title: cDNA_BR1_EEP_Fgf5_Run2


title: cDNA_BR3_EEP_Otx2_Run2

status: Public on Aug 10 2023

type: SRA

city: Amsterdam

first: Bas

last: van Steensel

address: Plesmanlaan 121

country: Netherlands

institute: The Netherlands Cancer Institute (NKI)

library_source: transcriptomic

data_processing:
Basecalling, filtering and demultiplexing were performed using standard software of the Illumina MiSeq, NextSeq or NovaSeq platforms
For cDNA and pDNA samples, the barcode sequence was extracted from the read (using a custom python scripts) and counted.
For iPCR samples the insert sequence was aligned to a custom genome (using bowtie2) containing only the target sequences extracted from the mm10 genome, and the barcode was extracted from the ead using a custom python script.
Barcodes were clustered using Starcode (version 1.1) {Zorita, 2015, 25638815} to remove errors from barcode sequencing.
barcodes  matched with multiple element combinations were removed from the data
Per  combination an activity value was calculated as the ratio of the mean readcount in the RNA samples and the mean readcount in the pDNA library samples across all barcodes assigned to the singlet or combination.
Baselines were calculated across control-control combinations and then used to calculate boost indices as the log2 ratio between the activity of a combination and the baseline
Assembly: mm10
Supplementary files format and content: tsv format, each row contains a unique EEP combination, with fragment identifiers, calculated activities, baselines and Boost indices

molecule: total RNA

organism: Mus musculus

growth_protocol: E14TG2a mouse embryonic stem cells (mESC) (ATCC CRL-1821) were cultured in 2i+LI. 2i+LIF was made according to the 4DN nucleome protocol for culturing mESCs (https://data.4dnucleome.org/protocols/cb03c0c6-4ba6-4bbe-9210-c430ee4fdb2c/).

extract_protocol:
For iPCR samples, each library was digested overnight with I-CeuI (#R0699S, NEB), barcode-insert fragments were then circularized, remaining linear fragments digested, and barcode-insert fragments were linearized again with I-SceI (#R0694S; NEB). Barcode-insert fragments were then amplified by PCR with Illumina adapters and sequenced on an Illumina NextSeq 550 platform using 150 bp paired-end sequencing. For cDNA samples, cells were collected 24 hours after transfection in 5 ml of TRIsure. From each sample, the aqueous phase containing the total RNA of the TRIsure solution was extracted and purified on an RNA extraction column (#K0732, Thermo Scientific). Total RNA was digested with 10 units of DNase I (#04716728001; Roche) for 30 minutes in 10-8 10 µl reactions containing 5 µg of RNA. DNase I was inactivated by addition of 1 µl of 25 mM EDTA and incubation at 70°C for 10 min. cDNA was produced in 20 µl reactions (1 per 10 µDNAse reaction) using Maxima Reverse transcriptase (#EP0743; ThermoFisher Scientific) and a gene specific (targeting the GFP reporter) primer (JvA304, see Supplementary table 3 for sequence). dNTPs and the primer were mixed with the DNAse digested RNA and incubated at 65°C for 5 minutes. The RT buffer, enzyme and RNAse inhibitor were added and the reaction was incubated at 50 °C for 1 hour. cDNA was then amplified by two nested PCRs to make it strand specific. The first PCR reaction uses (index variants of 285JvA containing the S2, index and p7 adaptor)  and 305JvA (targeting the adapter introduced by 304JvA). Each 20 µl RT reaction was amplified in a 100-µl PCR reaction with MyTaq Red mix (#BIO-25043; Bioline). The second PCR reaction was performed using 10 µl of the product of the previous reaction in a 100 µl reaction using the same index variant primer and index variants of 437JvA (containing the S1, index and p5 adaptor) (see Supplementary table 3 for primer sequences). The first PCR was run for 10 cycles and the second one for 8 cycles using the recommended Mytaq Red mix conditions ( for both PCRs: 1 min 96 °C, then each cycle 15 s 96 °C, 15 s 60 °C, 15 s 72 °C). PCR products were purified with magnetic beads and mixed for sequencing in an Illumina NovaSeq 6000 platform using 100bp single end reads. For pDNA samples, 1 µg of each plasmid library was digested with I-SceI in order to linearise the plasmid. Barcodes were amplified by PCR for 9 cycles from 50 ng of material. Primers and reaction conditions were the same as in the amplification of cDNA. PCR products were purified using magnetic beads and mixed for sequencing.
PCR

treatment_protocol: Each EEP library was transfected separatedly. Cells were transiently transfected using Amaxa nucleofector II, program A-30, and Mouse Embryonic Stem Cell NucleofectorTM Kit (#VPH-1001, Lonza). Three independent biological replicates were done for each library mix. For each biological replicate 20 million cells were transfected (5 million cells with 4 µg plasmid per cuvette).

tag: cell line

tag: cell type

value: mESCs


title: pDNA_EEP_Fgf5_Run2

library_source: genomic

molecule: genomic DNA


title: cDNA_BR2_EEP_Otx2_Run2


title: pDNA_EEP_Otx2_Run2


title: cDNA_BR3_EEP_Klf2_Run2


title: cDNA_BR2_EEP_Klf2_Run2


title: cDNA_BR1_EEP_Klf2_Run2


title: cDNA_BR1_EEP_Ap1m1_Run2


title: pDNA_EEP_Klf2_Run2


title: cDNA_BR3_EEP_Sox2_Run2


title: cDNA_BR2_EEP_Lefty1_Run2


title: cDNA_BR2_EEP_Fgf5_Run2


title: cDNA_BR2_EEP_Sox2_Run2


title: cDNA_BR1_EEP_Sox2_Run2


title: cDNA_BR1_EEP_Ap1m1_Run1


title: cDNA_BR1_EEP_Tbx3_Run1


title: cDNA_BR2_EEP_Ap1m1_Run1


title: cDNA_BR3_EEP_Ap1m1_Run1


title: cDNA_BR1_EEP_Otx2_Run2


title: pDNA_EEP_Sox2_Run2


title: pDNA_EEP_Ap1m1_Run1


title: pDNA_EEP_Tbx3_Run1


title: cDNA_BR1_EEP_Nanog_Run1


title: cDNA_BR2_EEP_Nanog_Run1


title: cDNA_BR2_EEP_Tbx3_Run1


title: cDNA_BR1_EEP_Lefty1_Run1


title: cDNA_BR3_EEP_Fgf5_Run1


title: pDNA_EEP_Lefty1_Run1


title: cDNA_BR3_EEP_Nanog_Run1


title: pDNA_EEP_Nanog_Run1


title: cDNA_BR3_EEP_Lefty1_Run1


title: cDNA_BR2_EEP_Otx2_Run1


title: cDNA_BR1_EEP_Otx2_Run1


title: pDNA_EEP_Fgf5_Run1


title: cDNA_BR3_EEP_Otx2_Run1


title: pDNA_EEP_Otx2_Run1


title: cDNA_BR3_EEP_Klf2_Run1


title: cDNA_BR3_EEP_Tbx3_Run1


title: cDNA_BR2_EEP_Klf2_Run1


title: cDNA_BR2_EEP_Lefty1_Run1


title: cDNA_BR1_EEP_Klf2_Run1


title: cDNA_BR2_EEP_Fgf5_Run1


title: cDNA_BR3_EEP_Sox2_Run1


title: cDNA_BR2_EEP_Sox2_Run1


title: pDNA_EEP_Sox2_Run1


title: DIRECT-mESC-4d-8

city: Xiamen

first: Xi

last: Zeng

state: Fujian

address: No.422, Siming South Road

country: China

institute: Xiamen University

description: DIRECT: single-cell DNA methylation and gene expression co-analysis in a single library

library_source: other

data_processing:
The original sequence data obtained contain 150bp paired-end sequences. Before alignment, Trim Galore version 0.6.7 was used for adapter removal. Trimmed clean sequences were aligned and de-duplicated with hg19/mm10 by the bisulfite alignment tool Bismark version v0.15.0. The obtained bam files were converted into sam files using samtools version 1.3.1.
For the sam files, after the initial comparison using Bismark, we obtained the methylation information of each read based on the XM-tag (methylation call string) in the sam files. We calculated the methylation levels of CHH and CHG for each read and distinguished the reads into methylomic reads and transcriptomic reads by the methylation levels. When the methylation level of the reads was greater than 90%, they were classified as transcriptomic reads, and when the methylation level of the reads was less than 50%, they were classified as methylomic reads. For the analysis of methylomic reads, we extracted CpG methylation site information using the common methylation analysis pipeline and the R package methylKit version 1.22.0. For the isolated transcriptomic reads, the transcriptome analysis pipeline was used, which mainly included realignment using STAR version 2.7.3a and obtaining the expression profiles of the cells using htseq version 0.12.4.
For clustering using transcriptome data, the expression matrix of cells was obtained from expression profiles and then clustered using R package SC3 version 1.24.0. For the clustering of the methylome data, the Pearson correlation coefficients for the methylation levels of the commonly covered methylation sites were calculated and clearer clusters could be seen on the correlation matrix.
We used the methylome data as DNA data to perform copy number variation analysis. We used the bwa tool version 0.7.17 to perform genomic alignment and analysis. The bam files after alignment were converted to bed files for input into ginkgo for copy number variation analysis. The length of a bin was set to the default of 1M. The cell copy number variance profiles were obtained by feeding the bed files to ginkgo.
We input the gene expression matrix obtained from the transcriptome analysis pipeline into the R package DESeq2 version 1.36.0, and obtained the differentially expressed genes. For the enriched pathway analysis, we input the gene list into the enrichGO function in the R package clusterProfiler version 4.4.4 to obtain the enriched pathways. The Gene Set Enrichment Analysis (GSEA) was performed using the python package gseapy version 1.0.5.
Assembly: hg19, mm10
Supplementary files format and content: CpG.txt file containing methylation information for CpG sites.
Supplementary files format and content: counts.txt file containing gene expression information.
Library strategy: DIRECT

molecule: other

growth_protocol: All cultures were maintained at 37ºC in a humidified incubator with 5% CO2. The human chronic myeloid leukemia cell line K562 and acute myeloid leukemia cell line HL60 were cultured in IMDM, supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin-Glutamine.

extract_protocol:
Single cells were isolated with the Digital microfluidics chip. By incorporating 2'-deoxy-5-methylcytidine 5'-triphosphate (5-methyl dCTP) during reverse transcription and cDNA amplification, the resulting library of RNA and gDNA sequencing reads exhibit drastically different methylation patterns. Single-cell DNA methylation and gene expression information were obtained simultaneously, without the need to physically separate DNA and RNA.
After single-cell isolation, droplets of lysis buffer are introduced to lyse the cells. The lysates are then subjected to RT and cDNA amplification using 5’-methyl-dCTP instead of dCTP, so that all cytosines in the synthesized cDNA are methylcytosines, while the genomic DNA remains unchanged during the process. Subsequently, both the cDNA and gDNA undergo bisulfite conversion concurrently, during which normal cytosines are converted to uracil, while methylated cytosines remain unchanged. Consequently, fully cytosine-methylated cDNA produced from RNA remains unaffected by the C-U transition. In contrast, less than 10% of mammalian genomic cytosines are endogenously methylated, resulting in a substantial conversion of gDNA cytosines to uracil, which will be identified as thymine in sequencing. After scBS-seq-based library construction, the DNA reads are distinguished from RNA reads based on the methylated cytosine density in reads, and thus both DNA methylation and gene expression information are obtained from a single-cell library.
smart-seq2 and scBS

treatment_protocol: Mouse embryonic stem cells (mESCs; E14, 129/Ola) were grown on 6-cm culture dishes with MEF feeder cells and cultured in a complete medium supplemented with leukemia inhibitory factor (LIF) to prevent differentiation. To induce undirectional differentiation, mESCs were dissociated and replated onto 0.1% gelatin-coated dishes, and cultured without LIF in a feeder-free manner for two passages.

