title: rna_C57_BMDM_KLA_2hr_rep2

description:
f0_tpm.txt
f0_raw.txt

data_processing:
Preprocessing: FASTQ files from sequencing experiments were mapped to the strain specific genome, mm10 was used for C57BL/6J (Link et al., 2018). MMARGE was used to shift strain specific alignment to the mm10 genome. STAR was used with default parameters was used to map RNA-seq experiments (Dobin et al., 2013). HOMER was used to convert aligned reads into “tag directories” for further analysis (Heinz et al., 2010).
Each experiment was quantified using the “analyzeRepeats” script of HOMER. To generate a table of raw read counts, the parameters -count exons -condenseGenes - noadj were used. To generate a table of TPM values, the parameters -count exons  -tpm were used. Differentially expressed genes were identified using raw sequencing read counts by DESeq2 (Love et al., 2014) analysis through the “getDifferentialExpression” HOMER command. Gene ontology analysis was performed using Metascape (Tripathi et al., 2015).
CB6F1/J data was mapped to both the C57BL/6J and BALB/cJ genome, strain specific mappings were shifted to the reference genome using MMARGE shift (Link et al., 2018). Finally, perfectly aligned reads overlying strain specific mutations were isolated using MMARGE allele specific reads command. As such, the processed data matrices for f1 data include two entries per sample, one initially aligned to the C57BL/6J genome and one initially aligned to the BALB/cJ genome, both then shifted to the mm10 genome.
Assembly: mm10
Supplementary files format and content: Tab delimited text files of the folllowing: Table of unnormalized read counts RNA-seq and table of TPM values for RNA-Seq

molecule: polyA RNA

source_name: BMDMs, C57BL6/J, LPS 2 hours

extract_protocol:
extract protocol, non-parenchymal cells (Kupffer cells, Stellate cells, liver sinusoidal endoethelial cells)
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.
extract protocol, hepatocyes
Hepatocytes were prepared by perfusion digestion in a retrograde fashion through the inferior vena cava to the portal vein. In brief, livers were blanched with clearing buffer, then digested with a collagenase solution at 39C. Livers were perfused for 18 minutes at 5 ml/min. The perfusion and digestion steps were performed in the presence of 1 mM flavopiridol to offset transcriptional changes associated with digestion. After digestion, individual livers were gently dissociated using forceps in Medium 199 supplemented with 5% FBS and penicillin/streptomycin/gentamycin. Crude hepatocyte preps were carefully strained through a 100-micron strainer into and isolated using a Percoll gradient.
extract protocol, BMDMs
Mice were humanely euthanized by exposure to CO2 and bone marrow from femur, tibia, and iliac bones was isolated as described previously (Link et al., 2018). Bone marrow cells were seeded innon- tissue culture plates in m-CSF enriched media. After an additional 4-5 days of culture, non-adherent cells were washed off with room temperature PBS, and adherent macrophages were obtained by scraping.
Performed as published previously (Gosselin et al., 2017; Oishi et al., 2016). Isolated cells were pelleted and put into 150 μl lysis/Oligo d(T) Magnetic Beads binding buffer and stored at -80°C until processing. mRNAs were enriched by incubation with Oligo d(T) Magnetic Beads (NEB, S1419S) and then fragmented/eluted by incubation at 94°C for 9 min. Poly A enriched mRNA was fragmented, in 2x Superscript III first-strand buffer with 10mM DTT (Invitrogen), by incubation at 94°C for 9 minutes, then immediately chilled on ice before the next step. The 10 μL of fragmented mRNA, 0.5 μL of Random primer (Invitrogen), 0.5 μL of Oligo dT primer (Invitrogen), 0.5 μL of SUPERase-In (Ambion), 1 μL of dNTPs (10 mM) and 1 μL of DTT (10 mM) were heated at 50°C for three minutes. At the end of incubation, 5.8 μL of water, 1 μL of DTT (100 mM), 0.1 μL Actinomycin D (2 μg/μL), 0.2 μL of 1% Tween-20 (Sigma) and 0.2 μL of Superscript III (Invitrogen) were added and incubated in a PCR machine using the following conditions: 25°C for 10 minutes, 50°C for 50 minutes, and a 4°C hold. The product was then purified with RNAClean XP beads according to manufacturer's instruction and eluted with 10 μL nuclease-free water. The RNA/cDNA double-stranded hybrid was then added to 1.5 μL of Blue Buffer (Enzymatics), 1.1 μL of dUTP mix (10 mM dATP, dCTP, dGTP and 20 mM dUTP), 0.2 μL of RNAse H (5 U/μL), 1.05 μL of water, 1 μL of DNA polymerase I (Enzymatics) and 0.15 μL of 1% Tween-20. The mixture was incubated at 16°C for 1 hour. The resulting dUTP-marked dsDNA was purified using 28 μL of Sera-Mag Speedbeads (Thermo Fisher Scientific), diluted with 20% PEG8000, 2.5M NaCl to final of 13% PEG, eluted with 40 μL EB buffer (10 mM Tris-Cl, pH 8.5) and frozen at -80°C. The purified dsDNA (40 μL) underwent end repair by blunting, A-tailing and adapter ligation as previously described (Heinz et al., 2010) using barcoded adapters (NextFlex, Bioo Scientific). Libraries were PCR-amplified for 9-14 cycles, size selected by gel extraction, quantified using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and sequenced on a Hi-seq 4000 or a NextSeq 500 (Illumina, San Diego, CA) according to the manufacturer's instructions.

