2015-3-2-2015_S5


title: 02-20-2015-3-2-2015_S2


title: 16-28-2015-3-30-2015_S16


title: 01-71-2015-3-2-2015_S1


title: 03-40-2015-3-2-2015_S3


title: PSC, KPC standard, 24 h control, rep 3

status: Public on Aug 14 2024

city: San Diego

first: Anais

middle: Aurelia

last: Zourelidis

address: 3855 Health Sciences Dr

institute: University of California San Diego

data_processing:
Data was processed as per Novogene standard protocols. CASAVA was used for base calling.
Raw reads were processed through fastq, and therefore, reads containing adapters, reads containing > 10 % bases that could not be determined, and low-quality reads (Qscore of over 50% bases of the read <= 5) were removed. CG content distribution and base error rate were determined for a quality check. Subsequent analyses were carried out with the clean data. For mapping the reads to the reference genome, mus musculus (Genom ID ensembl\_mus\_musculus\_grcm38\_p6\_gca\ _000001635\_8), the HISAT2 algorithm v2.0.5 was used. Reads were quantified using FeatureCounts v1.5.0-p3, and fragments per kilobase per million mapped reads (FPKM) of the genes were calculated.
For differential gene expression analysis, the DESeq2 R package v1.20.0 was used. This process involved the calculation of log2FoldChange, p-values, and adjusted p-values (padj), employing the Benjamini and Hochberg method to control the false discovery rate. Enrichment analysis, such as Gene Ontology (GO)- and Kyoto Encyclopedia of Genes and Genomes (KEGG)-analysis, was performed using the ClusterProfiler v3.8.1 R package.
Assembly: mm10
Supplementary files format and content: text file includes FPKM values for each sample

source_name: Pancreatic stellate cell

growth_protocol: PSC were cultured in DMEM low glucose medium (Sigma-Aldrich, Germany) supplemented with 40 % Nutrient mixture F12 (Gibco, UK), 16 % FBS (Sigma-Aldrich, Germany), 1 % Amphotericin B (Sigma-Aldrich, Germany) and 1 % penicillin/streptomycin (Gibco, UK).

extract_protocol:
RNA was harvested using direct-zol RNA Microprep Kit (Zymo Research, Germany). 450 ng (standard) or 120 ng (inverse) of total RNA was used for construction of sequencing libraries.
RNA libraries for RNA Seq were prepared by Novogene (Cambridge, United Kingdom) using Novogene NGS RNA Library Prep Set (PT042).

treatment_protocol: For standard co-culture, 1 x 105 KPC cells were seeded in the bottom of a 6-well dish and 1 x 105 PSC from wild-type C57BL/6J mice were seeded separately on top of the porous membrane of a culture-insert. After 24 h, the culture medium was changed, and the co-culture insert with PSC was transferred into the well with KPC cells. For inverse co-culture, culture inserts were turned around 180 °, and 1 x 105 KPC cells were seeded on the bottom of the membrane. After 24 h at 37 °C in 5 % CO2, the insert was turned around 180 ° again, and the attachment of the cancer cells was controlled via microscopy. Then, 1 x 105 PSC were seeded into the culture insert on top of the membrane. Another 24 hours later, the experiment was started. For all co-cultures, Advanced DMEM/F-12 medium (Gibco, UK) supplemented with 10 % FBS (Sigma-Aldrich, Germany) and 1 % penicillin/streptomycin (Gibco, UK) was used.

value: Pancreatic stellate cell

value: control

tag: time

value: 24 h control


title: PSC, KPC standard, 72 h control, rep 2

value: 72 h control


title: PSC, KPC standard, 72 h control, rep 3


title: IdU-treated, replicate 2

status: Public on Aug 28 2024

city: Miami

first: Binyamin

last: Zuckerman

state: Florida

department: Department of Cell and Systems Biology

institute: University of Miami

description: 10x Genomics

data_processing:
Reads were mapped to hg38 reference genome and further processed using 10x Cell Ranger (7.0.1). Intronic reads were excluded.
Seurat was used to filter for high-quality homogeneous cell population using the following filters: 3000 < detected genes < 5000, 8000 < UMI counts < 15000, and <10% reads mapping to mitochondrial genes per cell. The resulting count matrix was then further filtered by the BASiCS_Filter function function from the BASiCS R package with default parameters, which limited the analysis to genes with sufficient sequencing coverage for reliable noise quantification.
The output count matrix consisted of 5988 and 5786 cells for DMSO samples and 5148 and 4701 cells for IdU samples across 1107 genes. This output was then used to run 5 normalization pipelines according to their protocols: SCTransform, scran, Linnorm, BASiCS, and SCnorm.
BASiCS was run with default parameters and recommended settings (N=20000, Thin=20 and Burn=10000) using the horizontal integration strategy (no-spikes). The other packages were run with default parameters.
Assembly: hg38
Supplementary files format and content: tsv files and matrix files
Supplementary files format and content: Jurkat_scRNAseq_processed_data.csv contains summary statistics of the data as analyzed by above-mentioned methods. For each sample, we calculated separately for each biological replicate the mean, CV2, and Fano values per gene and then averaged the values of both replicates for further analysis.

source_name: Jurkat T Lymphocytes

growth_protocol: Jurkat T Lymphocytes were cultured in RPMI-1640 medium (supplemented with L-glutamine, 10% fetal bovine serum, and 1% penicillin-streptomycin), at 37°C, 5% CO2, in humidified conditions.

extract_protocol:
Two biological replicates (4 samples) of single-cell suspensions were loaded on a Chromium X instrument using a Chromium Next GEM Single Cell 3' HT Kit v3.1 (10x Genomics)
Libraries were constructed following manufacturer's protocol.

treatment_protocol: Cells were treated with either DMSO or 20 uM IdU (I7125, Millipore Sigma) for 48h.