tag: strain

value: mouse embryonic stem cell

tag: treatment

value: withdrawal of Leukemia inhibitory factor

tag: time


title: cDNA_BR1_EEP_Fgf5_Run1


title: DIRECT-mESC-0d-7


title: DIRECT-mESC-4d-6


title: DIRECT-mESC-4d-5


title: DIRECT-mESC-0d-8


title: DIRECT-mESC-0d-6


title: DIRECT-mESC-0d-5


title: DIRECT-mESC-4d-3


title: HTx020Mp12F, snATAC

city: Saint Louis

first: Li

last: Ding

state: MO

address: 660 S. Euclid Avenue

country: USA

institute: Washington University in St. Louis

description: 10x genomics

data_processing:
cellranger-arc-2.0.0 (https://support.10xgenomics.com/single-cell-multiome-atac-gex/software/pipelines/latest/installation)
Assembly: refdat a-cellranger-arc-mm10-2020-A-2.0.0
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Mammary Gland

extract_protocol:
dx.doi.org/10.17504/protocols.io.261gednx7v47/v1
Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Kits
snRNAseq/snATACseq Multiome Protocol

tag: tissue

value: Mammary Gland

value: B6.FVB-Tg(MMTV-PyVT) 634Mul/LellJ (Strain #:022974)

tag: Sex

value: female

tag: age

value: 12 weeks


title: cDNA_BR1_EEP_Sox2_Run1


title: DIRECT-mESC-0d-4


title: DIRECT-mESC-0d-3


title: DIRECT-mESC-0d-2


title: DIRECT-mESC-4d-1


title: DIRECT-mESC-4d-2


title: HTx020Mp12F, snRNA

description: 10x genomics


title: pDNA_EEP_Klf2_Run1


title: Young_Male_nmrHAS2_WAT_1

city: Rochester

first: JINLONG

middle: Yuyang

last: LU

state: NY

address: Hutchison Rd

institute: University of Rochester

data_processing:
Raw fastq reads files were cleaned by Trim Galore 0.6.5
Gene expression (reads counts) were calculated by salmon 1.4.0
Gencode M28 was used for the reference. The Has2 sequence of the naked mole rats was added manually.
Supplementary files format and content: tab-delimited text files includes raw counts for each Sample

extract_protocol: For preparing RNA from tissues, pulverized frozen tissues in the range of 10-15 mg were removed from samples kept at −80°C and extracted using Trizol reagent according to the supplier’s instructions. After recovery of total RNA from the Trizol reagent by isopropanol precipitation, RNA was digested with DNaseI for 30min at room temperature and further purified by RNeasy plus mini kit according to the instruction. For purifying RNA from cells, the RNeasy plus mini kit was used according to the user manual.

value: White adipose tissue

value: male

tag: genotype

value: 6 months


title: Young_Male_nmrHAS2_WAT_3


title: Young_Male_nmrHAS2_WAT_2


title: Young_Male_nmrHAS2_Spleen_3

value: Spleen


title: DIRECT-mESC-4d-7


title: Young_Male_nmrHAS2_Spleen_2


title: DIRECT-mESC-4d-4


title: Young_Male_nmrHAS2_Spleen_1


title: Young_Male_nmrHAS2_Muscle_2

value: Muscle


title: DIRECT-mESC-0d-1


title: Young_Male_nmrHAS2_Muscle_1


title: Young_Male_nmrHAS2_Liver_2

value: Liver


title: Young_Male_nmrHAS2_Liver_3


title: Young_Male_CreER_Muscle_2

value: CreER


title: Young_Male_CreER_Kidney_3

value: Kidney


title: Young_Male_CreER_Spleen_2


title: Young_Male_CreER_Liver_2


title: Young_Male_nmrHAS2_Kidney_1


title: Young_Male_nmrHAS2_intestine_3

value: Small intestine


title: Young_Male_CreER_Muscle_3


title: Young_Male_nmrHAS2_Kidney_2


title: Young_Male_CreER_WAT_1


title: Young_Male_nmrHAS2_intestine_2


title: Young_Male_CreER_Liver_3


title: Young_Male_CreER_WAT_3


title: Young_Male_nmrHAS2_Kidney_3


title: Young_Male_CreER_Kidney_2


title: Young_Male_CreER_Spleen_3


title: Young_Male_CreER_Muscle_1


title: Young_Male_CreER_intestine_3


title: Young_Male_nmrHAS2_intestine_1


title: Young_Male_CreER_Liver_1


title: Young_Male_CreER_WAT_2


title: Young_Male_CreER_Kidney_1


title: Young_Male_nmrHAS2_Muscle_3


title: Young_Male_CreER_intestine_2


title: Young_Female_nmrHAS2_WAT_3


title: Young_Female_nmrHAS2_WAT_2


title: Young_Male_nmrHAS2_Liver_1


title: Young_Male_CreER_intestine_1


title: Young_Female_nmrHAS2_WAT_1


title: Young_Female_nmrHAS2_spleen_3


title: Young_Male_CreER_Spleen_1


title: Young_Female_nmrHAS2_liver_2


title: Young_Female_nmrHAS2_liver_1


title: Young_Female_nmrHAS2_liver_3


title: Young_Female_nmrHAS2_intestine_3


title: Young_Female_CreER_WAT_3


title: Young_Female_CreER_spleen_2


title: Young_Female_nmrHAS2_intestine_1


title: Young_Female_CreER_WAT_1


title: Young_Female_nmrHAS2_muscle_2


title: Young_Female_nmrHAS2_intestine_2


title: Young_Female_CreER_spleen_1


title: Young_Female_CreER_spleen_3


title: Young_Female_nmrHAS2_spleen_1


title: Young_Female_nmrHAS2_muscle_3


title: Young_Female_CreER_liver_2


title: Young_Female_nmrHAS2_spleen_2


title: Young_Female_CreER_liver_1


title: Young_Female_CreER_muscle_1


title: Young_Female_CreER_kidney_2


title: Young_Female_CreER_kidney_1


title: Young_Female_CreER_WAT_2


title: Young_Female_nmrHAS2_kidney_3


title: Young_Female_nmrHAS2_kidney_1


title: Young_Female_CreER_muscle_2


title: Young_Female_CreER_liver_3


title: Young_Female_CreER_muscle_3


title: Young_Female_CreER_intestine_3


title: Young_Female_CreER_kidney_3


title: Young_Female_nmrHAS2_kidney_2


title: Young_Female_nmrHAS2_muscle_1


title: Young_Female_CreER_intestine_2


title: Old_Male_nmrHAS2_Spleen_2

value: 24 months


title: Old_Male_nmrHAS2_Spleen_3


title: Old_Male_nmrHAS2_muscle_3


title: Old_Male_nmrHAS2_WAT_2


title: Old_Male_nmrHAS2_WAT_1


title: Young_Female_CreER_intestine_1


title: Old_Male_nmrHAS2_muscle_2


title: Old_Male_nmrHAS2_Spleen_1


title: Old_Male_nmrHAS2_WAT_3


title: Old_Male_nmrHAS2_muscle_1


title: Old_Male_nmrHAS2_Kidney_1


title: Old_Male_nmrHAS2_intestine_1


title: Old_Male_nmrHAS2_Kidney_3


title: Old_Male_nmrHAS2_intestine_3


title: Old_Male_CreER_WAT_1


title: Old_Male_nmrHAS2_liver_3


title: Old_Male_nmrHAS2_liver_2


title: Old_Male_CreER_Spleen_2


title: Old_Male_nmrHAS2_intestine_2


title: Old_Male_nmrHAS2_Kidney_2


title: Old_Male_CreER_Spleen_3


title: Old_Male_nmrHAS2_liver_1


title: Old_Male_CreER_Spleen_1


title: Old_Male_CreER_WAT_2


title: Old_Male_CreER_WAT_3


title: Old_Male_CreER_muscle_3


title: Old_Male_CreER_liver_3


title: Old_Male_CreER_muscle_1


title: Old_Male_CreER_muscle_2


title: Old_Male_CreER_Kidney_2


title: Old_Male_CreER_liver_1


title: Old_Male_CreER_Kidney_1


title: Old_Male_CreER_intestine_2


title: Old_Male_CreER_intestine_1


title: Old_Male_CreER_Kidney_3


title: Old_Female_nmrHAS2_WAT_1


title: Old_Male_CreER_intestine_3


title: Old_Female_nmrHAS2_WAT_2


title: Old_Female_nmrHAS2_muscle_3


title: Old_Female_nmrHAS2_muscle_1


title: Old_Female_nmrHAS2_muscle_2


title: Old_Female_nmrHAS2_liver_2


title: Old_Male_CreER_liver_2


title: Old_Female_nmrHAS2_spleen_2


title: Old_Female_nmrHAS2_spleen_3


title: Old_Female_nmrHAS2_WAT_3


title: Old_Female_nmrHAS2_spleen_1


title: Old_Female_nmrHAS2_kidney_2


title: Old_Female_nmrHAS2_intestine_1


title: Old_Female_nmrHAS2_kidney_1


title: Old_Female_nmrHAS2_intestine_2


title: Old_Female_CreER_WAT_3


title: Old_Female_nmrHAS2_kidney_3


title: Old_Female_nmrHAS2_intestine_3


title: Old_Female_CreER_WAT_2


title: Old_Female_CreER_WAT_1


title: Old_Female_nmrHAS2_liver_3


title: Old_Female_CreER_spleen_2


title: Old_Female_CreER_spleen_1


title: Old_Female_CreER_muscle_2


title: Old_Female_nmrHAS2_liver_1


title: Old_Female_CreER_liver_3


title: Old_Female_CreER_muscle_1


title: Old_Female_CreER_liver_1


title: Old_Female_CreER_liver_2


title: Old_Female_CreER_kidney_2


title: Old_Female_CreER_kidney_3


title: Old_Female_CreER_intestine_2


title: Old_Female_CreER_intestine_1


title: Old_Female_CreER_intestine_3


title: Old_Female_CreER_kidney_1


title: Old_Female_CreER_spleen_3


title: Trypanosome-infected mice, bone marrow, scRNA-seq

city: Incheon

first: Magdalena

last: Radwanska

address: 119-5, Songdomunhwa-ro

country: South Korea

institute: Ghent University Global Campus

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were conducted using the Cell Ranger software v7.0.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Bone marrow

extract_protocol:
Bone marrow cells were collected from CO2 euthanized mice by isolating and flushing femurs, using a 26-gauge needle and 1 mL ice-cold DMEM (Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 1% FBS (Atlas Biologicals, CO, USA) and 1% penicillin/streptomycin (Capricorn Scientific, Ebsdorfergrund, Germany. Cell suspensions were centrifuged at 314 x g at 4oC for 7 mins. Cell pellets were re-suspended in 3 mL FACSFlow Sheath Fluid (BD Biosciences, CA, USA) containing 0.05% FBS (Atlas Biologicals, CO, USA) and total remaining live cells were counted using Trypan Blue (Sigma-Aldrich, Missouri, USA).
Library was performed according to the manufacter’s instructions (single cell 3’ v3 protocol, 10x Genomics). Briefly, bone marrow cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was reversetranscribed to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. These were loaded into single cell chips (10X Genomics) and partitioned using Gel Bead In-Emulsion (GEM) technology and a Chromium Controller (10X Genomics). Single cell libraries were prepared using the 10X Genomics Chromium Single Cell 3`reagent kit v3. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end P5 and P7 primers and a sample index were added.

value: Bone marrow cells

value: Bone marrow

value: 6-8 weeks old

tag: infection

value: Trypanosome-infected


title: Non-infected mice, bone marrow, scRNA-seq

description: 10x Genomics

value: Naive


title: CaBl: NPCs_WT1_RNAseq_rep3

city: Freiburg

first: Asifa

last: Akhtar

address: Stuebeweg 51

department: Chromatin Regulation

country: Germany

institute: Max Planck Institute of Immunobiology and Epigenetics

data_processing:
RNAseq and TTseq: The data are processed via snakePipes mRNA-seq pipeline (developing branch) (Bhardwaj et al., 2019). SnakePipes removed adapters and low quality bases (< Q20) using TrimGalore (v0.6.5) with parameters '-q 20 --trim-n'. The trimmed reads are aligned with STAR (v2.7.4) (Dobin et al., 2013) to an ‘N-masked’ genome, which is a version of the CAST(MAT)*C57BL(PAT) reference genome mm10 where all the polymorphicsites (SNPs) for Mus musculus CAST/EiJ and Mus musculus C57BL/6 (or 129S1/SvImJ) are masked by ambiguity nucleobase ‘N’ (Keane et al., 2011). The mapped reads are then passed to SNPSplit (0.3.4) (Krueger and Andrews, 2016) to generate allele-specific BAM files by separating the alignment into two distinct alleles (CAST/EiJ and C57BL/6 or 129S1/SvImJ) based on SNPs information downloaded from CAST(MAT)*C57BL(PAT) Genome Database (Keane et al., 2011). The aligned reads of standard and allele specific level are counted separately on Gencode GTF (m9) via featureCounts (v2.0.0) (Yang et al., 2014).
ChIPseq: The data are processed via snakePipes DNA-mapping and ChIP-seq pipeline (developing branch) (Bhardwaj et al., 2019). Adapters and low quality bases (< Q20) are removed using TrimGalore (v0.6.5) with parameters '-q 20 --trim-n'. For all samples, reads are then mapped to the ‘N-masked’’ genome with Bowtie2 (2.3.5) (Langmead and Salzberg, 2012). Reads mapped on blacklisted regions from Encode Consortium are discarded. Duplicated reads are also filtered with Picard MarkDuplicates (1.65) (https://broadinstitute.github.io/picard/). In the end, only properly paired mapped reads and reads with mapping quality over 3 are kept for further analysis. SNPSplit (0.3.4) (Krueger and Andrews, 2016) is then used to generate allele-specific BAM files and deepTools (v3.3.2) (Ramirez et al., 2014) is used to check data quality. In the ChIP-seq part, peak calling is performed with MACS2 (v2.2.6) (Zhang et al., 2008) with options [--qvalue 0.001]. It is done with input as control on total and allele specific ChIP-seq signals respectively. CSAW (v1.20.0) (Lun and Smyth, 2015) is additionally used to calculate log2FC on peak regions between MSL2 KO and WT or between allele2 and allele1. Bigwig files are created with deepTools bamCompare (v3.3.2) (Ramirez et al., 2014), using input normalization method and log2ratio and subtract option.
ATACseq: The data are processed via snakePipes DNA-mapping and ATAC-seq pipeline (developing branch) (Bhardwaj et al., 2019). In the DNA-mapping part, adapters and low quality bases (< Q20) are removed using TrimGalore (v0.6.5) with parameters '-q 20 --trim-n'. For all samples, reads are then mapped to the ‘N-masked’’ genome with Bowtie2 (v2.3.5) (Langmead and Salzberg, 2012). Reads mapped on blacklisted regions from Encode Consortium are discarded. Duplicated reads are also filtered with Picard MarkDuplicates (v1.65) (https://broadinstitute.github.io/picard/). In the end, only properly paired mapped reads and reads with mapping quality over 3 are kept for further analysis. SNPSplit (0.3.4) (Krueger and Andrews, 2016) is then used to generate allele-specific BAM files and deepTools (v3.3.2) (Ramirez et al., 2014) is used to check data quality. In the ATAC-seq part, the BAM files are filtered to include only properly paired reads with appropriate fragment sizes (<150 bases). To identify open/accessible regions, peak calling is done with MACS2 (v2.2.6) (Zhang et al., 2008) with options [--qvalue 0.001], on total and allele-specific ATAC-seq signal respectively. CSAW (v1.20.0) (Lun and Smyth, 2015) is additionally used to calculate log2FC on peak regions between MSL2 KO and WT or between allele2 and allele1. Bigwig files are created with deepTools bamCoverage (v3.3.2) (Ramirez et al., 2014), using size factor calculated by deepTools multiBamSummary (v3.3.2).
BSseq: Raw fastq files were aligned against the ‘N-masked’ genome and deduplicated using Bismark (v0.22.3) (Krueger and Andreas, 2011). Resulting Alignments were allele-separated using SNPsplit with flags '--paired' and '--bisulfite'. Relevant quality metrics (conversion rates, genome coverage and GC bias) were calculated using MethylDackel version 0.6.0 (https://github.com/dpryan79/MethylDackel) and DeepTools (v3.5.0). Allele-specific differentially CpG methylated sites between MSL2 KO and WT or between allele2 and allele1 were called with DSS version 2.34.0.
Assembly: mm10
Supplementary files format and content: bigWig files: log2 ratio or subtract value over the input sample (merged replicates) . bed: peak calling from MACS2 (merged replicates). RData: differential peak calling result from CSAW and differential CpG methylation sites from DSS. tsv: differential expression analysis from DESeq2. txt: Differentially expression genes further classification into five cateogories.