treatment_protocol: For in vivo LPS experiments 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. For in vitro BMDM LPS experiments macrophages were treated with 100 ng/ml Kdo2-lipid A (Avanti lipids), a highly purified E. coli lipopolysaccharide or an equivalent volume of PBS.

value: Bone Marrow Derived Macrophage


title: rna_cb6f1j_BMDM_KLA_2hr_rep1

status: Public on Aug 10 2023

type: SRA

city: La Jolla

first: Hunter

last: Bennett

state: California

address: 9500 Gilman Drive, MC 0651

department: Dept. of Cellular & Molecular Medicine

country: USA

institute: University of California, San Diego

description:
F1 data was processed to only consider perfectly aligned reads, then this perfectly aligned reads were filtered for reads overlying mutations to allow us to distinguish reads originating from C57BL/6J or BALB/cJ parental chromosomes. As such each F1 sample was processed into four distinct matrices for both raw and tpm counts. Two matrices were generated for reads that were perfectly aligned to the C57BL/6J genome and BALB/cJ genomes respectively ('perfect_reads'), and two matrices were generated for reads that perfectly aligned AND overlaid a mutation between C57BL/6J and BALB/cJ genomes ("only_muts").
f1_tpm_perfect_reads_balbcj.txt
f1_tpm_perfect_reads_c57bl6j.txt
f1_tpm_only_muts_balbcj.txt
f1_tpm_only_muts_c57bl6j.txt
f1_raw_perfect_reads_balbcj.txt
f1_raw_perfect_reads_c57bl6j.txt
f1_raw_only_muts_balbcj.txt
f1_raw_only_muts_c57bl6j.txt

library_source: transcriptomic

organism: Mus musculus

source_name: BMDMs, CB6F1/J, LPS 2 hours

tag: cell type

tag: strain

tag: treatment

value: LPS 2 hours


title: rna_cb6f1j_BMDM_PBS_rep2

source_name: BMDMs, CB6F1/J, PBS 2 hours

value: Control


title: rna_cb6f1j_BMDM_PBS_rep1


title: atac_AJ_Kupffer_Control_rep2

description:
atac_AJ_Kupffer_Control_rep2.peaks
atac_AJ_Kupffer_Control.idr
atac_f0_raw.txt

library_source: genomic

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.

molecule: genomic DNA

source_name: Kupffer Cells, A/J, 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.
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.