value: Jurkat T Lymphocytes

value: IdU


title: PSC, KPC standard, 72 h treatment, rep 1

value: WT

value: distant paracrine KPC co-culture

value: 72 h treatment


title: PSC, KPC standard, 72 h control, rep 1


title: PSC, KPC standard, 24 h treatment, rep 3

value: 24 h treatment


title: PSC, KPC standard, 24 h control, rep 2


title: PSC, KPC standard, 24 h control, rep 1


title: PSC, KPC standard, 72 h treatment, rep 3


title: PSC, KPC standard, 24 h treatment, rep 2


title: PSC, KPC standard, 24 h treatment, rep 1


title: Whole blood, ID57_Day60

status: Public on Jul 26 2024

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 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. Differential expression analysis was performed with edgeR v3.36.0, glmmSeq v0.2.0, and EBSeq-HMM v1.32.0.
Assembly: GCF_002263795.2
Supplementary files format and content: Comma-delimited text file of raw gene counts for each sample

extract_protocol:
Total RNA was isolated from Tempus Blood Tubes with Tempus Spin RNA Isolation Kit per product instruction.
RNA libraries were prepared for sequencing using Illumina Stranded mRNA Prep kits, using standard Illumina protocols.

treatment_protocol: All  samples were collected in Tempus blood RNA tubes (Applied Biosystem). Samples were stored per product recommendation.

value: Whole blood

tag: time point

value: COMBO

tag: pen

tag: individual id

tag: disease

value: No


title: Whole blood, ID43_Day60

tag: treatment

value: INR

value: Yes


title: Whole blood, ID54_Day60


title: Whole blood, ID49_Day60

value: AAC


title: Whole blood, ID39_Day60


title: Whole blood, ID50_Day60


title: Whole blood, ID48_Day60


title: Whole blood, ID42_Day60


title: PSC, KPC standard, 72 h treatment, rep 2

tag: cell type


title: Whole blood, ID40_Day60


title: Whole blood, ID34_Day60


title: Whole blood, ID46_Day60


title: Whole blood, ID30_Day60


title: Whole blood, ID29_Day60


title: Whole blood, ID41_Day60


title: Whole blood, ID27_Day60


title: Whole blood, ID10_Day60


title: Whole blood, ID16_Day60


title: Whole blood, ID23_Day60


title: Whole blood, ID3_Day60


title: Whole blood, ID21_Day60


title: Whole blood, ID31_Day60


title: Whole blood, ID7_Day60


title: Whole blood, ID49_Day45


title: Whole blood, ID17_Day60


title: hc29_ATAC

description: 10x Genomics snATAC-seq

value: hippocampus

tag: donor id


title: Whole blood, ID46_Day45


title: Whole blood, ID6_Day60


title: Whole blood, ID18_Day60


title: Whole blood, ID42_Day45


title: Whole blood, ID57_Day45


title: Whole blood, ID9_Day60


title: Whole blood, ID50_Day45


title: Whole blood, ID48_Day45


title: Whole blood, ID43_Day45


title: Whole blood, ID54_Day45


title: Whole blood, ID40_Day45


title: Whole blood, ID39_Day45


title: Whole blood, ID41_Day45


title: Whole blood, ID17_Day45


title: Whole blood, ID21_Day45


title: Whole blood, ID23_Day45


title: Whole blood, ID18_Day45


title: Whole blood, ID27_Day45


title: Whole blood, ID29_Day45


title: Whole blood, ID16_Day45


title: Whole blood, ID10_Day45


title: Whole blood, ID3_Day45


title: Whole blood, ID57_Day28


title: Whole blood, ID6_Day45


title: Whole blood, ID49_Day28


title: Whole blood, ID9_Day45


title: Whole blood, ID48_Day28


title: Whole blood, ID7_Day45


title: Whole blood, ID42_Day28


title: Whole blood, ID54_Day28


title: Whole blood, ID50_Day28


title: Whole blood, ID46_Day28


title: Whole blood, ID34_Day28


title: Whole blood, ID43_Day28


title: Whole blood, ID41_Day28


title: Whole blood, ID40_Day28


title: Whole blood, ID39_Day28


title: Whole blood, ID30_Day28


title: Whole blood, ID31_Day28


title: Whole blood, ID29_Day28


title: Whole blood, ID18_Day28


title: Whole blood, ID21_Day28


title: Whole blood, ID23_Day28


title: Whole blood, ID27_Day28


title: Whole blood, ID31_Day45


title: Whole blood, ID30_Day45


title: Whole blood, ID34_Day45


title: Whole blood, ID17_Day28


title: Whole blood, ID10_Day28


title: Whole blood, ID57_Day13


title: Whole blood, ID6_Day28


title: Whole blood, ID3_Day28


title: Whole blood, ID50_Day13


title: Whole blood, ID49_Day13


title: Whole blood, ID9_Day28


title: Whole blood, ID7_Day28


title: Whole blood, ID46_Day13


title: Whole blood, ID16_Day28


title: Whole blood, ID42_Day13


title: Whole blood, ID54_Day13


title: Whole blood, ID43_Day13


title: Whole blood, ID39_Day13


title: Whole blood, ID31_Day13


title: Whole blood, ID29_Day13


title: Whole blood, ID27_Day13


title: Whole blood, ID40_Day13


title: Whole blood, ID34_Day13


title: Whole blood, ID23_Day13


title: Whole blood, ID41_Day13


title: Whole blood, ID30_Day13


title: Whole blood, ID18_Day13


title: Whole blood, ID17_Day13


title: Whole blood, ID21_Day13


title: Whole blood, ID10_Day13


title: Whole blood, ID9_Day13


title: Whole blood, ID16_Day13


title: Whole blood, ID7_Day13


title: Whole blood, ID3_Day13


title: Whole blood, ID54_Day0


title: Whole blood, ID6_Day13


title: Whole blood, ID49_Day0


title: Whole blood, ID48_Day0


title: Whole blood, ID57_Day0


title: Whole blood, ID50_Day0


title: Whole blood, ID41_Day0


title: Whole blood, ID46_Day0


title: Whole blood, ID43_Day0


title: Whole blood, ID34_Day0


title: Whole blood, ID31_Day0


title: Whole blood, ID40_Day0


title: Whole blood, ID39_Day0


title: Whole blood, ID30_Day0


title: Whole blood, ID27_Day0


title: Whole blood, ID21_Day0


title: Whole blood, ID18_Day0


title: Whole blood, ID17_Day0


title: Whole blood, ID42_Day0


title: Whole blood, ID23_Day0


title: Whole blood, ID16_Day0


title: Whole blood, ID10_Day0


title: Whole blood, ID7_Day0


title: Growth plate chondrocytes, control con116, 4

status: Public on Jun 28 2024

city: Madison

first: Jianhong

last: Ou

state: Wisconsin

address: 330 N Orchard Street

institute: Morgridge Institute For Research

description: single cell RNA

data_processing:
Raw base call demultiplex into Fastq Files
Matrix generation 10X cell ranger software 3.0.1
PCA analysis, clustering, and gene expression analysis Seurat version 3
Monocle trajectory analysis Monocle3
Assembly: mm10
Supplementary files format and content: h5 files provided the output of cellRanger count; RDS file provided after PCA and clustering, from which gene expression was taken and monocle3 was performed

source_name: E18.5 Growth plate chondrocytes

growth_protocol: Animals were bred in accordance to Duke University IACUC protocols and aged to 2 months.