source_name: Neural progenitor cells

growth_protocol: Male CAST(MAT)*C57BL(PAT) ES cells, obtained from Anne Ferguson-Smith (University of Cambridge), were cultured in NDiff 227 medium (Takara) supplemented with LIF (Millipore), 2i inhibitors (PD0325901 and CHIR99021, Stemgent). Male CAST(MAT)*C57BL(PAT) ES cells, obtained from Edith Heard (EMBL Heidelberg), were cultured in KnockOut-DMEM (KO-DMEM, Gibco) supplemented with 15% KnockOut Serum Replacement (KOSR, Gibco), GlutaMAX (Gibco), non-essential amino acids (Gibco), sodium pyruvate (Gibco), 2-Mercaptoethanol (Gibco), LIF, and 2i inhibitors. Male and Male ES cells were differentiated into NPCs as previously described (Conti et al. 2005; Splinter et al. 2011). In brief, 1 million male or Male ES cells were plated on 0.1% gelatin-coated plates in N2B27 medium, consisting of 1:1 DMEM/F-12 (Gibco) and Neurobasal medium (Gibco), supplemented with B27 (Gibco), GlutaMAX, 2-Mercaptoethanol, Apo-transferrin (Sigma), bovine serum albumin (Sigma), insulin (Sigma), putrescine (Sigma), progesterone (Sigma), and sodium selenite (Sigma). After 7 days, cells were dissociated and 3 million cells were plated on non-adherent culture dishes in the presence of 10ng/ml epidermal growth factor (EGF, Peprotech) and 10ng/ml basic fibroblast growth factor (FGF, Peprotech) to induce formation of embryoid bodies. After 3 days, the embryoid bodies were transferred to gelatin-coated plates to allow the expansion of NPCs.

extract_protocol:
RNAseq: Total RNA was extracted using the Direct-zol RNA Miniprep Plus Kit (Zymo Research) according to the manufacturers’ instructions. Maxima First Strand cDNA Synthesis Kit (Thermo Scientific) was used to synthesize cDNA from total RNA according to the manufacturers’ instructions. Quantitative reverse transcription PCR was carried out on Roche LightCycler II using the Faststart SYBR Green Master (Rox) mix (Roche) at a final volume of 10μL.
TT-seq: In brief, 10 million NPCs were incubated with N2B27 medium supplemented with 500 uM 4- thiouridine (4sU, Sigma) for 5 minutes at 37°C. Total RNA was isolated, fragmented using Bioruptor Plus for 1 minute (30s ON, 30s OFF, high setting), and incubated with Biotin-HPDP (Thermo Scientific) for 2 hours. MyOne C1 Streptavidin magnetic beads (Thermo Scientific) were used to immunoprecipitate labelled RNA before elution in 5% beta-mercaptoethanol (Roth). FInal RNA was cleaned up using the Oligo Clean and Concentrator Kit (Zymo Research) according to the manufacturer’s instructions. Libraries were prepared from 100 ng of high quality RNA using the Ovation Universal RNA-Seq System (Nugen) according to the manufacturer’s instructions.
ChIPseq: MSL2 WT and KO NPCs were covered with 1% formaldehyde in culture media on the plate and incubated at room temperature (RT) on a rocking plate for 10 min. Media was removed and cells were washed twice with 1xPBS supplemented with 1x protease inhibitor cocktail (Roche). Cells were scraped off the plate, collected in ice-cold 1xPBS and centrifuged at 500g for 5 minutes. After an additional 1xPBS wash, cell pellets were stored at -80°C until usage. Nuclei were isolated using the NEXSON based nuclei isolation protocol. Cell pellets were thawed on ice and resuspended in ice cold 1ml of lysis buffer (10mM Tris-HCl pH 8, 10mM NaCl, 0.2% Igepal, 1x protease inhibitor cocktail). The cell suspension was then transferred into 1ml milliTUBE (Covaris) and sonicated in Covaris instrument (E220) for 30 sec at peak power 75W, duty factor 2% and 200 cycles/burst. Nuclei were pelleted at 1000g at 20°C for 5min. The cytoplasmic fraction in the supernatant was discarded and nuclear pellets were carefully resuspended in 0.5% SDS and incubated at room temperature for 10min. Sample was diluted 1:5 in the digestion buffer (Final concentration: 1.1% Triton, 1x CutSmart buffer, nuclease free H2O, 1x protease inhibitor cocktail). Chromatin of up to 1 million nuclei were digested in 250ul digestion buffer with Cvik1 (5U/100.000 cells, New England Biolabs) at 20°C for 16hrs, shaking at 800rpm. After the chromatin digestion, the nuclei were pelleted for 5min at 1000g and washed in cold nuclei wash solution (10mM Tris-HCl pH 8, 0.25% Triton, 0.2mg/ml BSA) and stored on ice. To confirm the enzymatic chromatin shearing efficiency, 5% of the nuclei solution was incubated together with a decrosslinking solution consisting of 2ul proteinase K, 1ul RNaseA and 4ul 5M NaCl at 37°C for 30min followed by a 2hr incubation at 65°C. DNA was purified using MinElute PCR purification kit (Qiagen) and analyzed on a Agilent Tapestation system (G2964AA) using the Agilent D1000 Screentape reagents.
ATACseq: In brief, nuclei of 50,000 cells per replicate were isolated in 50 μl of cold ATAC resuspension buffer (0.1% NP-40, 0.1% Tween-20, and 0.01% digitonin (Promega)) by incubating on ice for 3 minutes. After lysis, 1 ml of cold ATAC resuspension buffer containing 0.1% Tween-20 (without NP-40 or digitonin) was added, and nuclei were then centrifuged at 500g for 10 minutes. Transposition was carried out using 50 μl of transposition mix (25 μl 2X TD buffer (Illumina), 2.5 μl transposase (Illumina), 16.5 μl 1X PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, and 5 μl water) at 37°C for 30 minutes in a thermomixer with shaking at 1000 rpm. Transposed DNA was collected using Zymo DNA Clean and Concentrator (Zymo Research) according to the manufacturer's instructions, and added to the PCR mix containing 25 μl of NEBNext Master Mix (New England Biolabs) and 5 μl of Index adapters (Illumina). Libraries were amplified using the following PCR program: 72°C for 5 minutes, 98°C for 30 seconds, then 10 cycles of: 98°C for 10 seconds, 63°C for 30 seconds, and 72°C for 1 minute. Library quality was analyzed using the Fragment analyzer (Agilent Technologies) prior to sequencing.
BSseq: Bisulfite-Seq was performed using the NEBNext Enzymatic Methyl-seq Kit (E7120S) according to the manufacturer’s instructions. In brief, genomic DNA (gDNA) was extracted from wild-type and MSL2 knockout NPCs using the QIAamp DNA Mini kit (Qiagen) according to the manufacturer’s instructions.Subsequently, 50ng of high quality gDNA was sheared for 120 seconds using an E220 Covaris to obtain fragments between around 300 base pairs in length. Following end prep and EM-seq adapter ligation, DNA was oxidised and denatured using sodium hydroxide. Libraries were amplified for 6 PCR cycles, and sequenced with 2x100bp paired-end reads on an Illumina NovaSeq6000 sequencer.

treatment_protocol: For the MSL2 knockout generation, two guide RNAs (gRNAs) were cloned into a PX-459 derivative (pSpCas9(BB)-2A-Puro) plasmid (Addgene 62988), and a split puromycin resistance or GFP-positive approach for selection was used (Flemr and Bühler 2015). Male and Male ES cells and NPCs were transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. After 48 hours, the cells were sorted based on their GFP expression status using a MoFlo XDP Cell Sorter (Beckman Coulter). Genomic DNA from single clones was isolated by resuspending cells in lysis buffer (10 mM Tris pH 8.0, 0.5 mM Ethylenediaminetetraacetic acid (EDTA), 0.5% Triton X-100) supplemented with Proteinase K (Thermo Scientific), and incubated at 55°C for 1 hour. Genomic DNA was collected after centrifugation at 16,000 g for 10 minutes. Polymerase chain reaction (PCR) was carried out using the 2X PCR Master Mix (Qiagen) at a final volume of 10μL. Successful knockout was verified using CloneJet PCR Cloning (Thermo Scientific) and BigDye Direct Sanger Sequencing (Thermo Scientific) kits according to the manufacturer’s instructions.

value: Neural progenitor cells

tag: gender

value: Male

value: WT

tag: library type

value: RNAseq

tag: replicate


title: CaBl: NPCs_MSL2KO2_Pol2_rep1


title: CaBl: NPCs_WT2_RNAseq_rep3


title: CaBl: NPCs_WT2_RNAseq_rep2


title: CaBl: NPCs_MSL2KO2_MOF_rep2


title: CaBl: NPCs_WT2_RNAseq_rep1


title: CaBl: NPCs_MSL2KO2_Pol2_rep2


title: CaBl: NPCs_WT3_RNAseq_rep3


title: CaBl: NPCs_WT3_RNAseq_rep1


title: Old_Female_CreER_muscle_3

value: female


title: CaBl: NPCs_WT1_RNAseq_rep1


title: CaBl: NPCs_WT3_RNAseq_rep2


title: CaBl: NPCs_MSL2KO2_MOF_rep1


title: CaBl: NPCs_MSL2KO2_KANSL3_rep2


title: CaBl: NPCs_MSL2KO2_SP1_rep1


title: CaBl: NPCs_MSL2KO2_SP1_rep2


title: CaBl: NPCs_MSL2KO2_KANSL3_rep1


title: CaBl: NPCs_MSL2KO2_NRF1_rep1


title: CaBl: NPCs_WT1_RNAseq_rep2


title: CaBl: NPCs_MSL2KO2_BRD4_rep2


title: CaBl: NPCs_MSL2KO2_BRD4_rep1


title: CaBl: NPCs_MSL2KO2_Input3_rep2

description: Same batch as MSL2, KANSL3, SP1, Pol2,  MOF


title: CaBl: NPCs_MSL2KO2_Input3_rep1


title: CaBl: NPCs_MSL2KO2_H4K5ac_rep2


title: CaBl: NPCs_MSL2KO2_H4K5ac_rep1


title: CaBl: NPCs_MSL2KO2_Input2_rep1

description: Same batch as NRF1, BRD4


title: CaBl: NPCs_MSL2KO2_Input1_rep1

description: Same batch as H3K4me1, H3K4me3, H3K27ac, H3K27me3, H3K36me3, H3K9me3, H4K5ac, H4K12ac