tag: source

value: Sorted non-parenchymal cells

value: Kupffer cell

value: A/J


title: rna_cb6f1j_BMDM_KLA_2hr_rep2


title: atac_cb6f1j_kupffer_Control_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_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_rep4

description:
atac_AJ_Kupffer_Control_rep4.peaks
atac_AJ_Kupffer_Control.idr
atac_f0_raw.txt


title: rna_C57_BMDM_KLA_2hr_rep1

description:
f0_tpm.txt
f0_raw.txt


title: rna_C57_BMDM_PBS_rep2

description:
f0_tpm.txt
f0_raw.txt

source_name: BMDMs, C57BL6/J, PBS 2 hours


title: rna_C57_BMDM_PBS_rep1

description:
f0_tpm.txt
f0_raw.txt


title: rna_BALBcJ_BMDM_KLA_2hr_rep2

description:
f0_tpm.txt
f0_raw.txt

source_name: BMDMs, BALB/cJ, LPS 2 hours

value: BALB/cJ


title: rna_BALBcJ_BMDM_KLA_2hr_rep1

description:
f0_tpm.txt
f0_raw.txt


title: rna_BALBcJ_BMDM_PBS_rep2

description:
f0_tpm.txt
f0_raw.txt

source_name: BMDMs, BALB/cJ, PBS 2 hours


title: rna_BALBcJ_BMDM_PBS_rep1

description:
f0_tpm.txt
f0_raw.txt


title: rna_AJ_BMDM_KLA_2hr_rep1

description:
f0_tpm.txt
f0_raw.txt

source_name: BMDMs, A/J, LPS 2 hours


title: rna_AJ_BMDM_KLA_2hr_rep2

description:
f0_tpm.txt
f0_raw.txt


title: rna_AJ_BMDM_PBS_rep2

description:
f0_tpm.txt
f0_raw.txt

source_name: BMDMs, A/J, PBS 2 hours


title: rna_AJ_BMDM_PBS_rep1

description:
f0_tpm.txt
f0_raw.txt


title: rna_cb6f1j_Kupffer_LPS_2hr_rep5

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


title: rna_cb6f1j_Kupffer_LPS_2hr_rep4


title: rna_cb6f1j_Kupffer_LPS_2hr_rep3


title: rna_cb6f1j_Kupffer_LPS_2hr_rep2


title: rna_cb6f1j_Kupffer_LPS_2hr_rep1


title: rna_cb6f1j_kupffer_Control_rep2


title: rna_cb6f1j_kupffer_Control_rep1


title: rna_cb6f1j_kupffer_Control_rep4


title: rna_cb6f1j_kupffer_Control_rep3


title: h3k27ac_cb6f1j_kupffer_Control_rep1

description:
Kupffer Cells, CB6F1/J, Control
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.
h327ac_f1_raw.txt

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 cell

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: antibody

value: anti-H3K27Ac Active Motif 39133 RRID:AB_2561016


title: input_cb6f1j_kupffer_Control_rep3

description: Kupffer Cells, CB6F1/J, Control


title: input_cb6f1j_kupffer_Control_rep2

description: Kupffer Cells, CB6F1/J, Control


title: input_cb6f1j_kupffer_Control_rep1

description: Kupffer Cells, CB6F1/J, Control


title: h3k27ac_cb6f1j_kupffer_Control_rep3


title: h3k27ac_cb6f1j_kupffer_Control_rep2


title: input_C57_Kupffer_Control_rep1

description: Kupffer Cells, C57BL/6J, Control


title: h3k27ac_C57_Kupffer_Control_rep2

description:
Kupffer Cells, C57BL/6J, Control
h327ac_f0_raw.txt


title: input_BALBcJ_Kupffer_Control_rep3

description: Kupffer Cells, BALB/cJ, Control


title: h3k27ac_BALBcJ_Kupffer_Control_rep2

description:
Kupffer Cells, BALB/cJ, Control
h327ac_f0_raw.txt


title: input_BALBcJ_Kupffer_Control_rep1

description: Kupffer Cells, BALB/cJ, Control


title: input_BALBcJ_Kupffer_Control_rep2

description: Kupffer Cells, BALB/cJ, Control


title: Leukemic Bone Marrow cells from a Pax5+/- mouse, biological replicate 5

type: RNA

city: Salamanca

first: Isidro

last: Sánchez García

address: Campus Miguel de Unamuno

department: Centro de inv. del Cáncer (CIC).

country: Spain

institute: Inst. de Inv. Biomédica de Salamanca (IBSAL). H. U. de Salamanca. Inst. de Biología Molecular y Celular del Cáncer (IBMCC).