extract_protocol:
Animals were euthanized according to IACUC protocol and hind limbs were isolated. Surrounding tissues were dissected off growth plates (GP) from distal femur and proximal tibia were washed with PBS. GP chondrocytes were digested using 2mg/ml Pronase I at 37C for 1 hour with agitation followed by two times, 3mg/ml Collagenase IV digestion at 37C. Cells were washed in PBS. Live cells were FACS sorted followed by GEM generation and RT using for 10X Genomics single cell isolation and barcoding.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: Idh1 LSL/+

value: distal femur and proximal tibia

value: chondrocytes


title: Growth plate chondrocytes, control con116, 3

description: single cell RNA


title: Whole blood, ID29_Day0


title: Growth plate chondrocytes, Idh1 mutant 119-1

description: single cell RNA

value: Col2a1-Cre; Idh1 LSL/+


title: Whole blood, ID9_Day0


title: Growth plate chondrocytes, control con119-3

description: single cell RNA


title: Growth plate cartilage, control rep3

data_processing:
RNA-seq reads were trimmed by Trim Galore (v 0.6.4) and mapped with STAR [ref]  (v 2.6.1d, with parameters --twopassMode Basic --runDirPerm All_RWX and supplying the Ensembl GRCm38 annotation) to mouse genome (GRCm38).
The mapped reads were counted using featureCounts [ref]  (v 1.6.4).
Bioconductor package DESeq2 [ref]  (v 1.28.1) was employed to analyze differential expressions (DE) with genotype information.
Assembly: GRCm38
Supplementary files format and content: text, raw counts

source_name: E18.5 Growth plate cartilage

extract_protocol: Animals were euthanized according to IACUC protocol and hind limbs were isolated. Surrounding tissues were dissected off growth plates (GP) from distal femur and proximal tibia were washed with PBS and snap frozen.

value: Growth plate chondrocytes


title: Growth plate cartilage, Idh1 mutant rep1


title: Growth plate chondrocytes, Idh1 mutant 124-3

description: single cell RNA


title: Whole blood, ID3_Day0


title: Whole blood, ID6_Day0


title: Growth plate cartilage, Idh1 mutant rep3


title: Growth plate cartilage, control rep2


title: Growth plate chondrocytes, Idh1 mutant 119-2

description: single cell RNA


title: Growth plate cartilage, Idh1 mutant rep2


title: Growth plate cartilage, control rep1

data_processing:
RNA-seq reads were trimmed by Trim Galore (v 0.6.4) and mapped with STAR (v 2.6.1d, with parameters --twopassMode Basic --runDirPerm All_RWX and supplying the Ensembl GRCm38 annotation) to mouse genome (GRCm38).
The mapped reads were counted using featureCounts  (v 1.6.4).
Bioconductor package DESeq2  (v 1.28.1) was employed to analyze differential expressions (DE) with genotype information.
Assembly: GRCm38
Supplementary files format and content: text, raw counts


title: WS234 H3K9me3 Rep 1

status: Public on Jun 25 2024

first: Jeffrey

last: Dilworth

email: jdilworth@ohri.ca, fdilworth@wisc.edu

state: WI

address: 1111 Highland Ave - WIMR II 4518

department: Cell and Regenerative Biology

institute: University of Wisconsin - Madison

data_processing:
Nextera adapters were trimmed from sequenced reads with Cutadapt 4.4 (Python 3.9.6).
Trimmed sequences were aligned to mm10 using Bowtie2 version 2.4.4 with the following parameters: --local --very-sensitive-local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700.
Duplicates, non-uniquely aligned reads were marked with Picard (3.1.1) and filtered from BAMs using Samtools v1.10 (htslib 1.10). Retained mapped and properly paired reads with greater than 10 Q-score.
Genome coverage was calculated using bamCoverage v3.5.3 (deepTools v3.5.3) with the following parameters: --effectiveGenomeSize 2467481108 --binSize 1 --normalizeUsing 'RPKM' --ignoreForNormalization 'chrM' --extendReads.
Peaks were called using MACS (3.0.0) with parameters: -f BAMPE -g 2805636331 -q 1e-5.
Assembly: GRCh38_hg38
Supplementary files format and content: bigWig, bigBed
Library strategy: CUT&Tag

source_name: Skeletal muscle

growth_protocol: WS234 cells were cultured on 0.1% gelatin-coated plates in LHNC media (4:1 DMEM:Medium 199, 15% BGS (v/v), 0.03 ug/ml Zinc Sulfate, 1.4 ug/ml vitamin B12, 0.055 ug/ml dexamethasone, 2.5 ng/ml HGF, 10 ng/ml FGF, 0.6X penicillin/streptomycin, 20 mM HEPES (pH 7.4)). Cells were collected under proliferating conditions.

extract_protocol:
Tn5-PA tagmented DNA was treated with proteinase K, extracted with phenol chloroform, and precipitated with ethanol.
Extracted DNA was amplified with barcoded i5 and i7 compatible primers
CUT&Tag libraries were sequenced on the Illumina NextSeq 6000 instrument

treatment_protocol: CUT&Tag experiements were performed on 75,000 cells

value: Skeletal muscle

tag: cell line

value: muscle progenitor

value: Wildtype

value: proliferative conditions

tag: antibody

value: Rabbit anti-Histone H3 (tri methyl K9) abcam ab8898


title: WS234 H3K4me1 Rep 2

tag: tissue

value: Rabbit anti-Histone H3 (mono methyl K4) abcam ab8895


title: WS234 H3K27ac Rep 2

tag: genotype

value: Rabbit anti-Histone H3 (acetyl K27) abcam ab177178


title: WS234 H3K9me3 Rep 2


title: WS234 H3K4me3 Rep 2

value: Rabbit anti-Histone H3 (tri methyl K4) abcam ab8580


title: DMSO-treated, replicate 2

description: 10x Genomics

value: Jurkat T Lymphocytes

value: DMSO


title: WS234 H3K27me3 Rep 2

value: Rabbit anti-Tri-Methyl-Histone H3 (Lys27) Cell Signaling 9733


title: WS234 H3K27me3 Rep 1


title: WS234 H3K4me3 Rep 1


title: 324C+R

status: Public on May 24 2024

data_processing:
The fastq files were aligned to the mouse reference GRCm38/mm10 sequence using bwa (v 0.7.15) mem with default parameters.
The technique duplicates were merged and library PCR duplicates were removed with picard (v 2.18.7).
SNPs were called from the individual strain BAM files with Genome Analysis Toolkit (v4.0.5.1).
The SNPs were filtered with expression “QD < 2.0 || FS > 60.0 || MQ < 40.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0”
and the Indels were filtered with expression "QD < 2.0 || FS > 200.0 || ReadPosRankSum < -20.0"
Assembly: GRCm38/mm10
Supplementary files format and content: vcf files with merged SNPs and Indels
Library strategy: Whole genome sequencing (WGS)