title: CaBl: NPCs_MSL2KO2_H3K9me3_rep2


title: CaBl: NPCs_MSL2KO2_H3K9me3_rep1


title: CaBl: NPCs_MSL2KO2_H4K12ac_rep2


title: CaBl: NPCs_MSL2KO2_H4K12ac_rep1


title: CaBl: NPCs_MSL2KO2_H3K4me1_rep2


title: CaBl: NPCs_MSL2KO2_H3K4me1_rep1


title: CaBl: NPCs_MSL2KO2_H3K27me3_rep1


title: CaBl: NPCs_MSL2KO2_H3K36me3_rep1


title: CaBl: NPCs_MSL2KO2_H3K36me3_rep2


title: CaBl: NPCs_MSL2KO2_H3K27me3_rep2


title: CaBl: NPCs_MSL2KO2_NRF1_rep2


title: CaBl: NPCs_MSL2KO2_H3K4me3_rep2


title: CaBl: NPCs_MSL2KO2_H3K27ac_rep2


title: CaBl: NPCs_MSL2KO2_H3K27ac_rep1


title: CaBl: NPCs_MSL2KO1_Pol2_rep2


title: CaBl: NPCs_MSL2KO2_Input2_rep2

description: Same batch as NRF1, BRD4


title: CaBl: NPCs_MSL2KO1_Pol2_rep1


title: CaBl: NPCs_MSL2KO2_Input1_rep2


title: CaBl: NPCs_MSL2KO1_MOF_rep1


title: CaBl: NPCs_MSL2KO1_KANSL3_rep2


title: CaBl: NPCs_MSL2KO1_SP1_rep1


title: CaBl: NPCs_MSL2KO1_SP1_rep2


title: CaBl: NPCs_MSL2KO1_NRF1_rep2


title: CaBl: NPCs_MSL2KO1_NRF1_rep1


title: CaBl: NPCs_MSL2KO1_BRD4_rep1


title: CaBl: NPCs_MSL2KO1_Input3_rep2


title: CaBl: NPCs_MSL2KO1_BRD4_rep2


title: CaBl: NPCs_MSL2KO2_H3K4me3_rep1


title: CaBl: NPCs_MSL2KO1_Input3_rep1


title: CaBl: NPCs_MSL2KO1_Input1_rep2


title: CaBl: NPCs_MSL2KO1_Input2_rep1

description: Same batch as NRF1, BRD4


title: CaBl: NPCs_MSL2KO1_Input1_rep1


title: CaBl: NPCs_MSL2KO1_MOF_rep2


title: CaBl: NPCs_MSL2KO1_H4K5ac_rep2


title: CaBl: NPCs_MSL2KO1_KANSL3_rep1


title: CaBl: NPCs_MSL2KO1_H4K12ac_rep1


title: CaBl: NPCs_MSL2KO1_H3K9me3_rep2


title: CaBl: NPCs_MSL2KO1_H4K12ac_rep2


title: CaBl: NPCs_MSL2KO1_H3K4me3_rep2


title: CaBl: NPCs_MSL2KO1_H3K4me1_rep1


title: CaBl: NPCs_MSL2KO1_H3K4me1_rep2


title: CaBl: NPCs_MSL2KO1_H3K4me3_rep1


title: CaBl: NPCs_MSL2KO1_H3K9me3_rep1


title: CaBl: NPCs_MSL2KO1_H3K36me3_rep2


title: CaBl: NPCs_MSL2KO1_H3K36me3_rep1


title: CaBl: NPCs_MSL2KO1_H3K27me3_rep2


title: CaBl: NPCs_MSL2KO1_H3K27me3_rep1


title: CaBl: NPCs_MSL2KO1_H3K27ac_rep2


title: CaBl: NPCs_MSL2KO1_Input2_rep2

description: Same batch as NRF1, BRD4


title: CaBl: NPCs_Control_MSL2_rep1


title: CaBl: NPCs_Control_MOF_rep2


title: CaBl: NPCs_Control_MSL2_rep2


title: CaBl: NPCs_Control_Pol2_rep1


title: CaBl: NPCs_Control_Pol2_rep2


title: CaBl: NPCs_Control_KANSL3_rep2


title: CaBl: NPCs_MSL2KO1_H4K5ac_rep1


title: CaBl: NPCs_Control_MOF_rep1


title: CaBl: NPCs_Control_SP1_rep2


title: CaBl: NPCs_Control_KANSL3_rep1


title: CaBl: NPCs_Control_SP1_rep1


title: CaBl: NPCs_Control_NRF1_rep2


title: CaBl: NPCs_Control_NRF1_rep1


title: CaBl: NPCs_Control_BRD4_rep2


title: CaBl: NPCs_Control_BRD4_rep1


title: CaBl: NPCs_Control_Input2_rep2

description: Same batch as NRF1, BRD4


title: CaBl: NPCs_Control_Input3_rep2


title: CaBl: NPCs_Control_Input2_rep1

description: Same batch as NRF1, BRD4


title: CaBl: NPCs_MSL2KO1_H3K27ac_rep1


title: CaBl: NPCs_Control_Input1_rep1


title: CaBl: NPCs_Control_H4K5ac_rep1


title: CaBl: NPCs_Control_Input1_rep2


title: CaBl: NPCs_Control_H4K12ac_rep2


title: CaBl: NPCs_Control_H4K12ac_rep1


title: CaBl: NPCs_Control_H3K4me3_rep1


title: CaBl: NPCs_Control_H3K4me3_rep2


title: CaBl: NPCs_Control_H3K9me3_rep2


title: CaBl: NPCs_Control_H3K4me1_rep1


title: CaBl: NPCs_Control_Input3_rep1


title: CaBl: NPCs_Control_H3K36me3_rep1


title: CaBl: NPCs_Control_H3K27ac_rep2


title: CaBl: NPCs_Control_H3K27ac_rep1


title: CaBl: NPCs_Control_H3K27me3_rep1


title: CaBl: NPCs_Control_H3K27me3_rep2


title: CaBl: NPCs_Control_H4K5ac_rep2


title: BlCa: NPCs_WT3_RNAseq_rep2

data_processing:
RNAseq and TTseq: The data are processed via snakePipes mRNA-seq pipeline (developing branch) (Bhardwaj et al., 2019). SnakePipes removed adapters and low quality bases (< Q20) using TrimGalore (v0.6.5) with parameters '-q 20 --trim-n'. The trimmed reads are aligned with STAR (v2.7.4) (Dobin et al., 2013) to an ‘N-masked’ genome, which is a version of the C57BL(MAT)*CAST(PAT) reference genome mm10 where all the polymorphicsites (SNPs) for Mus musculus CAST/EiJ and Mus musculus C57BL/6 (or 129S1/SvImJ) are masked by ambiguity nucleobase ‘N’ (Keane et al., 2011). The mapped reads are then passed to SNPSplit (0.3.4) (Krueger and Andrews, 2016) to generate allele-specific BAM files by separating the alignment into two distinct alleles (CAST/EiJ and C57BL/6 or 129S1/SvImJ) based on SNPs information downloaded from C57BL(MAT)*CAST(PAT) Genome Database (Keane et al., 2011). The aligned reads of standard and allele specific level are counted separately on Gencode GTF (m9) via featureCounts (v2.0.0) (Yang et al., 2014).
ChIPseq: The data are processed via snakePipes DNA-mapping and ChIP-seq pipeline (developing branch) (Bhardwaj et al., 2019). Adapters and low quality bases (< Q20) are removed using TrimGalore (v0.6.5) with parameters '-q 20 --trim-n'. For all samples, reads are then mapped to the ‘N-masked’’ genome with Bowtie2 (2.3.5) (Langmead and Salzberg, 2012). Reads mapped on blacklisted regions from Encode Consortium are discarded. Duplicated reads are also filtered with Picard MarkDuplicates (1.65) (https://broadinstitute.github.io/picard/). In the end, only properly paired mapped reads and reads with mapping quality over 3 are kept for further analysis. SNPSplit (0.3.4) (Krueger and Andrews, 2016) is then used to generate allele-specific BAM files and deepTools (v3.3.2) (Ramirez et al., 2014) is used to check data quality. In the ChIP-seq part, peak calling is performed with MACS2 (v2.2.6) (Zhang et al., 2008) with options [--qvalue 0.001]. It is done with input as control on total and allele specific ChIP-seq signals respectively. CSAW (v1.20.0) (Lun and Smyth, 2015) is additionally used to calculate log2FC on peak regions between MSL2 KO and WT or between allele2 and allele1. Bigwig files are created with deepTools bamCompare (v3.3.2) (Ramirez et al., 2014), using input normalization method and log2ratio and subtract option.
ATACseq: The data are processed via snakePipes DNA-mapping and ATAC-seq pipeline (developing branch) (Bhardwaj et al., 2019). In the DNA-mapping part, adapters and low quality bases (< Q20) are removed using TrimGalore (v0.6.5) with parameters '-q 20 --trim-n'. For all samples, reads are then mapped to the ‘N-masked’’ genome with Bowtie2 (v2.3.5) (Langmead and Salzberg, 2012). Reads mapped on blacklisted regions from Encode Consortium are discarded. Duplicated reads are also filtered with Picard MarkDuplicates (v1.65) (https://broadinstitute.github.io/picard/). In the end, only properly paired mapped reads and reads with mapping quality over 3 are kept for further analysis. SNPSplit (0.3.4) (Krueger and Andrews, 2016) is then used to generate allele-specific BAM files and deepTools (v3.3.2) (Ramirez et al., 2014) is used to check data quality. In the ATAC-seq part, the BAM files are filtered to include only properly paired reads with appropriate fragment sizes (<150 bases). To identify open/accessible regions, peak calling is done with MACS2 (v2.2.6) (Zhang et al., 2008) with options [--qvalue 0.001], on total and allele-specific ATAC-seq signal respectively. CSAW (v1.20.0) (Lun and Smyth, 2015) is additionally used to calculate log2FC on peak regions between MSL2 KO and WT or between allele2 and allele1. Bigwig files are created with deepTools bamCoverage (v3.3.2) (Ramirez et al., 2014), using size factor calculated by deepTools multiBamSummary (v3.3.2).
BSseq: Raw fastq files were aligned against the ‘N-masked’ genome and deduplicated using Bismark (v0.22.3) (Krueger and Andreas, 2011). Resulting Alignments were allele-separated using SNPsplit with flags '--paired' and '--bisulfite'. Relevant quality metrics (conversion rates, genome coverage and GC bias) were calculated using MethylDackel version 0.6.0 (https://github.com/dpryan79/MethylDackel) and DeepTools (v3.5.0). Allele-specific differentially CpG methylated sites between MSL2 KO and WT or between allele2 and allele1 were called with DSS version 2.34.0.
Assembly: mm10
Supplementary files format and content: bigWig files: log2 ratio or subtract value over the input sample (merged replicates) . bed: peak calling from MACS2 (merged replicates). RData: differential peak calling result from CSAW and differential CpG methylation sites from DSS. tsv: differential expression analysis from DESeq2. txt: Differentially expression genes further classification into five cateogories.

growth_protocol: Male C57BL(MAT)*CAST(PAT) ES cells, obtained from Anne Ferguson-Smith (University of Cambridge), were cultured in NDiff 227 medium (Takara) supplemented with LIF (Millipore), 2i inhibitors (PD0325901 and CHIR99021, Stemgent). Male C57BL(MAT)*CAST(PAT) ES cells, obtained from Edith Heard (EMBL Heidelberg), were cultured in KnockOut-DMEM (KO-DMEM, Gibco) supplemented with 15% KnockOut Serum Replacement (KOSR, Gibco), GlutaMAX (Gibco), non-essential amino acids (Gibco), sodium pyruvate (Gibco), 2-Mercaptoethanol (Gibco), LIF, and 2i inhibitors. Male and Male ES cells were differentiated into NPCs as previously described (Conti et al. 2005; Splinter et al. 2011). In brief, 1 million male or Male ES cells were plated on 0.1% gelatin-coated plates in N2B27 medium, consisting of 1:1 DMEM/F-12 (Gibco) and Neurobasal medium (Gibco), supplemented with B27 (Gibco), GlutaMAX, 2-Mercaptoethanol, Apo-transferrin (Sigma), bovine serum albumin (Sigma), insulin (Sigma), putrescine (Sigma), progesterone (Sigma), and sodium selenite (Sigma). After 7 days, cells were dissociated and 3 million cells were plated on non-adherent culture dishes in the presence of 10ng/ml epidermal growth factor (EGF, Peprotech) and 10ng/ml basic fibroblast growth factor (FGF, Peprotech) to induce formation of embryoid bodies. After 3 days, the embryoid bodies were transferred to gelatin-coated plates to allow the expansion of NPCs.