description: Gene expression data from a Pax5+/- leukemic bone marrow cells

hyb_protocol: Samples were hybridized using Affymetrix hybridization kit materials • Washing and scanning were performed using GeneChip System of Affymetrix (GeneChip Hybridization Oven 640, GeneChip Fluidics Station 450)

scan_protocol: Affymetrix Gene ChIP Scanner 3000 7G

data_processing: Affymetrix Transcriptome Analysis Console (TAC) Software Version 4.0.1 was used for the normalization, summarization and quality control of the microarray data.

label: biotin

molecule: total RNA

label_protocol: Samples were enzymatically fragmented and biotinylated using the WT Terminal Labeling Kit (Ambion)

growth_protocol: Animals were bred and maintained in the SPF (specific pathogen-free) area of the animal house until the age of 2 months when they were moved to a conventional facility where they have been exposed to common pathogens.

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions.

treatment_protocol: No treatement was performed.

tag: tissue

value: Bone marrow


title: Bone Marrow proB and preB cells from a healthy wild type mouse, biological replicate 4

description: Gene expression data from Bone Marrow proB and preB cells from a healthy wild type mouse

source_name: Wild type-7

value: Bone marrow:sorted B220low IgM- cells


title: Leukemic Bone Marrow cells from a Pax5+/- mouse, biological replicate 2


title: Leukemic Bone Marrow cells from a Pax5+/- mouse, biological replicate 3


title: Leukemic Bone Marrow cells from a Pax5+/- mouse, biological replicate 6


title: input_AJ_Kupffer_Control_rep2

description: Kupffer Cells, A/J, Control


title: input_AJ_Kupffer_Control_rep1

description: Kupffer Cells, A/J, Control


title: Leukemic Bone Marrow cells from a Pax5+/- mouse, biological replicate 4


title: h3k27ac_AJ_Kupffer_Control_rep2

description:
Kupffer Cells, A/J, Control
h327ac_f0_raw.txt


title: Bone Marrow proB and preB cells from a healthy wild type mouse, biological replicate 2

source_name: Wild type-5


title: Bone Marrow proB and preB cells from a healthy wild type mouse, biological replicate 3

source_name: Wild type-6


title: Bone Marrow proB and preB cells from a healthy wild type mouse, biological replicate 1

source_name: Wild type-4


title: Leukemic Bone Marrow cells from a Pax5+/- mouse, biological replicate 1


title: Hippocampus, NG-KO, rep4

city: xi'an

first: Xin-shang

last: Wang

state: China

address: Changle road No.169

country: China

institute: Fourth Military Medical University

data_processing:
Reads obtained from the sequencing machines includes raw reads containing adapters or low quality bases which will affect the following assembly and analysis. Thus, to get high quality clean reads, reads were further filtered by Cutadapt (https://cutadapt.readthedocs.io/en/stable/,version:cutadapt-1.9)
We aligned reads of all samples to the research species reference genome using HISAT2 (https://daehwankimlab.github.io/hisat2/,version:hisat2-2.0.4) package, which initially remove a portion of the reads based on quality information accompanying each read and then maps the reads to the reference genome. And then by removing sequence-dependent bias and amplification noise using UMI-tools.
The mapped reads of each sample were assembled using StringTie (http://ccb.jhu.edu/software/stringtie/,version:stringtie-1.3.4d) with default parameters. Then, all transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using gffcompare software(http://ccb.jhu.edu/software/stringtie/gffcompare.shtml,version:gffcompare-0.9.8). After the final transcriptome was generated, StringTie and ballgown (http://www.bioconductor.org/packages/release/bioc/html/ballgown.html) were used to estimate the expression levels of all transcripts and perform expression abundance for mRNAs by calculating FPKM (fragment per kilobase of transcript per million mapped reads) value.
Genes differential expression analysis was performed by DESeq2 software between two different groups (and by edgeR between two samples). The genes with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change ≥ 2 were considered differentially expressed genes. Differentially expressed genes were then subjected to enrichment analysis of GO functions and KEGG pathways.
Assembly: GRCm38
Supplementary files format and content: tab-delimited text file includes raw gene counts and FPKM values for each sample

source_name: hippocampus

extract_protocol:
Total RNA was extracted using Trizol reagent following the manufacturer's procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit, high-quality RNA samples with RIN number > 7.0 were used to construct sequencing library.
After total RNA was extracted, mRNA was purified from total RNA (5ug) using Dynabeads Oligo (dT) (Thermo Fisher, CA, USA) with two rounds of purification. Following purification, the mRNA was fragmented into short fragments using divalent cations under elevated temperature (Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 94℃ 5-7min). Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA). An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters, which including custom Unique Molecular Identifiers for minimizing sequence-dependent bias and amplification noise according to (Shiroguchi et al. 2012). Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA librarys were 300±50 bp. At last, we performed the 2×150bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 following the vendor's recommended protocol.