source_name: sarcoma

extract_protocol:
genomic DNA extraction using Qiagen Dneasy kit
We do not know, need input from Core

value: sarcoma

value: Sorted sarcoma cells

value: FACS sorted from primary mouse sarcoma


title: WS234 H3K27ac Rep 1


title: genomic DNA input

data_processing:
The reads were aligned to the human hg38 genome using bowtie2 (v2.3.5.1).
Peak calling: MACS2 using a q value cutoff = 0.05
Genome_build: hg38

source_name: Immortalized non-FSHD human myoblast cell line WS234 (donor 15VBic)

growth_protocol: Cells were cultured in LHCN medium on 0.1% (w/v) gelatin coated plates at 37oC in 5% CO2 incubator.

extract_protocol:
Cells were rinsed with PBS, collected using cell scrapers, centrifuged at 500 x g for 5 minutes and resuspended in swelling buffer (50 mM Tris pH 8, 2.5 mM EDTA and 30% (v/v) glycerol). Cells were then incubated for 30 minutes in swelling buffer at 4oC, sonicated using Bioruptor (Diagenode) for 2 cycles (HI power, cycles of 30 sec on / 1 min off). Nuclei were spun down at 1000 x g for 5 mins at 4oC, resuspended in sonication buffer (50 mM Hepes pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and 1% SDS), and sonicated for 13 cycles using the same settings as the previous step. 7.5 ug of SMCHD1 antibody (Abcam # ab31865) was added to 250 ug of precleared chromatin and incubated for 3 hours at RT.
Library was constructed using KAPA HyperPrep Kit (Roche #07962363001).

treatment_protocol: Proliferating myoblasts were fixed in 1% (v/v) formaldehyde for 20 minutes at RT and quenched with 125 mM glycine for 10 minutes at RT.

tag: chip antibody

tag: cell tpye

value: human myoblast cell line WS234 (donor 15VBic, female non-FSHD sample)


title: shSMCHD1 B

data_processing:
Reads are mapped to the hg38 genome using STAR.
Counts were summarized using featureCounts function in Rsubread.
DESeq2 was used to identify the differentially expressed genes.
Genome_build: hg38
Supplementary_files_format_and_content: FeatureCount Table

extract_protocol:
5 days after SMCHD1 and non-silencing lentiviral transduction, RNA was collected using a PureLink RNA mini kit (Thermo Fisher Scientific #12183018A) according to the manufacturer’s protocol.
rRNA depletionwas performed with a Ribo-Zero Magnetic Gold Kit (Illumina cat. No. MRZG12324). RNA library was prepared with a KAPA stranded RNA-seq kit (roche #07962169001).

treatment_protocol: For preparing the lentivirus, HEK293T cells were transfected with the pMD2.G, psPAX2, and shRNA vector plasmids using polyethylenimine. Lentivirus-containing medium supernatant  was centrifuged at 126086 x g for 2.5 hours at 4 oC using Optima L-100 XP ultracentrifuge, and the pellet was resuspended in LHCN medium. To transduce the cells, purified lentivirus was added to culture medium for 16 hours and fresh LHCN medium was replaced.


title: 324RFP


title: WS234 IgG Rep 2

value: Rabbit IgG Isotype Control ThermoFisher 10500C


title: non-silencing scramble shRNA control replicate 2

value: non-silencing scramble shRNA control


title: non-silencing scramble shRNA control replicate 1


title: WS234 H3K4me1 Rep 1

value: Skeletal muscle


title: WS234 IgG Rep 1

value: muscle progenitor


title: shSMCHD1 C


title: 732RFP


title: 324CFP


title: 324YFP


title: 732YFP


title: SMCHD1 CHIP pull down

value: SMCHD1 antibody (Abcam #ab31865)


title: 732CFP


title: 277GFP


title: 277RFP


title: Metastatic mouse UPS cell, IgG, rep3

data_processing:
ChIP-seq reads were trimmed using TrimGalore (v 0.6.6) and then aligned to GRCm38/mm10 reference genome by bwa (v 0.7.17-r1188).
Unmapped reads reads aligned to multiple locations were removed using samtools (v 1.11).
Duplicated reads were marked by picard (v 2.26.0).
The genomic coverage was saved in bigwig format and normalized by CPM method by bedTools (v 2.29.2) and UCSC Utilities bedGraphToBigWig (v 377).
Assembly: GRCm38/mm10
Supplementary files format and content: bigWig normalized by total reads

source_name: mouse UPS cell

growth_protocol: DMEM, 10% fetal bovine serum, 1x penicillin/streptomycin. Cells were cultured at 37C at 5% CO2.