title: BlCa: NPCs_WT3_RNAseq_rep1


title: BlCa: NPCs_WT2_RNAseq_rep2


title: BlCa: NPCs_WT2_RNAseq_rep3


title: BlCa: NPCs_WT1_RNAseq_rep3


title: CaBl: NPCs_Control_H3K9me3_rep1


title: BlCa: NPCs_WT1_RNAseq_rep1


title: BlCa: NPCs_WT1_RNAseq_rep2


title: BlCa: NPCs_MSL2KO1_Pol2_rep1


title: BlCa: NPCs_MSL2KO1_Pol2_rep2


title: BlCa: NPCs_MSL2KO1_MOF_rep2


title: BlCa: NPCs_MSL2KO1_SP1_rep2


title: BlCa: NPCs_WT2_RNAseq_rep1


title: BlCa: NPCs_MSL2KO1_MOF_rep1


title: CaBl: NPCs_Control_H3K36me3_rep2


title: CaBl: NPCs_Control_H3K4me1_rep2


title: BlCa: NPCs_MSL2KO1_NRF1_rep2


title: BlCa: NPCs_MSL2KO1_BRD4_rep2


title: BlCa: NPCs_MSL2KO1_NRF1_rep1


title: BlCa: NPCs_MSL2KO1_Input2_rep2

description: Same batch as MSL2, KANSL3, NRF1, SP1, Pol2, BRD4, MOF


title: BlCa: NPCs_MSL2KO1_BRD4_rep1


title: BlCa: NPCs_MSL2KO1_Input2_rep1


title: BlCa: NPCs_MSL2KO1_Input1_rep2


title: BlCa: NPCs_MSL2KO1_Input1_rep1


title: BlCa: NPCs_MSL2KO1_H4K5ac_rep2


title: BlCa: NPCs_MSL2KO1_H4K5ac_rep1


title: BlCa: NPCs_MSL2KO1_H4K12ac_rep2


title: BlCa: NPCs_MSL2KO1_H3K9me3_rep2


title: BlCa: NPCs_MSL2KO1_H3K4me3_rep2


title: BlCa: NPCs_MSL2KO1_H3K9me3_rep1


title: BlCa: NPCs_MSL2KO1_H3K4me1_rep2


title: BlCa: NPCs_MSL2KO1_H3K4me3_rep1


title: BlCa: NPCs_MSL2KO1_SP1_rep1


title: BlCa: NPCs_MSL2KO1_H3K4me1_rep1


title: BlCa: NPCs_MSL2KO1_H3K36me3_rep2


title: BlCa: NPCs_MSL2KO1_H3K27ac_rep2


title: BlCa: NPCs_MSL2KO1_H3K27me3_rep2


title: BlCa: NPCs_MSL2KO1_H3K36me3_rep1


title: BlCa: NPCs_Control_Pol2_rep2


title: BlCa: NPCs_MSL2KO1_H3K27ac_rep1


title: BlCa: NPCs_Control_Pol2_rep1


title: BlCa: NPCs_Control_MSL2_rep1


title: BlCa: NPCs_Control_MOF_rep2


title: BlCa: NPCs_Control_MOF_rep1


title: BlCa: NPCs_MSL2KO1_H4K12ac_rep1


title: BlCa: NPCs_Control_BRD4_rep2


title: BlCa: NPCs_Control_NRF1_rep2


title: BlCa: NPCs_Control_NRF1_rep1


title: BlCa: NPCs_Control_SP1_rep2


title: BlCa: NPCs_Control_BRD4_rep1


title: BlCa: NPCs_Control_Input2_rep1


title: BlCa: NPCs_Control_H4K5ac_rep2


title: BlCa: NPCs_Control_Input1_rep1


title: BlCa: NPCs_Control_Input1_rep2


title: BlCa: NPCs_MSL2KO1_H3K27me3_rep1


title: BlCa: NPCs_Control_H4K12ac_rep2


title: BlCa: NPCs_Control_H3K9me3_rep2


title: BlCa: NPCs_Control_H4K12ac_rep1


title: BlCa: NPCs_Control_MSL2_rep2


title: BlCa: NPCs_Control_H3K9me3_rep1


title: BlCa: NPCs_Control_H3K4me1_rep1


title: BlCa: NPCs_Control_H3K27me3_rep1


title: BlCa: NPCs_Control_H3K4me3_rep1


title: BlCa: NPCs_Control_H3K36me3_rep1


title: BlCa: NPCs_Control_SP1_rep1


title: BlCa: NPCs_Control_H3K36me3_rep2


title: BlCa: NPCs_Control_H3K27ac_rep2


title: BlCa: NPCs_Control_H3K4me1_rep2


title: BlCa: NPCs_Control_H3K27ac_rep1


title: BlCa: NPCs_Control_H3K27me3_rep2


title: BlCa: NPCs_Control_Input2_rep2


title: 9sCa: NPCs_MSL2KO2_NRF1_rep2

data_processing:
RNAseq and TTseq: The data are processed via snakePipes mRNA-seq pipeline (developing branch) (Bhardwaj et al., 2019). SnakePipes removed adapters and low quality bases (< Q20) using TrimGalore (v0.6.5) with parameters '-q 20 --trim-n'. The trimmed reads are aligned with STAR (v2.7.4) (Dobin et al., 2013) to an ‘N-masked’ genome, which is a version of the mouse reference genome mm10 where all the polymorphicsites (SNPs) for Mus musculus CAST/EiJ and Mus musculus C57BL/6 (or 129S1/SvImJ) are masked by ambiguity nucleobase ‘N’ (Keane et al., 2011). The mapped reads are then passed to SNPSplit (0.3.4) (Krueger and Andrews, 2016) to generate allele-specific BAM files by separating the alignment into two distinct alleles (CAST/EiJ and C57BL/6 or 129S1/SvImJ) based on SNPs information downloaded from Mouse Genome Database (Keane et al., 2011). The aligned reads of standard and allele specific level are counted separately on Gencode GTF (m9) via featureCounts (v2.0.0) (Yang et al., 2014).
ChIPseq: The data are processed via snakePipes DNA-mapping and ChIP-seq pipeline (developing branch) (Bhardwaj et al., 2019). Adapters and low quality bases (< Q20) are removed using TrimGalore (v0.6.5) with parameters '-q 20 --trim-n'. For all samples, reads are then mapped to the ‘N-masked’’ genome with Bowtie2 (2.3.5) (Langmead and Salzberg, 2012). Reads mapped on blacklisted regions from Encode Consortium are discarded. Duplicated reads are also filtered with Picard MarkDuplicates (1.65) (https://broadinstitute.github.io/picard/). In the end, only properly paired mapped reads and reads with mapping quality over 3 are kept for further analysis. SNPSplit (0.3.4) (Krueger and Andrews, 2016) is then used to generate allele-specific BAM files and deepTools (v3.3.2) (Ramirez et al., 2014) is used to check data quality. In the ChIP-seq part, peak calling is performed with MACS2 (v2.2.6) (Zhang et al., 2008) with options [--qvalue 0.001]. It is done with input as Control2 on total and allele specific ChIP-seq signals respectively. CSAW (v1.20.0) (Lun and Smyth, 2015) is additionally used to calculate log2FC on peak regions between MSL2 KO and WT or between allele2 and allele1. Bigwig files are created with deepTools bamCompare (v3.3.2) (Ramirez et al., 2014), using input normalization method and log2ratio and subtract option.
ATACseq: The data are processed via snakePipes DNA-mapping and ATAC-seq pipeline (developing branch) (Bhardwaj et al., 2019). In the DNA-mapping part, adapters and low quality bases (< Q20) are removed using TrimGalore (v0.6.5) with parameters '-q 20 --trim-n'. For all samples, reads are then mapped to the ‘N-masked’’ genome with Bowtie2 (v2.3.5) (Langmead and Salzberg, 2012). Reads mapped on blacklisted regions from Encode Consortium are discarded. Duplicated reads are also filtered with Picard MarkDuplicates (v1.65) (https://broadinstitute.github.io/picard/). In the end, only properly paired mapped reads and reads with mapping quality over 3 are kept for further analysis. SNPSplit (0.3.4) (Krueger and Andrews, 2016) is then used to generate allele-specific BAM files and deepTools (v3.3.2) (Ramirez et al., 2014) is used to check data quality. In the ATAC-seq part, the BAM files are filtered to include only properly paired reads with appropriate fragment sizes (<150 bases). To identify open/accessible regions, peak calling is done with MACS2 (v2.2.6) (Zhang et al., 2008) with options [--qvalue 0.001], on total and allele-specific ATAC-seq signal respectively. CSAW (v1.20.0) (Lun and Smyth, 2015) is additionally used to calculate log2FC on peak regions between MSL2 KO and WT or between allele2 and allele1. Bigwig files are created with deepTools bamCoverage (v3.3.2) (Ramirez et al., 2014), using size factor calculated by deepTools multiBamSummary (v3.3.2).
BSseq: Raw fastq files were aligned against the ‘N-masked’ genome and deduplicated using Bismark (v0.22.3) (Krueger and Andreas, 2011). Resulting Alignments were allele-separated using SNPsplit with flags '--paired' and '--bisulfite'. Relevant quality metrics (conversion rates, genome coverage and GC bias) were calculated using MethylDackel version 0.6.0 (https://github.com/dpryan79/MethylDackel) and DeepTools (v3.5.0). Allele-specific differentially CpG methylated sites between MSL2 KO and WT or between allele2 and allele1 were called with DSS version 2.34.0.
scMultiomics: A unified dataset of both scATAC-seq and scRNA-seq was processed with the count function in cellranger-arc (v2.0.0) (https://support.10xgenomics.com) using the reference mm10-2020-A-2.0.0 (10X). The filtered matrix of WT and MSL2 KO were merged together with Signac (v1.5.0) (131) and Seurat (v4.1.0) (132). The merged dataset was then centered, dimensionally reduced with principal-component analysis using 20 dimensions and embedded with UMAP clustering. Three clustering techniques from Seurat were applied for the merged dataset: independent RNA, independentATAC and WNN method. Chromatin accessibility (TSS +- 200bp) for each gene from the scATAC-seq data was calculated by Signac (v1.5.0) (131) GeneActivities function. Gene counts from all cells were merged together for each gene and compared to the bulk RNA-seq data to calculate pearson correlation.  For allele-specific analysis, the raw fastq files of chromatin accessibility and gene expression were aligned against an N-masked genome (Bowtie2 v2.2.5 (116) for chromatin accessibility data, STAR v2.7.9a (110) for gene expression data) and split into allele-specific BAM files, similar to the analysis of the bulk data. Read headers were then extracted from the allele-specific BAM files and used to create allele-specific fastq files which were subsequently processed using the count function in cellranger-arc (v2.0.0). The workflow to generate the allele-specific fastq files is available under https://github.com/Akhtar-Lab-MPI-IE/multiomePhaser. Note that only the cells identified in the bulk analysis were kept in the allele-specific analysis. Allele-specific gene counts from scRNA-seq data and gene activity from scATAC-seq data were then projected on the UMAP clusters in the featureplot with Seurat (v4.1.0) (132) Featureplot function.
H3K4me3 HiChIP: HiChIP-seq data was processed with MAPS (130) (downloaded from github on May 21th, 2021). In summary, MAPS aligned the FASTQ-files with BWA to a mm10 reference genome. Low mapping quality reads, invalid pairs of alignments and PCR duplicates were filtered sequentially and only valid read pairs were kept for downstream analysis. Filtered reads were binned at 10 kb size to generate the chromatin contact matrix. MAPS normalizes chromatin contact frequencies anchored at genomic regions at the merged H3K4me3 peaks to identify long-range chromatin interactions at 10 kb resolution. A binomial test was then used to determine significant chromatin interactions with FDR corrected p-value cutoff of 0.01.
Assembly: mm10
Supplementary files format and content: bigWig files: log2 ratio or subtract value over the input sample (merged replicates) . bed: peak calling from MACS2 (merged replicates). RData: differential peak calling result from CSAW and differential CpG methylation sites from DSS. tsv: differential expression analysis from DESeq2. txt: Differentially expression genes further classification into five cateogories.

growth_protocol: Male mouse ES cells, obtained from Anne Ferguson-Smith (University of Cambridge), were cultured in NDiff 227 medium (Takara) supplemented with LIF (Millipore), 2i inhibitors (PD0325901 and CHIR99021, Stemgent). Female mouse ES cells, obtained from Edith Heard (EMBL Heidelberg), were cultured in KnockOut-DMEM (KO-DMEM, Gibco) supplemented with 15% KnockOut Serum Replacement (KOSR, Gibco), GlutaMAX (Gibco), non-essential amino acids (Gibco), sodium pyruvate (Gibco), 2-Mercaptoethanol (Gibco), LIF, and 2i inhibitors. Male and female ES cells were differentiated into NPCs as previously described (Conti et al. 2005; Splinter et al. 2011). In brief, 1 million male or female ES cells were plated on 0.1% gelatin-coated plates in N2B27 medium, consisting of 1:1 DMEM/F-12 (Gibco) and Neurobasal medium (Gibco), supplemented with B27 (Gibco), GlutaMAX, 2-Mercaptoethanol, Apo-transferrin (Sigma), bovine serum albumin (Sigma), insulin (Sigma), putrescine (Sigma), progesterone (Sigma), and sodium selenite (Sigma). After 7 days, cells were dissociated and 3 million cells were plated on non-adherent culture dishes in the presence of 10ng/ml epidermal growth factor (EGF, Peprotech) and 10ng/ml basic fibroblast growth factor (FGF, Peprotech) to induce formation of embryoid bodies. After 3 days, the embryoid bodies were transferred to gelatin-coated plates to allow the expansion of NPCs.