value: hippocampus

tag: genotype

value: neuron-specific GPR30 KO

tag: age

value: 10 weeks old

tag: Sex

value: female


title: Bone Marrow B220+ cells from a healthy Pax5+/- Myd88-/- mouse, biological replicate 4

description: Gene expression data from a healthy Pax5+/-Myd88-/- B220+ bone marrow cells

value: Bone marrow:MACS-sorted B220+ cells


title: Hippocampus, NG-KO, rep3


title: Hippocampus, NG-KO, rep2


title: Bone Marrow B220+ cells from a healthy Pax5+/- Myd88-/- mouse, biological replicate 3


title: Bone Marrow B220+ cells from a healthy Pax5+/- Myd88-/- mouse, biological replicate 2


title: Bone Marrow B220+ cells from a healthy Pax5+/- Myd88+/- mouse, biological replicate 4

description: Gene expression data from a healthy Pax5+/-Myd88+/- B220+ bone marrow cells


title: Bone Marrow B220+ cells from a healthy Pax5+/- Myd88-/- mouse, biological replicate 1


title: input_AJ_Kupffer_Control_rep3

description: Kupffer Cells, A/J, Control


title: Hippocampus, NG-KO, rep1


title: Bone Marrow B220+ cells from a healthy Pax5+/- Myd88+/- mouse, biological replicate 1


title: Hippocampus,  AG-KO, rep4

value: astrocyte-specific GPR30 KO


title: Bone Marrow B220+ cells from a healthy wild type mouse, biological replicate 4

description: Gene expression data from a healthy wild-type B220+ bone marrow cells

source_name: Wild type  (4)


title: Bone Marrow B220+ cells from a healthy Pax5+/- Myd88+/- mouse, biological replicate 2


title: Bone Marrow B220+ cells from a healthy Pax5+/- Myd88+/- mouse, biological replicate 3


title: Bone Marrow B220+ cells from a healthy wild type mouse, biological replicate 3

source_name: Wild type  (3)


title: Hippocampus,  AG-KO, rep3


title: Hippocampus,  AG-KO, rep2


title: Bone Marrow B220+ cells from a healthy wild type mouse, biological replicate 2

source_name: Wild type (2)


title: Hippocampus, Δ3102, rep1

value: global GPR30 KO


title: Hippocampus, Δ3102, rep3


title: Hippocampus, Δ3102, rep2


title: Hippocampus, wild type, rep1

value: WT


title: Hippocampus, wild type, rep3


title: Hippocampus, wild type, rep2


title: mouse endothelial lung cells, endothelial Sp1/Sp3 knockout mice, rep6

city: Jinan

first: Hanlin

last: Lu

address: No. 107, Wen Hua Xi Rd, Jinan, Shandong, China

institute: Qilu Hospital of Shandong University

data_processing:
illumina Casava1.7 software used for basecalling
（1）Remove adapter polluted reads (remove reads contain adapter more than 5bp. For paired-end sequencing, both reads will be filtered if either end is polluted )（2）Remove low-quality reads (low-quality reads refer to reads in which more than 15% bases with base calling quality Q≤19. For paired-end sequencing, both reads will be filtered if either end is low-quality).（3）Remove reads which contain N higher than 5% (For paired-end sequencing, both reads will be filtered if either end has N beyond the offset 5%).
The reference genomes and the annotation file were downloaded from ENSEMBL database (http://www.ensembl.org/index.html). Bowtie2 v2.2.3 was used for building the genome index, and Clean Data was then aligned to the reference genome using HISAT2 v2.1.0. HISAT2 is the successor to TopHat2, which uses a modified BWT algorithm to convert reference genomes to index for faster speed and fewer resources.
Reads Count for each gene in each sample was counted by HISAT v2.1.0, and FPKM (Fragments Per Kilobase Millon Mapped Reads) was then calculated to estimate the expression level of genes in each sample.
DESeq2 was designed for differential gene expression analysis between two samples with biological replicates. Genes with q≤0.05 and |log2_Foldchange|≥1 are identified as differentially expressed genes.
Assembly: mm39
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