extract_protocol:
Cells were fixed with 1% formaldehyde for 10 minutes. After quenching and cell lysis, chromatin was sonicated using a E220 focused-ultrasonicator (Covaris).
ChIP-seq libraries were prepared using the SimpleChIP Plus Sonication Chromatin IP kit (CST). 10 ug of DNA was used as starting material for input and ip samples. Libraries were amplified using 10 cycles on the thermocycler. Libraries were validated using the Agilent High Sensitivity DNA Kit and 2% Agarose gel

value: mouse UPS cell

value: IgG


title: Metastatic mouse UPS cell, H3K4Me3, rep1


title: 277YFP


title: Metastatic mouse UPS cell lacking Setd1a, IgG, rep1

source_name: Mouse Setd1a KO UPS cell

value: Mouse Setd1a KO UPS cell


title: Metastatic mouse UPS cell, H3K4Me3, rep2


title: Metastatic mouse UPS cell lacking Setd1a, H3K4Me3, rep2


title: Metastatic mouse UPS cell, IgG, rep2


title: Metastatic mouse UPS cell, IgG, rep1


title: Metastatic mouse UPS cell lacking Setd1a, IgG, rep3


title: Metastatic mouse UPS cell lacking Setd1a, H3K4Me3, rep1


title: Setd1a KO-Metastatic cell  [RNA_seq_2_1350]

data_processing:
The RNA-seq datasets were processed using the RNA-seq pipeline implemented in snakePipe (Bhardwaj et al. 2019).
Specifically, paired-end reads were mapped to the mouse genome (mm10) using STAR aligner (Dobin et al. 2013) with default settings.
Reads mapped to genes were counted using featureCounts (Liao et al. 2014) to obtain the gene-level counts, and differential expression was done using DESeq in R (Love et al. 2014).
Assembly: mm10
Supplementary files format and content: tab-delimited text files include raw counts for each Sample by featureCounts, text file

source_name: Setd1a KO-Metastatic cell

growth_protocol: mouse UPS cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
RNA was harvested using Direct-zol RNA mini prep kit (ZYMO RESEARCH). Total RNA ≥200ng, Concentration ≥20ng/μl of total RNA was used for the construction of sequencing libraries.
RNA libraries were prepared for RNA sequencing using standard BGI protocols.

value: mouse Undifferentiated Pleomorphic Sarcoma (UPS) cell

value: Sarcoma cell line

value: Setd1a Knock Out


title: Setd1a KO-Metastatic cell  [RNA_seq_3_1350]


title: Setd1a KO-Metastatic cell  [RNA_seq_1_1350]


title: Control-Metastatic cell  [RNA_seq_2_AAV2]

source_name: Control-Metastatic cell

value: WT


title: Control-Metastatic cell  [RNA_seq_1_AAV2]


title: Metastatic mouse UPS cell lacking Setd1a, IgG, rep2


title: Control-Metastatic cell  [RNA_seq_3_AAV2]


title: RNA-Seq of mus musculus: adult female brain,10μm

status: Public on May 01 2024

city: Changchun

first: Zhengyang

last: Jin

state: Jilin

address: No. 1163 Xinmin Street

country: China

institute: Jilin University

description: Well-ST-seq

data_processing:
*library strategy: Well-ST-seq
The filtered data was mapped to the mouse genome (mm10 mouse) using SpaceRanger V1.1.0.
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Brain

extract_protocol:
Tissue was permeabilized with tissue permeabilizing enzyme for 12 minutes
RNA libraries for RNA-seq were prepared using Kapa HIFI HotStart ReadyMix PCR Kit following manufacturer's protocols.

value: Brain

tag: Sex

value: Female

tag: age

value: 12 weeks

value: Cryosection


title: RNA-Seq of mus musculus: adult female brain,20μm

description: Well-ST-seq


title: ATAC-seq_MCD rep 2

status: Public on Apr 26 2024

data_processing:
Quality-control with FastQC
Adapters were trimmed from quality-controlled paired-end reads with Cutadapt v2.6;
Reads were aligned to mm10 using Bowtie2 v2.4.4; BigWigs were generated using BamCoverage (deeptools v3.5.1);
Peaks were called using MACS3 v3.0.0;
Differential peaks were found using DiffBind (R 4.0.2)
Genome_build: mm10
Supplementary_files_format_and_content: BigWigs (.bw) as a .tar.gz file - Counts per million (CPM) normalization; differential peaks (.csv)

source_name: RAW  267.4 macrophagte

extract_protocol:
50,000 RAW 267.4 macrophages were lysed in the presence of 10 mM NaCl 0.1% IGEPAL CA-630. Tagementation was performed usign TDE1 transposase as per manufacturer recommendations.Tagmented DNA was prepared using Zymo-spin concentrator 5 columns
Purified tagmented DNA was amplified with KAPA HIFI polymerase (12 PCR cycles) and Unique Dual Primers from Illumina (Cat number 20332088). Size-selection (L:1.1; R :0.6) was performed with KAPA Pure beads and size distribution of the final libraries was assessed on bioanalyzer (Agilent). Libraries were quantified by qPCR and loaded equimolarly on a S4 Novaseq flowcell. Each library was sequenced with a coverage of 50M paired-end reads (PE100).

treatment_protocol: RAW 267.4 macrophage cells were maintained in DMEM supplemented with 10% FCS at 37 °C in a 5% CO2 incubator. Cells were treated with MCD (5 mM) in 1 mg/ml BSA for 1 hour.

value: tissue culture

value: macrophages

value: MCD


title: Ctrl rep 1

data_processing:
Read alignment to mm10 was performed with STAR v2.5.3;
HTSeq v0.9.1 was used for transcript quantification;
DESeq2 v1.18.1 was used for differential expression.
Genome_build: mm10
Supplementary_files_format_and_content: HTSeq counts + tabluar format with DESeq2 output

source_name: RAW macrophages

extract_protocol:
Total RNA was isolated using a total RNA isolation kit (ThermoFisher cat. No. 12183020)
Total RNA (approx. 350 ng per sample) underwent rRNA depletion with a Ribo-Zero Magnetic Gold Kit (Human/Mouse/Rat) (Illumina cat. No. MRZG12324). RNA library preparation proceeded with a KAPA stranded RNA-seq kit (roche 07962169001-KAPA KK8401). Fragmentation/1st strand / 2nd strand end-repair and A-tailing steps proceeded, followed with ligation to  Illumina TruSeq LT Indexes (Illumina FC-121-2001 / FC-121-2002) followed by 8 cycles of PCR enrichment.

treatment_protocol: RAW 267.4 macrophage cells were maintained in DMEM supplemented with 10% FCS at 37 °C in a 5% CO2 incubator. Cells were treated with MCD (5 mM) in 1 mg/ml BSA for 1 hour or statin (7 υM) plus lipoprotein deficient serum (LPDS).  For LPS, cells were stimulated with 100 ng/ml LPS in DMEM supplemented with 1 mg/ml BSA for 3 h.

value: RAW macrophages


title: statin LPS rep 2

growth_protocol: RAW 267.4 macrophage cells were maintained in DMEM supplemented with 10% FCS at 37 °C in a 5% CO2 incubator.