extract_protocol:
RNAseq: Total RNA was extracted using the Direct-zol RNA Miniprep Plus Kit (Zymo Research) according to the manufacturers’ instructions. Maxima First Strand cDNA Synthesis Kit (Thermo Scientific) was used to synthesize cDNA from total RNA according to the manufacturers’ instructions. Quantitative reverse transcription PCR was carried out on Roche LightCycler II using the Faststart SYBR Green Master (Rox) mix (Roche) at a final volume of 10μL.
TT-seq: In brief, 10 million NPCs were incubated with N2B27 medium supplemented with 500 uM 4- thiouridine (4sU, Sigma) for 5 minutes at 37°C. Total RNA was isolated, fragmented using Bioruptor Plus for 1 minute (30s ON, 30s OFF, high setting), and incubated with Biotin-HPDP (Thermo Scientific) for 2 hours. MyOne C1 Streptavidin magnetic beads (Thermo Scientific) were used to immunoprecipitate labelled RNA before elution in 5% beta-mercaptoethanol (Roth). FInal RNA was cleaned up using the Oligo Clean and Concentrator Kit (Zymo Research) according to the manufacturer’s instructions. Libraries were prepared from 100 ng of high quality RNA using the Ovation Universal RNA-Seq System (Nugen) according to the manufacturer’s instructions.
ChIPseq: MSL2 WT and KO NPCs were covered with 1% formaldehyde in culture media on the plate and incubated at room temperature (RT) on a rocking plate for 10 min. Media was removed and cells were washed twice with 1xPBS supplemented with 1x protease inhibitor cocktail (Roche). Cells were scraped off the plate, collected in ice-cold 1xPBS and centrifuged at 500g for 5 minutes. After an additional 1xPBS wash, cell pellets were stored at -80°C until usage. Nuclei were isolated using the NEXSON based nuclei isolation protocol. Cell pellets were thawed on ice and resuspended in ice cold 1ml of lysis buffer (10mM Tris-HCl pH 8, 10mM NaCl, 0.2% Igepal, 1x protease inhibitor cocktail). The cell suspension was then transferred into 1ml milliTUBE (Covaris) and sonicated in Covaris instrument (E220) for 30 sec at peak power 75W, duty factor 2% and 200 cycles/burst. Nuclei were pelleted at 1000g at 20°C for 5min. The cytoplasmic fraction in the supernatant was discarded and nuclear pellets were carefully resuspended in 0.5% SDS and incubated at room temperature for 10min. Sample was diluted 1:5 in the digestion buffer (Final concentration: 1.1% Triton, 1x CutSmart buffer, nuclease free H2O, 1x protease inhibitor cocktail). Chromatin of up to 1 million nuclei were digested in 250ul digestion buffer with Cvik1 (5U/100.000 cells, New England Biolabs) at 20°C for 16hrs, shaking at 800rpm. After the chromatin digestion, the nuclei were pelleted for 5min at 1000g and washed in cold nuclei wash solution (10mM Tris-HCl pH 8, 0.25% Triton, 0.2mg/ml BSA) and stored on ice. To confirm the enzymatic chromatin shearing efficiency, 5% of the nuclei solution was incubated together with a decrosslinking solution consisting of 2ul proteinase K, 1ul RNaseA and 4ul 5M NaCl at 37°C for 30min followed by a 2hr incubation at 65°C. DNA was purified using MinElute PCR purification kit (Qiagen) and analyzed on a Agilent Tapestation system (G2964AA) using the Agilent D1000 Screentape reagents.
ATACseq: In brief, nuclei of 50,000 cells per replicate were isolated in 50 μl of cold ATAC resuspension buffer (0.1% NP-40, 0.1% Tween-20, and 0.01% digitonin (Promega)) by incubating on ice for 3 minutes. After lysis, 1 ml of cold ATAC resuspension buffer containing 0.1% Tween-20 (without NP-40 or digitonin) was added, and nuclei were then centrifuged at 500g for 10 minutes. Transposition was carried out using 50 μl of transposition mix (25 μl 2X TD buffer (Illumina), 2.5 μl transposase (Illumina), 16.5 μl 1X PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, and 5 μl water) at 37°C for 30 minutes in a thermomixer with shaking at 1000 rpm. Transposed DNA was collected using Zymo DNA Clean and Concentrator (Zymo Research) according to the manufacturer's instructions, and added to the PCR mix containing 25 μl of NEBNext Master Mix (New England Biolabs) and 5 μl of Index adapters (Illumina). Libraries were amplified using the following PCR program: 72°C for 5 minutes, 98°C for 30 seconds, then 10 cycles of: 98°C for 10 seconds, 63°C for 30 seconds, and 72°C for 1 minute. Library quality was analyzed using the Fragment analyzer (Agilent Technologies) prior to sequencing.
BSseq: Bisulfite-Seq was performed using the NEBNext Enzymatic Methyl-seq Kit (E7120S) according to the manufacturer’s instructions. In brief, genomic DNA (gDNA) was extracted from wild-type and MSL2 knockout NPCs using the QIAamp DNA Mini kit (Qiagen) according to the manufacturer’s instructions.Subsequently, 50ng of high quality gDNA was sheared for 120 seconds using an E220 Covaris to obtain fragments between around 300 base pairs in length. Following end prep and EM-seq adapter ligation, DNA was oxidised and denatured using sodium hydroxide. Libraries were amplified for 6 PCR cycles, and sequenced with 2x100bp paired-end reads on an Illumina NovaSeq6000 sequencer.
scMultiomics: WT and MSL2 KO NPCs were harvested using Accutase (Sigma), resuspended in fresh medium, and placed on ice. Viability was estimated using trypan blue and a hemocytometer. Nuclei were prepared according to 10x Genomics guidelines (manual CG000365 revB) using the recipes indicated in the manual with few modifications. In brief, cell pellets were resuspended in 1X cell lysis buffer supplemented with RNase A inhibitor (Sigma 3335399001), incubated for 8 minutes on ice, and then washed twice with a wash buffer (supplemented with RNase A inhibitor). Nuclei were resuspended in a diluted nuclei buffer (supplemented with RNase inhibitors) and filtered through a 40 mm tip strainer (Flowmi Cell Strainer, H13680-0040, Bel-Art). Nuclei were quantified using a hemocytometer and adjusted to 3220 nuclei/ml. 5 ml of diluted nuclei were subsequently used for the transposase reaction.
H3K4me3 HiChIP: HiChIP was performed as previously described (108) with slight modifications. In brief, 5 million WT and MSL2 KO cells were fixed with 1% formaldehyde for 15 minutes at room temperature, followed by 5 minutes quenching with 0.2M glycine. Fixed cell pellets were resuspended in a cold lysis buffer (10 mM Tris, pH 8.0, 10 mM NaCl, 0.2% IGEPAL CA-630 with proteinase inhibitor). Nuclei isolation was performed by sonication using a Covaris E220 for 30 secs according to the following settings: 75 peak power, 200 cycles per burst, 3% duty factor, temperature 4°C. Successful nuclei isolation was confirmed using a bright-field microscope. Nuclei were then resuspended in 50 µl 0.5% of SDS and incubated at 37 degrees for 10 min. Permeabilization was quenched by adding a master mix containing 25 µl 10% Triton X-100 , 135 µl water, 25 µl 10X CutSmart Buffer, and 4 µl MboI enzyme (25U/µl, New England Biolabs), followed by digestion for 2 hours at 37 degrees in a thermomixer, shaking at 800 rpm. Biotin fill-in was performed by the addition of 10mM each of dATP, dGTP, dTTP, biotin14-dCTP (Thermo Fisher Scientific), and 25 units of Klenow, and incubation at 25 degrees for 1 hour in a thermomixer, shaking at 600 rpm. Proximity ligation was performed at room temperature in 1× T4 DNA ligase buffer (New England Biolabs), 0.1mg/ml BSA, 1% Triton X-100 and 4,000 U T4 DNA Ligase (New England Biolabs). The nuclei were harvested and then resuspended in 1ml chromatin shearing buffer (10mM Tris, pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.5% sodium deoxycholate, protease inhibitors). Chromatin shearing was performed by sonication using Covaris E220 for 20 minutes with the following settings: 104 peak power, 200 cycles per purst, 3% duty factor. The samples were centrifuged at 10,000g for 10 minutes, and the supernatant was collected. Chromatin immunoprecipitation was performed overnight by the addition of 5ug of H3K4me3 antibody (Diagenode) to the sheared chromatin. The next day, 40 µl per sample of Magnetic Dynabeads G were reclaimed using a magnet, washed twice with chromatin shearing buffer, and then added to the immunoprecipitation samples and left rotating for 3 hours in a cold room. After incubation, the beads were washed with chromatin shearing buffer three times, LiCl buffer once (10mM Tris, pH 8.0, 250mM LiCl, 1mM EDTA, 0.5% IGEPAL CA-630, 0.1% sodium deoxycholate), and TE buffer twice (10mM Tris, pH 8.0, 0.1mM EDTA). To elute DNA, washed beads were resuspended in elution buffer (10mM Tris, pH 8.0, 350mM NaCl, 1% SDS) with 10μg RNase A and 20μg proteinase K, and were incubated at 37 degrees for 1 hour and then at 65 degrees overnight. DNA was purified using the Zymo DNA Clean & Concentrator kit. Biotin pull-down was performed using Dynabeads MyOne Streptavidin T1 beads, and libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina according to manufacturer’s instructions (New England Biolabs, #E7645).

treatment_protocol: For the MSL2 knockout generation, two guide RNAs (gRNAs) were cloned into a PX-459 derivative (pSpCas9(BB)-2A-Puro) plasmid (Addgene 62988), and a split puromycin resistance or GFP-positive approach for selection was used (Flemr and Bühler 2015). Male and female ES cells and NPCs were transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. After 48 hours, the cells were sorted based on their GFP expression status using a MoFlo XDP Cell Sorter (Beckman Coulter). Genomic DNA from single clones was isolated by resuspending cells in lysis buffer (10 mM Tris pH 8.0, 0.5 mM Ethylenediaminetetraacetic acid (EDTA), 0.5% Triton X-100) supplemented with Proteinase K (Thermo Scientific), and incubated at 55°C for 1 hour. Genomic DNA was collected after centrifugation at 16,000 g for 10 minutes. Polymerase chain reaction (PCR) was carried out using the 2X PCR Master Mix (Qiagen) at a final volume of 10μL. Successful knockout was verified using CloneJet PCR Cloning (Thermo Scientific) and BigDye Direct Sanger Sequencing (Thermo Scientific) kits according to the manufacturer’s instructions.

value: Female


title: 9sCa: NPCs_Control_NRF1_rep1


title: BlCa: NPCs_Control_H4K5ac_rep1


title: 9sCa: NPCs_Control_NRF1_rep2


title: 9sCa: NPCs_WT3_RNAseq_rep3


title: 9sCa: NPCs_MSL2KO1_NRF1_rep2


title: 9sCa: NPCs_MSL2KO1_NRF1_rep1


title: BlCa: NPCs_Control_H3K4me3_rep2


title: 9sCa: NPCs_MSL2KO2_NRF1_rep1


title: 9sCa: NPCs_WT3_RNAseq_rep2


title: 9sCa: NPCs_WT3_RNAseq_rep1


title: 9sCa: NPCs_WT2_RNAseq_rep3


title: 9sCa: NPCs_WT2_RNAseq_rep1


title: 9sCa: NPCs_WT1_RNAseq_rep3


title: 9sCa: NPCs_WT1_RNAseq_rep2


title: 9sCa: NPCs_Control2_scRNAseq

value: scRNAseq


title: 9sCa: NPCs_MSL2KO1_scRNAseq


title: 9sCa: NPCs_MSL2KO1_scATACseq_c

value: scATACseq


title: 9sCa: NPCs_MSL2KO1_scATACseq_d


title: CaBlCaXi: NPCs_MSL2KO1_RNAseq_rep3


title: 9sCa: NPCs_Control2_scATACseq_d


title: 9sCa: NPCs_Control2_scATACseq_c


title: 9sCa: NPCs_Control2_scATACseq_b


title: 9sCa: NPCs_MSL2KO1_scATACseq_b


title: 9sCa: NPCs_MSL2KO1_scATACseq_a


title: CaBlCaXi: NPCs_MSL2KO1_RNAseq_rep2


title: CaBlCaXi: NPCs_Control_RNAseq_rep3


title: CaBlCaXi: NPCs_MSL2KO1_RNAseq_rep1


title: CaBlCaXi: NPCs_Control_RNAseq_rep2


title: DMSO Mouse 2

city: Madison

first: Jack

middle: M

last: Shireman

state: WI

address: 1500 Highland Ave

department: Neurological Surgery

institute: UW-Madison

description:
DMSO control mouse at death date
AA2

data_processing:
fastq data obtained from sequencer
https://github.com/aselewa/dropseq_pipeline is run on samples
dropseq pipeline outputs are imported to Seurat
all samples merged into single seurat object
analysis continued as standard within seurat pipelines
Assembly: hg19

source_name: brain tumor

extract_protocol: extraction protocol is published (https://mccarrolllab.org/dropseq/)