source_name: lung

extract_protocol:
Retinas were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries
RNA libraries were prepared for sequencing using standard Illumina protocols

value: lung

value: mouse endothelial lung cells

value: endothelial Sp1/Sp3 knockout

value: tamoxifen-infection


title: mouse endothelial lung cells, endothelial Sp1/Sp3 knockout mice, rep5


title: Bone Marrow B220+ cells from a healthy wild type mouse, biological replicate 1

source_name: Wild type (1)


title: Bone Marrow B220+ cells from a healthy Pax5+/- mouse, biological replicate 1

description: Gene expression data from a healthy Pax5+/- B220+ bone marrow cells


title: Bone Marrow B220+ cells from a healthy Pax5+/- mouse, biological replicate 4


title: Leukemic Bone Marrow cells from a Pax5+/- Myd88+/- mouse, biological replicate 6

description: Gene expression data from a Pax5+/- Myd88+/- leukemic bone marrow cells


title: Leukemic Bone Marrow cells from a Pax5+/- Myd88+/- mouse, biological replicate 5


title: mouse endothelial lung cells, endothelial Sp1/Sp3 knockout mice, rep2


title: Leukemic Bone Marrow cells from a Pax5+/- Myd88+/- mouse, biological replicate 3


title: mouse endothelial lung cells, endothelial Sp1/Sp3 knockout mice, rep3


title: Leukemic Bone Marrow cells from a Pax5+/- Myd88+/- mouse, biological replicate 2


title: Bone Marrow B220+ cells from a healthy Pax5+/- mouse, biological replicate 3


title: Bone Marrow B220+ cells from a healthy Pax5+/- mouse, biological replicate 2


title: mouse endothelial lung cells, endothelial Sp1/Sp3 knockout mice, rep1


title: mouse endothelial lung cells, control mice, rep6


title: mouse endothelial lung cells, control mice, rep5


title: Hippocampus,  AG-KO, rep1


title: mouse endothelial lung cells, control mice, rep4


title: Leukemic Bone Marrow cells from a Pax5+/- Myd88+/- mouse, biological replicate 1


title: mouse endothelial lung cells, control mice, rep3


title: mouse endothelial lung cells, control mice, rep2


title: mouse endothelial lung cells, control mice, rep1


title: BlCa: NPCs_MSL2KO_scRNAseq_rep2

city: Freiburg

first: Asifa

last: Akhtar

address: Stuebeweg 51

department: Chromatin Regulation

country: Germany

institute: Max Planck Institute of Immunobiology and Epigenetics

description:
raw.counts.object.cluster.total.rds
raw.counts.object.cluster.allelic.rds

library_source: transcriptomic single cell

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 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.
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.

source_name: Neural progenitor cells

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.

tag: gender

value: Male

tag: library type

value: scRNAseq

tag: replicate


title: BlCa: NPCs_MSL2KO_scRNAseq_rep1


title: BlCa: NPCs_Control_scRNAseq_rep2


title: mouse endothelial lung cells, endothelial Sp1/Sp3 knockout mice, rep4


title: BlCa: NPCs_MSL2KO_scATACseq_rep2_d

library_source: genomic single cell

value: scATACseq


title: BlCa: NPCs_MSL2KO_scATACseq_rep2_b


title: BlCa: NPCs_MSL2KO_scATACseq_rep2_c


title: BlCa: NPCs_MSL2KO_scATACseq_rep2_a


title: BlCa: NPCs_MSL2KO_scATACseq_rep1_b


title: BlCa: NPCs_MSL2KO_scATACseq_rep1_d


title: BlCa: NPCs_Control_scATACseq_rep2_c


title: BlCa: NPCs_Control_scATACseq_rep2_a


title: sh2_3

city: Pamplona

first: Silvestre

last: Vicent

state: Outside the US or Canada

address: 55 PIO XII Ave

department: Oncology

institute: Center for Applied Medical Research (CIMA)

data_processing:
The quality of the samples was verified using FastQC v0.11.8 software.
FASTQ files were mapped to GRCh38 human assembly using STAR v2.6.1c with default parameters.
featureCounts v1.6.0 software and Gencode v35 release were used to obtain read counts at gene level considering only those genes aligned to exonic regions
Obtained gene counts were normalized using edgeR and voom function of limma
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited text files include gene counts obtained with featureCounts for each sample.