value: statin + LPS


title: MCD-LPS rep2

value: MCD + LPS


title: ctl rep2


title: ATAC-seq_ctl rep 1


title: statin rep 2

value: statin


title: ctl LPS rep 1

value: LPS


title: MCD rep 1


title: LPS rep 2


title: Ctrl rep 2


title: LPS rep 1


title: MCD rep 2


title: ATAC-seq_ctl rep 2


title: ATAC-seq_MCD rep 1


title: hnRNPL_KO rep1

status: Public on Apr 24 2024

city: Montrreal

first: Javier

middle: M

last: Di Noia

address: 110 Av des Pins Ouest

institute: Institut de Recherches Cliniques de Montreal

data_processing:
Sequencing adapters were trimmed with fastp (0.23.1) with (parameter- --detect_adapter_for_pe).
Trimmed means were mapped to mm10 genome with STAR (2.7.9a) following manual instructions for multi-sample 2-pass mapping.
Gene expression counts were determined with featureCounts (Rsubread 2.12.2). DESeq2 (1.38.2) was used for differential gene expression. rMATS (4.2.2) was used for differential splicing analysis.
Mapped reads to mouse genome were filtered for mapping quality (MAPQ>=1) with using samtools (1.16.1) to generate bigwig files using bamCoverage and bamCompare from deepTools (3.5.0) with a bin size of 1bp.
Assembly: mm10
Supplementary files format and content: bigwig (forward and reverse strands)

source_name: spleen

growth_protocol: Cells were cultured in RPMI 1640 media (Wisent) supplemented with 10% fetal bovine serum (Wisent), 1% penicillin/streptomycin (Wisent) and 0.1 mM 2-mercaptoethanol (Bioshop), with 5μg/ml LPS + 5ng/ml IL-4.

extract_protocol:
RNA was extracted from  2 million cells per replicate using Qiagen RNeasy Mini kit.
mRNA was enriched using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) and library was prepared using KAPA RNA HyperPrep kit (Roche Diagnostics) as per manufacturer's instructions.

value: spleen

value: activated B cells

value: hnRNPL F/F CD21-cre

value: LPS+IL4 (24h)


title: WT rep3


title: ctl rep1


title: hnRNPL_KO rep2


title: ctl LPS rep 2


title: statin LPS rep 1


title: statin rep 1


title: MCD-LPS rep1


title: hnRNPL_KO rep3


title: WT rep2


title: WT rep1


title: Foxp1/2-cKO_P56 #3

status: Public on Apr 03 2024

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

description: 10x Genomics

data_processing:
cellranger mkfastq
cellranger count
cellbender
doubletfinder
seurat
Assembly: mm10 (GRCm38p6)
Supplementary files format and content: barcodes.tsv' a tab-delimited text files include list of cell-barcodes and 'features.tsv' a tab-delimited text file include list of genes and 'matrix.mtx' file in a sparse matrix format containing raw counts per gene per cell-barcode.

source_name: Dorsal Striatum

extract_protocol:
P56 mice were sacrificed by rapid dissection and brains were quickly removed and placed in ice-cold 1X PBS. Using a brain matrix with 1mm markings, striatal sections were obtained, as determined by visual cues indicating the presence of  the striatum. Using forceps, the striatum was separated from the cortex, flash frozen, and stored in -80C. Nuclei isolation for snRNA-Seq was done as follows: tissue was homogenized in a glass Dounce tissue grinder (25 times with pestle A, 25 times with pestle B; Sigma, Cat#D8938) in 2mL ice-cold EZ Nuclei Lysis Buffer (Sigma, Cat #NUC-101). Samples were incubated on ice for 5 minutes with an additional 2mL ice-cold EZ lysis buffer and then centrifuged at 500 X g for 5 minutes at 4C, washed with 2mL ice-cold EZ lysis buffer, and incubated on ice for another 5 minutes before being spun again. Nuclei were then washed in 500uL Nuclei Suspension Buffer (NSB; 1X PBS, 0.01% UltraPure BSA, and 0.1% RNase inhibitor) and mixed with 900uL nuclei sucrose cushion buffer (Sigma, Cat #NUC-201). This mixture was layered on top of another 500uL of sucrose cushion buffer and spun at 13,000 X g for 45 minutes at 4C. The supernatant was then discarded, the pellet was resuspended in 60uL NSB, and filtered through FLOWMI tip strainer (Bel-Art Products, Cat# H13680-0040); more NSB was added as needed to help samples go through the FLOWMI tip. An aliquot was stained with Trypan Blue and counted under a microscope to determine concentration (targeting 700-1,200 nuclei/uL). Striatal tissue for P9 mice was collected following rapid decapitation and brains were removed and placed in ice-cold artificial cerebrospinal fluid (ACSF; 126 mM NaCl, 20 mM NaHCO3, 20 mM D-Glucose, 3 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl2, 2 mM MgCl2) bubbled with 95% O2 and 5% CO2. 500uM coronal sections were made in ACSF using a VF-200 Compresstome (Precisionary Instruments) and transferred to another chamber filled with ACSF. From 3 striatal sections, 6 punches of striatal tissue were collected (2 per hemisphere), flash frozen, and stored in -80C. Nuclei were then isolated in a similar fashion as adults, with only one 5 minute centrifugation in EZ Nuclei Lysis buffer.  No sucrose cushion was used. Again, samples were filtered through the FLOWMI tip strainer, stained with Trypan Blue, counted under a microscope, and stored on ice until library generation.
Library was performed according to the manufacter’s instructions (single cell 3’ v3 and v3.1 protocol, 10x Genomics). Isolated nuclei were mixed with the 10X  master mix and loaded with partitioning oil and gel beads onto the provided chip. This results in generation of gel bead-in-emulsion (GEMs).  GEMs were then retrotranscripted to cDNA; these contained  Ilumina R1 primer sequence, Unique Molecular Identifier (UMIs) and the 10x Barcode. Silane DynaBeads were used to purify cDNA which were then amplified by PCR. SPRIselect beads were used to select proper sized fragments. Samples were cleaned with 80% ethanol after every amplification step as well. Finally, libraries were constructed and an Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added during these final steps.