value: brain tumor

value: b6 wt

value: DMSO


title: 9sCa: NPCs_WT2_RNAseq_rep2


title: DMSO Mouse 1

description:
DMSO control mouse at death date
AA1


title: TMZ Mouse 1

description:
TMZ treated mouse at death date
AA3

value: TMZ end


title: CaBlCaXi: NPCs_Control_RNAseq_rep1


title: TMZ Mouse 2

description:
TMZ treated mouse at death date
AA4


title: h3k27ac_BALBcJ_Kupffer_Control_rep3

city: La Jolla

first: Hunter

last: Bennett

state: California

address: 9500 Gilman Drive, MC 0651

department: Dept. of Cellular & Molecular Medicine

institute: University of California, San Diego

data_processing:
Sequencing data were assessed for quality using fastqc. ChIP-seq data were mapped using Bowtie2 (Langmead and Salzberg, 2012). Strain specific genomes for BALB/cJ and A/J were generated from by replacing invariant positions of mm10 sequence with alleles reported in the Mouse Genome Project strain specific VCF files. Samples from parental strains of mice were mapped to the strain specific genome.  Mapped reads were shifted to the chromosome coordinates of the mm10 genome build using MARGE.pl shift with -ind set to balbcj or aj for reads mapped to the BALB/cJ or A/J genome, respectively (Link et al., 2018).
For samples from CB6F1/J samples, reads were mapped to the mm10 and BALB/cJ genome builds. Then the BALB/cJ mapped reads were shifted to the mm10 build with MMARGE as above. Perfectly mapped reads spanning genetic mutations between BALB/cJ and mm10 were identified using the MMARGE.pl allele_specific_reads command with -ind set to BALB/cJ and a second time with -ind set to mm10 resulting in two SAM files for each biological sample: one SAM file containing reads perfectly mapped to the mm10 genome that spanned known DNA sequence differences relative to the BALB/cJ genome; and a second SAM file containing reads perfectly mapped to the BALB/cJ genome that spanning known DNA sequence differences relative to the reference mm10 genome.
In the F0 mice, H3K27Ac ChIP-seq tags were quantified for differential peak analysis by annotating merged ATAC-seq peaks with ChIP-seq tag directories using the HOMER command “annotatePeaks.pl” parameters -size 1000 -raw (Heinz et al., 2010). H3K27Ac tag counts were quantified for visualization in heatmaps using “annotatePeaks.pl” with the following parameters -size 1000 -norm 1e7 (Heinz et al., 2010). In F1 mice, strain specific SAM files were used to generate strain specific H3K27Ac tag directories that only contained perfectly aligned reads spanning a mutation between the intercrossed strains. H3K27Ac reads spanning mutations were aggregated over IDR peaks using a 2000bp window centered on the IDR peak. Alterations in allelic tag counts from pooled peaks were detected using DESeq2 with the following thresholds normalized average tag depth > 16; absolute log2 fold-change > 1; and adjusted p-value < 0.05.
Assembly: mm10
Supplementary files format and content: Tab delimited text files of H3K27Ac peaks
Supplementary files format and content: Tab delimited file of normalized and raw tag counts over ATAC-seq IDR peaks.

source_name: Kupffer Cells, BALB/cJ, Control

extract_protocol:
Mice were humanely euthanized by exposure to CO2 and liver non-parenchymal cells processed for fluorescence activated cell sorting of Kupffer cells, with modifications from published methodology (Seidman et al., 2020; Sakai et al., 2019). In brief, livers were retrograde perfused for 3 min at a rate of 5-7 ml/min through the inferior vena cava with HBSS without Ca++ or Mg++ supplemented with 0.5 mM EGTA, 0.5 mM EDTA, and 20 mM HEPES. Perfusions were then switched to 40 ml of a digestion buffer, held at 37C, comprised of HBSS with Ca++ and Mg++ supplemented with 0.033 mg/ml of Liberase TM (Roche), 20 μg/ml DNaseI (Worthington), and 20 mM HEPES. Livers were then excised, minced, and digested for an additional 20 minutes in vitro at 37C with gentle rotation in 20 ml of fresh digestion buffer. The perfusion and digestion steps were performed in the presence of 1 μM flavopiridol to offset transcriptional changes associated with digestion. After tissue digestion, cells were passed through a 70-micron cell strainer and hepatocytes removed by 2 low-speed centrifugation steps at 50 X G for 2 min. Nonparenchymal cells in the supernatant were further separated from debris by pelleting for 15 min at 600 X G in 50 ml of 20% isotonic Percoll (Sigma Aldrich) at room temperature. Cells were then washed from Percoll containing buffer and suspended in 10 ml 28% OptiPrep (Sigma Aldrich) and carefully underlaid beneath 3 ml of wash buffer. The resulting gradient was centrifuged at 1,400 X G for 25 minutes at 4C with no break and cells enriched at the interface were saved and subjected to isotonic erythrocyte lysis. Enriched non-parenchymal cells were then washed, suspended in PBS, then stained for 10 minutes with Zombie NIR (BioLegend) and purified anti-CD16/32 (93, BioLegend) to label dead cells and block Fc receptors. Cells were then immunolabeled with specific antibodies of interest, washed, and sorted using a Beckman Coulter MoFlo Astrios EQ configured with spatially separated 355 nm, 405 nm, 488 nm, 561 nm, and 642 nm lasers.
Chromatin immunoprecipitation and sequencing libraries were generated as previously described (Texari et al., 2021) with the modifications to lysis, immunoprecipitation buffer, and washing buffer as described (Eichenfield et al., 2016; Seidman et al., 2020). In brief, FACS purified cells were fixed with 1% paraformaldehyde for 10 min at room temperature. Next, 2.625Mglycine was added to 125mM to quench fixation and cells were collected by centrifugation with the addition of 0.01% Tween-20 at 1,200 X G for 10 min at 4°C . Cells were washed once with 0.01% Tween-20 in PBS and collected by centrifugation at 1,200 XG for 10 min at 4°C . Cell pellets were then snap frozen and stored at -80C. For ChIP reactions, cell pellets were thawed on ice and lysed in 80 ml LB3 (10mMTris/HCl pH 7.5, 100mMNaCl, 1mMEDTA, 0.5mM EGTA, 0.1% deoxycholate, 0.5% sarkosyl, 1 x protease inhibitor cocktail, and 1 mM sodium butyrate). Lysate was sonicated using a Covaris for 12 cycles with the following setting: time, 60 s; duty, 5.0; PIP, 140; cycles, 200; amplitude, 0.0; velocity, 0.0; dwell, 0.0. Samples were collected and 10% Triton X-100 was added to 1% final concentration. One percent of the sonicated lysate was saved as a ChIP input. For each chromatin immunoprecipitation, aliquots of ~500,000 cells were added to 20 µl Dynabeads Protein A with 2 µg anti-H3K27ac (Active Motif) and incubated with slow rotation at 4°C overnight. The following day, beads were collected using a magnet and washed three times each with wash buffer I (20 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, and 1 x protease inhibitor cocktail) and wash buffer III (10 mM Tris/HCl pH 7.5, 250 mM LiCl, 1% Triton X-100, 0.7% Deoxycholate, 1 mM EDTA, and 1 x protease inhibitor cocktail). Beads were then washed twice with ice cold 10 mM Tris/HCl pH 7.5, 1 mM EDTA, 0.2% Tween-20.
Sequencing libraries were prepared for ChIP products while bound to the Dynabeads Protein A as in (Texari et al., 2021). For F1 samples, dual indexed libraries were pooled for a targeted depth of 100 million reads per sample.

tag: source

value: Sorted non-parenchymal cells

value: Kupffer cell

value: BALB/cJ

value: Control

tag: antibody

value: anti-H3K27Ac Active Motif 39133 RRID:AB_2561016


title: 9sCa: NPCs_WT1_RNAseq_rep1

value: Neural progenitor cells


title: h3k27ac_C57_Kupffer_Control_rep1

source_name: Kupffer Cells, C57BL/6J, Control


title: h3k27ac_AJ_Kupffer_Control_rep3

source_name: Kupffer Cells, A/J, Control

value: A/J


title: h3k27ac_C57_Kupffer_Control_rep3


title: h3k27ac_BALBcJ_Kupffer_Control_rep1


title: h3k27ac_AJ_Kupffer_Control_rep1


title: atac_cb6f1j_Kupffer_LPS_2hr_rep5

description:
F1 Hybrid: For samples from CB6F1/J samples, reads were mapped to the mm10 and BALB/cJ genome builds. Then the BALB/cJ mapped reads were shifted to the mm10 build with MMARGE as above. Perfectly mapped reads spanning genetic mutations between BALB/cJ and mm10 were identified using the MMARGE.pl allele_specific_reads command with -ind set to BALB/cJ and a second time with -ind set to mm10 resulting in two SAM files for each biological sample: one SAM file containing reads perfectly mapped to the mm10 genome that spanned known DNA sequence differences relative to the BALB/cJ genome; and a second SAM file containing reads perfectly mapped to the BALB/cJ genome that spanning known DNA sequence differences relative to the reference mm10 genome.
atac_cb6f1j_kupffer_chow_C57BL6J_only_muts_C57BL6J_rep5.peaks
atac_cb6f1j_kupffer_chow_BALBcJ_only_muts_BALBcJ_rep5.peaks
atac_cb6f1j_Kupffer_LPS_2hr_C57BL6J_only_muts.idr
atac_cb6f1j_Kupffer_LPS_2hr_BALBcJ_only_muts.idr
atac_f1_raw.txt

data_processing:
FASTQ files: Sequencing data were assessed for quality using fastqc. ATAC-seq data was mapped using Bowtie2 (Langmead and Salzberg, 2012). ATAC-seq data were trimmed to 30 bp to remove sequencing adapters, which improved mapping efficiency.  Strain specific genomes for BALB/cJ and A/J were generated from by replacing invariant positions of mm10 sequence with alleles reported in the Mouse Genome Project strain specific VCF files. mm10 was used as the C57BL/6J strain specific genome. Samples from parental strains of mice were mapped to the strain specific genome.  Mapped reads were shifted to the chromosome coordinates of the mm10 genome build using MARGE.pl shift with -ind set to balbcj or aj for reads mapped to the BALB/cJ or A/J genome, respectively (Link et al., 2018b)
F1 Hybrid: For samples from CB6F1/J samples, reads were mapped to the mm10 and BALB/cJ genome builds. Then the BALB/cJ mapped reads were shifted to the mm10 build with MMARGE as above. Perfectly mapped reads spanning genetic mutations between BALB/cJ and mm10 were identified using the MMARGE.pl allele_specific_reads command with -ind set to BALB/cJ and a second time with -ind set to mm10 resulting in two SAM files for each biological sample: one SAM file containing reads perfectly mapped to the mm10 genome that spanned known DNA sequence differences relative to the BALB/cJ genome; and a second SAM file containing reads perfectly mapped to the BALB/cJ genome that spanning known DNA sequence differences relative to the reference mm10 genome.
IDR: Strain specific ATAC-seq SAM files were used to generate HOMER tag directories and allelic IDR peaks were identified using each biological replicate. Alterations in allelic signals from pooled IDR peaks were detected using DeSeq2 and required the following thresholds: minimum normalized average tag depth > 16; absolute log2 fold-change > 1; and adjusted p-value < 0.05.
Assembly: mm10
Supplementary files format and content: Tab delimited file of IDR peaks
Supplementary files format and content: Tab delimited file of normalized and raw tag counts over IDR peaks.

source_name: Kupffer Cells, CB6F1/J, LPS 2 hours

extract_protocol:
Mice were humanely euthanized by exposure to CO2 and liver non-parenchymal cells processed for fluorescence activated cell sorting of Kupffer cells, with modifications from published methodology (Seidman et al., 2020; Sakai et al., 2019). In brief, livers were retrograde perfused for 3 min at a rate of 5-7 ml/min through the inferior vena cava with HBSS without Ca++ or Mg++ supplemented with 0.5 mM EGTA, 0.5 mM EDTA, and 20 mM HEPES. Perfusions were then switched to 40 ml of a digestion buffer, held at 37C, comprised of HBSS with Ca++ and Mg++ supplemented with 0.033 mg/ml of Liberase TM (Roche), 20 μg/ml DNaseI (Worthington), and 20 mM HEPES. Livers were then excised, minced, and digested for an additional 20 minutes in vitro at 37C with gentle rotation in 20 ml of fresh digestion buffer. The perfusion and digestion steps were performed in the presence of 1 μM flavopiridol to offset transcriptional changes associated with digestion. After tissue digestion, cells were passed through a 70-micron cell strainer and hepatocytes removed by 2 low-speed centrifugation steps at 50 X G for 2 min. Nonparenchymal cells in the supernatant were further separated from debris by pelleting for 15 min at 600 X G in 50 ml of 20% isotonic Percoll (Sigma Aldrich) at room temperature. Cells were then washed from Percoll containing buffer and suspended in 10 ml 28% OptiPrep (Sigma Aldrich) and carefully underlaid beneath 3 ml of wash buffer. The resulting gradient was centrifuged at 1,400 X G for 25 minutes at 4C with no break and cells enriched at the interface were saved and subjected to isotonic erythrocyte lysis. Enriched non-parenchymal cells were then washed, suspended in PBS, then stained for 10 minutes with Zombie NIR (BioLegend) and purified anti-CD16/32 (93, BioLegend) to label dead cells and block Fc receptors. Cells were then immunolabeled with specific antibodies of interest, washed, and sorted using a Beckman Coulter MoFlo Astrios EQ configured with spatially separated 355 nm, 405 nm, 488 nm, 561 nm, and 642 nm lasers.
Transposase reactions and sequencing libraries were generated as described previously (Buenrostro et al., 2013; Sakai et al., 2019; Seidman et al., 2020) using 25,000 to 50,000 FACS purified Kupffer cells. Tagmented DNA was cleaned up using Zymo ChIP Clean & Concentrate columns and PCR amplified for 14 cycles using barcoding primers. Libraries were size selected to 175-225 bp using gel excision and purified as described (Texari et al., 2021). For F1 samples, dual indexed libraries were pooled for a targeted depth of 100 million reads per sample.

treatment_protocol: Male mice were fasted overnight and treated for 2 hours by intraperitoneal injection of 0.1 mg/kg E. coli O114:B4 lipopolysaccharide. Control mice were equivalently fasted but uninjected.

value: LPS 2 hours


title: 9sCa: NPCs_Control2_scATACseq_a

value: WT


title: atac_cb6f1j_Kupffer_LPS_2hr_rep4

description:
F1 Hybrid: For samples from CB6F1/J samples, reads were mapped to the mm10 and BALB/cJ genome builds. Then the BALB/cJ mapped reads were shifted to the mm10 build with MMARGE as above. Perfectly mapped reads spanning genetic mutations between BALB/cJ and mm10 were identified using the MMARGE.pl allele_specific_reads command with -ind set to BALB/cJ and a second time with -ind set to mm10 resulting in two SAM files for each biological sample: one SAM file containing reads perfectly mapped to the mm10 genome that spanned known DNA sequence differences relative to the BALB/cJ genome; and a second SAM file containing reads perfectly mapped to the BALB/cJ genome that spanning known DNA sequence differences relative to the reference mm10 genome.
atac_cb6f1j_kupffer_chow_C57BL6J_only_muts_C57BL6J_rep4.peaks
atac_cb6f1j_kupffer_chow_BALBcJ_only_muts_BALBcJ_rep4.peaks
atac_cb6f1j_Kupffer_LPS_2hr_C57BL6J_only_muts.idr
atac_cb6f1j_Kupffer_LPS_2hr_BALBcJ_only_muts.idr
atac_f1_raw.txt


title: atac_cb6f1j_Kupffer_LPS_2hr_rep3

description:
F1 Hybrid: For samples from CB6F1/J samples, reads were mapped to the mm10 and BALB/cJ genome builds. Then the BALB/cJ mapped reads were shifted to the mm10 build with MMARGE as above. Perfectly mapped reads spanning genetic mutations between BALB/cJ and mm10 were identified using the MMARGE.pl allele_specific_reads command with -ind set to BALB/cJ and a second time with -ind set to mm10 resulting in two SAM files for each biological sample: one SAM file containing reads perfectly mapped to the mm10 genome that spanned known DNA sequence differences relative to the BALB/cJ genome; and a second SAM file containing reads perfectly mapped to the BALB/cJ genome that spanning known DNA sequence differences relative to the reference mm10 genome.
atac_cb6f1j_kupffer_chow_C57BL6J_only_muts_C57BL6J_rep3.peaks
atac_cb6f1j_kupffer_chow_BALBcJ_only_muts_BALBcJ_rep3.peaks
atac_cb6f1j_Kupffer_LPS_2hr_C57BL6J_only_muts.idr
atac_cb6f1j_Kupffer_LPS_2hr_BALBcJ_only_muts.idr
atac_f1_raw.txt


title: atac_cb6f1j_Kupffer_LPS_2hr_rep2

description:
F1 Hybrid: For samples from CB6F1/J samples, reads were mapped to the mm10 and BALB/cJ genome builds. Then the BALB/cJ mapped reads were shifted to the mm10 build with MMARGE as above. Perfectly mapped reads spanning genetic mutations between BALB/cJ and mm10 were identified using the MMARGE.pl allele_specific_reads command with -ind set to BALB/cJ and a second time with -ind set to mm10 resulting in two SAM files for each biological sample: one SAM file containing reads perfectly mapped to the mm10 genome that spanned known DNA sequence differences relative to the BALB/cJ genome; and a second SAM file containing reads perfectly mapped to the BALB/cJ genome that spanning known DNA sequence differences relative to the reference mm10 genome.
atac_cb6f1j_kupffer_chow_C57BL6J_only_muts_C57BL6J_rep2.peaks
atac_cb6f1j_kupffer_chow_BALBcJ_only_muts_BALBcJ_rep2.peaks
atac_cb6f1j_Kupffer_LPS_2hr_C57BL6J_only_muts.idr
atac_cb6f1j_Kupffer_LPS_2hr_BALBcJ_only_muts.idr
atac_f1_raw.txt


title: atac_cb6f1j_Kupffer_LPS_2hr_rep1

description:
F1 Hybrid: For samples from CB6F1/J samples, reads were mapped to the mm10 and BALB/cJ genome builds. Then the BALB/cJ mapped reads were shifted to the mm10 build with MMARGE as above. Perfectly mapped reads spanning genetic mutations between BALB/cJ and mm10 were identified using the MMARGE.pl allele_specific_reads command with -ind set to BALB/cJ and a second time with -ind set to mm10 resulting in two SAM files for each biological sample: one SAM file containing reads perfectly mapped to the mm10 genome that spanned known DNA sequence differences relative to the BALB/cJ genome; and a second SAM file containing reads perfectly mapped to the BALB/cJ genome that spanning known DNA sequence differences relative to the reference mm10 genome.
atac_cb6f1j_kupffer_chow_C57BL6J_only_muts_C57BL6J_rep1.peaks
atac_cb6f1j_kupffer_chow_BALBcJ_only_muts_BALBcJ_rep1.peaks
atac_cb6f1j_Kupffer_LPS_2hr_C57BL6J_only_muts.idr
atac_cb6f1j_Kupffer_LPS_2hr_BALBcJ_only_muts.idr
atac_f1_raw.txt


title: atac_C57_Kupffer_Control_rep1

description:
atac_C57_Kupffer_Control_rep1.peaks
atac_C57_Kupffer_Control.idr
atac_f0_raw.txt


title: atac_C57_Kupffer_Control_rep2

description:
atac_C57_Kupffer_Control_rep2.peaks
atac_C57_Kupffer_Control.idr
atac_f0_raw.txt


title: atac_C57_Kupffer_Control_rep4

description:
atac_C57_Kupffer_Control_rep4.peaks
atac_C57_Kupffer_Control.idr
atac_f0_raw.txt


title: atac_C57_Kupffer_Control_rep3

description:
atac_C57_Kupffer_Control_rep3.peaks
atac_C57_Kupffer_Control.idr
atac_f0_raw.txt


title: atac_BALBcJ_Kupffer_Control_rep3

description:
atac_BALBcJ_Kupffer_Control_rep3.peaks
atac_BALBcJ_Kupffer_Control.idr
atac_f0_raw.txt


title: atac_BALBcJ_Kupffer_Control_rep4

description:
atac_BALBcJ_Kupffer_Control_rep4.peaks
atac_BALBcJ_Kupffer_Control.idr
atac_f0_raw.txt


title: atac_BALBcJ_Kupffer_Control_rep2

description:
atac_BALBcJ_Kupffer_Control_rep2.peaks
atac_BALBcJ_Kupffer_Control.idr
atac_f0_raw.txt


title: atac_BALBcJ_Kupffer_Control_rep1

description:
atac_BALBcJ_Kupffer_Control_rep1.peaks
atac_BALBcJ_Kupffer_Control.idr
atac_f0_raw.txt


title: atac_AJ_Kupffer_Control_rep3

description:
atac_AJ_Kupffer_Control_rep3.peaks
atac_AJ_Kupffer_Control.idr
atac_f0_raw.txt


title: atac_cb6f1j_kupffer_Control_rep4

description:
F1 Hybrid: For samples from CB6F1/J samples, reads were mapped to the mm10 and BALB/cJ genome builds. Then the BALB/cJ mapped reads were shifted to the mm10 build with MMARGE as above. Perfectly mapped reads spanning genetic mutations between BALB/cJ and mm10 were identified using the MMARGE.pl allele_specific_reads command with -ind set to BALB/cJ and a second time with -ind set to mm10 resulting in two SAM files for each biological sample: one SAM file containing reads perfectly mapped to the mm10 genome that spanned known DNA sequence differences relative to the BALB/cJ genome; and a second SAM file containing reads perfectly mapped to the BALB/cJ genome that spanning known DNA sequence differences relative to the reference mm10 genome.
atac_cb6f1j_kupffer_chow_C57BL6J_only_muts_C57BL6J_rep4.peaks
atac_cb6f1j_kupffer_chow_BALBcJ_only_muts_BALBcJ_rep4.peaks
atac_cb6f1j_kupffer_chow_C57BL6J_only_muts.idr
atac_cb6f1j_kupffer_chow_BALBcJ_only_muts.idr
atac_f1_raw.txt

source_name: Kupffer Cells, CB6F1/J, Control


title: atac_AJ_Kupffer_Control_rep1

description:
atac_AJ_Kupffer_Control_rep1.peaks
atac_AJ_Kupffer_Control.idr
atac_f0_raw.txt


title: atac_cb6f1j_kupffer_Control_rep3

description:
F1 Hybrid: For samples from CB6F1/J samples, reads were mapped to the mm10 and BALB/cJ genome builds. Then the BALB/cJ mapped reads were shifted to the mm10 build with MMARGE as above. Perfectly mapped reads spanning genetic mutations between BALB/cJ and mm10 were identified using the MMARGE.pl allele_specific_reads command with -ind set to BALB/cJ and a second time with -ind set to mm10 resulting in two SAM files for each biological sample: one SAM file containing reads perfectly mapped to the mm10 genome that spanned known DNA sequence differences relative to the BALB/cJ genome; and a second SAM file containing reads perfectly mapped to the BALB/cJ genome that spanning known DNA sequence differences relative to the reference mm10 genome.
atac_cb6f1j_kupffer_chow_C57BL6J_only_muts_C57BL6J_rep3.peaks
atac_cb6f1j_kupffer_chow_BALBcJ_only_muts_BALBcJ_rep3.peaks
atac_cb6f1j_kupffer_chow_C57BL6J_only_muts.idr
atac_cb6f1j_kupffer_chow_BALBcJ_only_muts.idr
atac_f1_raw.txt


title: atac_cb6f1j_kupffer_Control_rep2

description:
F1 Hybrid: For samples from CB6F1/J samples, reads were mapped to the mm10 and BALB/cJ genome builds. Then the BALB/cJ mapped reads were shifted to the mm10 build with MMARGE as above. Perfectly mapped reads spanning genetic mutations between BALB/cJ and mm10 were identified using the MMARGE.pl allele_specific_reads command with -ind set to BALB/cJ and a second time with -ind set to mm10 resulting in two SAM files for each biological sample: one SAM file containing reads perfectly mapped to the mm10 genome that spanned known DNA sequence differences relative to the BALB/cJ genome; and a second SAM file containing reads perfectly mapped to the BALB/cJ genome that spanning known DNA sequence differences relative to the reference mm10 genome.
atac_cb6f1j_kupffer_chow_C57BL6J_only_muts_C57BL6J_rep2.peaks
atac_cb6f1j_kupffer_chow_BALBcJ_only_muts_BALBcJ_rep2.peaks
atac_cb6f1j_kupffer_chow_C57BL6J_only_muts.idr
atac_cb6f1j_kupffer_chow_BALBcJ_only_muts.idr
atac_f1_raw.txt


title: T. brucei 14dpi mouse spleen, scRNAseq

first: Hien

middle: Thi Thu

last: Pham

address: Songdomunhwa-ro 119

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v5.0.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Spleen

extract_protocol:
Single-cell suspensions were prepared from freshly collected mouse spleensby homogenizing spleens in 4 mL of Dulbecco’s Modified Eagle Medium (DMEM) (Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% Fetal Bovine Serum (FBS) (Atlas Biologicals, CO, USA) and 1% penicillin/streptomycin (Capricorn Scientific, Ebsdorfergrund, Gemany), using gentleMACS™ Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). After passing the homogenate through a 70 µm cell strainer (SPL Life Sciences, Gyeongi-do, Korea), cells were centrifuged at 314 x g at 4oC for 7 minutes. Cell pellets were resuspended in RBC lysis buffer (Biolegend, CA, USA) at 4oC and incubated for 5 minutes. Cells were washed and re-suspended in PBS supplemented with 0.04% BSA. Total remaining live cells were counted by Trypan Blue (Sigma-Aldrich, Missouri, USA).
Library was performed according to the manufacter’s instructions (single cell 3’ v3 protocol, 10x Genomics). Briefly, splenocytes were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. were loaded into single cell chips (10X Genomics) and partitioned using Gel Bead In-Emulsion (GEM) technology and a Chromium Controller (10X Genomics). Single cell libraries were prepared using the 10X Genomics Chromium Single Cell 3`reagent kit v3. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.

value: Whole splenocytes

value: Spleen

value: 6-8 weeks old


title: Non-infected mouse spleen spleen scRNAseq

description: 10x Genomics