value: F

value: Dorsal Striatum

tag: strain

tag: prep batch

tag: sequencing batch


title: PVN,ZT20 treatment, replicate 1,scRNA-seq

status: Public on Apr 16 2024

city: Houston

first: Yulin

last: Dai

address: 7000 fannin, suite 600

department: School of Biomedical Informatics

institute: University of Texas health science center at Houston

description: 10x Genomics

data_processing:
The Cell Ranger™ Single Cell Software Suite v.7.0.0 was used and The reads were aligned to the human transcriptome reference (cellranger mm10-2020-A) with STAR 2.6
Assembly: cellranger-mm10-2020-A
Supplementary files format and content: standard output of cellranger with gzipped barcode, features (gene), and Unique Molecular Identifier (UMI)

source_name: PVN

extract_protocol:
PVN ncueli was obtained from compiling 5 micorpunched PVNs together and then carrying out nuceli isolation using a sucrose gradient. The nuceli morpholohy and quantity were checked under the microscope by staining
Single cell Gene Expression Library was prepared according toChromium Next GEM Single Cell 3' Reagent Kits v3(10x Genomics). In Brief, single nuclei suspension, reverse transcription (RT) reagents, Gel Beads containing barcoded oligonucleotides, and oil were loaded on a Chromium controller (10x Genomics) to generate single cell GEMS (Gel Beads-In-Emulsions) where full length cDNA was synthesized and barcoded for each single cell. Subsequently the GEMS are broken and cDNA from each single cell are pooled. Following cleanup using Dynabeads MyOne Silane Beads, cDNA is amplified by PCR. The amplified product is fragmented to optimal size before end-repair, A-tailing, and adaptor ligation. Final library was generated by amplification.

value: PVN

value: PVN cells

value: Male


title: Foxp1-cKO_P56 #3

description: 10x Genomics

value: M


title: Foxp1/2-cKO_P56 #1

description: 10x Genomics


title: Foxp2-cKO_P56 #1

description: 10x Genomics


title: Control_P56 #1

description: 10x Genomics

value: Control


title: Foxp2-cKO_P56 #3

description: 10x Genomics


title: Foxp1/2-cKO_P9 #1

description: 10x Genomics


title: Foxp2-cKO_P56 #2

description: 10x Genomics


title: Foxp1-cKO_P56 #1

description: 10x Genomics


title: Foxp2-cKO_P9 #1

description: 10x Genomics


title: Foxp1/2-cKO_P9 #2

description: 10x Genomics


title: Foxp1/2-cKO_P9 #3

description: 10x Genomics


title: Control_P56 #2

description: 10x Genomics


title: Foxp1-cKO_P9 #3

description: 10x Genomics


title: Foxp1/2-cKO_P56 #2

description: 10x Genomics


title: Foxp1-cKO_P56 #2

description: 10x Genomics


title: Foxp2-cKO_P56 #4

description: 10x Genomics


title: Foxp2-cKO_P9 #2

description: 10x Genomics


title: Control_P56 #3

description: 10x Genomics


title: Foxp1-cKO_P9 #2

description: 10x Genomics


title: Foxp2-cKO_P9 #3

description: 10x Genomics


title: Control_P9 #1

description: 10x Genomics


title: Foxp1-cKO_P9 #1

description: 10x Genomics


title: Foxp1/2-cKO #3

description: 10x Genomics

data_processing:
cellranger-atac mkfastq
cellranger-atac count
macs2
seurat
signac
Assembly: mm10 (GRCm38p6)
Supplementary files format and content: barcodes.tsv' a tab-delimited text files include list of cell-barcodes and 'features.tsv' a tab-delimited text file include list of peaks and 'matrix.mtx' file in a sparse matrix format containing raw counts per peak per cell-barcode.

extract_protocol:
Striatal tissue for P9 mice was collected following rapid decapitation and brains were removed and placed in ice-cold artificial cerebrospinal fluid (ACSF; 126 mM NaCl, 20 mM NaHCO3, 20 mM D-Glucose, 3 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl2, 2 mM MgCl2) bubbled with 95% O2 and 5% CO2. 500uM coronal sections were made in ACSF using a VF-200 Compresstome (Precisionary Instruments) and transferred to another chamber filled with ACSF. From 3 striatal sections, 6 punches of striatal tissue were collected (2 per hemisphere), flash frozen, and stored in -80C. Nuclei isolation for snATAC-Seq was done as follows: tissue was homogenized in a glass Dounce tissue grinder (25 times with pestle A, 25 times with pestle B; Sigma, Cat#D8938) in 1mL ice-cold EZ Nuclei Lysis Buffer (Sigma, Cat #NUC-101). Samples were incubated on ice for 3 minutes with an additional 1mL ice-cold EZ lysis buffer and then centrifuged at 500 X g for 5 minutes at 4C. Nuclei were then washed in 500uL Nuclei Wash Buffer (20uL of 1M Tris pH 7.4, 4uL of 5M NaCl, 6uL 1M MgCl2, 0.01% BSA, 20uL of 10% Tween-20 + 1.75mL H2O) and spun at 500 X g for 5 minutes again. The supernatant was then discarded, the pellet was resuspended in 150uL nuclei wash buffer, and filtered through FLOWMI tip strainer (Bel-Art Products, Cat# H13680-0040); more wash bufer was added as needed to help samples go through the FLOWMI tip. An aliquot was stained with Trypan Blue and counted under a microscope to determine concentration (targeting 4,000 nuclei/uL). Nuclei were then diluted to desired concentration, using 20X Nuclei buffer provided by 10X.
Library was performed according to the manufacter’s instructions (single cell ATAC v1.1 and v2 protocol, 10x Genomics). Isolated nuclei were mixed with the transposition mix which was followed by transposition. Then, 10X  master mix was and samples were loaded onto chip alongside  partitioning oil and gel beads. This results in generation of gel bead-in-emulsion (GEMs).  GEMs were then cleaned with Silane DynaBeads  followed by size-sleion using SPRIselect beads. Samples were cleaned with 80% ethanol  as well. Finally, libraries were constructed and  paired-end constructs with P5 and P7 sequences and a sample index were added during these final steps.


title: Foxp2-cKO #2

description: 10x Genomics


title: Foxp2-cKO #1

description: 10x Genomics


title: Control #2

description: 10x Genomics


title: Foxp1/2-cKO #2

description: 10x Genomics


title: D2 Foxp1cKO + ControlAAV #2 (WPRE)

status: Public on Feb 20 2024

data_processing:
cellranger mkfastq
cellranger count
cellbender
doubletfinder
harmony
seurat
Assembly: mm10 (GRCm38p6)
Supplementary files format and content: matrix.mtx.gz' has raw counts for all samples
Supplementary files format and content: barcodes.tsv' has list of barcodes for all samples matching with raw counts
Supplementary files format and content: features.tsv' has list of genes matching to raw counts
Supplementary files format and content: meta.tsv' has all meta information including assigned celltypes

extract_protocol:
We sacrificed P18 mice by rapid decapitation and quickly dissected the brain and transferred it into ice-cold nACSF that contained (mM): 125 NaCl, 25 NaHCO3, 10 Dextrose, 3 KCl, 1.25 NaH2PO4, 2 CaCl2.2H2O, 2 MgCl2 with 7.3-7.4 pH and 300-305 mOsm osmolality. We placed the brain onto an ice-cold 1.0 mm stainless steel brain matrix (Stoelting, Cat#51386) and obtained three 1.0 mm thick striatal sections which were then transferred into a petri-dish containing cold nACSF. Using forceps and under a dissection microscope, striatum was dissected out from these slices, flash frozen in liquid N2, and stored at -80°C. The striatal tissue was collected from P18 mice of required genotypes at different time points and stored. Protocol for isolating nuclei was adapted and modified from our previously published work6. Briefly, tissue was homogenized 20 times with pestle A, followed by 20 times with pestle B, in a glass Dounce tissue grinder (Sigma, Cat#D8938) in 2 ml ice-cold EZ Nuclei Lysis Buffer (Sigma, Cat #NUC-101). Homogenized samples were transferred into 15 ml tube with an additional 2 ml ice-cold EZ Nuclei Lysis Buffer, incubated on ice for 3 min and centrifuged at 500 X g for 5 min at 4°C. The supernatant was discarded, and nuclei pellet was resuspended in 3 ml ice-cold Nuclei Suspension Buffer (NSB; 1X PBS, 0.01% Ultrapure BSA, and 0.1% RNase inhibitor) and again centrifuged at 500 X g for 5 min at 4°C. The supernatant was then discarded, and nuclei pellet was resuspended in 300-400 µl NSB, and filtered through 40 µm size Flowmi cell strainer (Bel-Art Products, Cat# H13680-0040). An aliquot of the filtered nuclei suspension was mixed with Trypan Blue and transferred into a disposable cell counting chamber nuclei to visualize and count the healthy nuclei under a light microscope to determine desired nuclei concentration (targeting 700-1,200 nuclei/µl).
Libraries from a total of 9 mice (3 mice/genotype) were prepared using the Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.1 (10X Genomics, Cat#1000121) targeting 10,000 nuclei per sample as per the manufacturer’s instructions. All mice were injected either with ControlAAV or Foxp1AAV in their striatum at P1 age where only a fraction of neurons was infected that were identified based on the presence of AAV transcripts. However, the proportion of AAV transcripts is exceptionally small in the pool of total transcripts that are captured by snRNA-seq experiment and therefore we used a modified approach 12 where we amplified the viral transcript specific sequences and prepared separate libraries. Briefly, we used 5% of the total cleaned up cDNA (amplified from the GEMs that were generated from each sample for the targeted 10,000 nuclei) and amplified the viral transcripts with reverse primer 10X_Enrich_R – 5’CTACACGACGCTCTTCCG3’ and forward primers 10X_WPRE_F1 – 5’ACGAGTCGGATCTCCCTTT3’ (for ControlAAV infected mice) and 10X_mCherry_F1 – 5’GCATGGACGAGCTGTACA3’ (for Foxp1AAV infected mice) for 25 cycles using Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB, Cat# M0531S). We used different forward primers for ControlAAV and Foxp1AAV infected mice since ControlAAV vector has a WPRE sequence at the 3’end whereas we had to remove WPRE sequence from the AAVFoxp1 vector to accommodate Foxp1 and mCherry into the vector and thus have the mCherry sequence at 3’end of Foxp1AAV vector. The amplified PCR product was run on an agarose gel, where a ~250 bp product was excised from the gel and eluted using QIAquick Gel Extraction Kit (Qiagen, Cat#28706) in 20 µl elution buffer (NEB). 5% of this first round amplified product was used to perform a second round of amplification using the reverse primer, i. e. 10X_WPRE_F1 and forward primers 10X_WPRE_F2 – 5’GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACGAGTCGGATCTCCCTTT3’ (for AAVControl infected mice) and 10X_mCherry_F2 –5’GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTACAAGTAGACCCAGCTT3’ (for AAVFoxp1 infected mice) for 20 cycles using the same Phusion polymerase. Once again, this amplified PCR product was loaded on an agarose gel where similar size product (~250 bp) was excised and eluted in 30 µl elution buffer. The eluted PCR products were once again cleaned up using SPRIselect Reagent and subjected to sample-index PCR amplification according to the manufacturer’s instructions (10X Genomics) to generate cDNA libraries. Finally, both (total and AAV_transcript) libraries were subjected to quality control using 4200 TapeStation System (Agilent, Cat# G2991BA) and quantified using Qubit 2.0 Fluorometer (Invitrogen). For each sample, 5 nM of the total libraries and 0.5 nM of the AAV transcript libraries (10% of the total library) were pooled and sequenced on a NovaSeq 6000 (Illumina) platform on S4 flow cell.

value: D2 Foxp1cKO


title: D2 Foxp1cKO + Foxp1AAV #2 (mCherry)


title: D2 Foxp1cKO + Foxp1AAV #3


title: Foxp1-cKO #2

description: 10x Genomics


title: D2 Foxp1cKO + Foxp1AAV #3 (mCherry)


title: D2 Foxp1cKO + ControlAAV #3 (WPRE)


title: Foxp1-cKO #1

description: 10x Genomics


title: Control_P9 #2

description: 10x Genomics


title: D2 Foxp1CTL + ControlAAV #2 (WPRE)

value: D2 Foxp1CTL


title: Foxp1-cKO #3

description: 10x Genomics


title: Control #1

description: 10x Genomics


title: D2 Foxp1cKO + Foxp1AAV #2


title: D2 Foxp1cKO + Foxp1AAV #1 (mCherry)


title: D2 Foxp1CTL + ControlAAV #3 (WPRE)


title: Foxp2-cKO #3

description: 10x Genomics


title: Control #3

description: 10x Genomics


title: Foxp1/2-cKO #1

description: 10x Genomics


title: Control_P9 #3

description: 10x Genomics


title: D2 Foxp1cKO + ControlAAV #1 (WPRE)


title: D2 Foxp1cKO + ControlAAV #1


title: D2 Foxp1cKO + Foxp1AAV #1