organism: Homo sapiens

source_name: pancreatic cancer cells (HPAFII)

growth_protocol: HPAFII cell lines was grown in DMEM (10% serum and 1% antibiotics) and transduced with constitutive shRNAs (GFPsh or LAMC2sh) for 48 h. Cells were selected with 2 ug/ml puromycin for 3 days and then plated overnight before RNA was harvested.

extract_protocol:
Trizol
Illumina protocol

tag: cell line

value: HPAFII


title: sh2_1


title: BlCa: NPCs_Control_scATACseq_rep1_c


title: BlCa: NPCs_Control_scATACseq_rep1_d


title: BlCa: NPCs_Control_scATACseq_rep2_d


title: GFP_3


title: BlCa: NPCs_MSL2KO_scATACseq_rep1_c


title: CaBl: ESCs_MSL2KO3_RNAseq_rep1

description:
ESCs_DEgenes_classification.txt
ESCs_DEseq_allelic_DEresults.tsv
ESCs_DEseq_basic_DEresults.tsv

source_name: Embryonic stem cells

value: RNAseq


title: Leukemic Bone Marrow cells from a Pax5+/- Myd88+/- mouse, biological replicate 4

value: Bone marrow


title: BlCa: NPCs_Control_scATACseq_rep1_a


title: CaBl: ESCs_MSL2KO3_RNAseq_rep2


title: BlCa: NPCs_MSL2KO_scATACseq_rep1_a


title: GFP_2


title: sh2_2


title: CaBl: ESCs_MSL2KO3_RNAseq_rep3


title: BlCa: NPCs_Control_scRNAseq_rep1


title: CaBl: NPCs_MSL2KO2_RNAseq_rep3

description:
NPCs_DEgenes_classification.txt
NPCs_DEseq_allelic_DEresults.tsv
NPCs_DEseq_basic_DEresults.tsv


title: CaBl: NPCs_MSL2KO2_RNAseq_rep2


title: Placenta_Female18_KO_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 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.

value: Female


title: Placenta_Female18_KO_RNAseq_rep1


title: CaBl: NPCs_MSL2KO2_RNAseq_rep1


title: BlCa: NPCs_Control_scATACseq_rep2_b


title: BlCa: NPCs_Control_scATACseq_rep1_b


title: Placenta_Female18_Control_RNAseq_rep3


title: Placenta_Female18_Control_RNAseq_rep1


title: Placenta_Female18_Control_RNAseq_rep2


title: CaBl: NPCs_MSL2KO_scRNAseq_rep1


title: CaBl: NPCs_Control_scRNAseq_rep2


title: genomic DNA from NC-69

type: genomic

first: chuanxin

last: wang

address: 247 Beiyuan Street, Jinan 250033, Shandong, China,

institute: The Second Hospital of Shandong University

description: normal PBMCs sample

hyb_protocol: bisulphite converted DNA was amplified, fragmented and hybridised to Illumina Infinium Human Methylation850 Beadchip using standard Illumina protocol

scan_protocol: Arrays were imaged using BeadArray Reader using standard recommended Illumina scanner setting

data_processing: BeadStudio software v3.2

label: Cy3 and Cy5

label_protocol: Standard Illumina Protocol

extract_protocol: genomic DNA was extracted and purified from PBMC samples using Qiagen DNeasy Kit according to standard instructions

tag: sample type

value: PBMCs

tag: disease state

value: normal


title: genomic DNA from NC-67

description: normal PBMCs sample


title: CaBl: NPCs_Control_scRNAseq_rep1


title: Wholebrain_Female18_KO_RNAseq_rep2


title: Wholebrain_Female18_KO_RNAseq_rep3


title: Wholebrain_Female18_KO_RNAseq_rep1


title: Wholebrain_Female18_Control_RNAseq_rep3


title: GFP_1


title: Wholebrain_Female18_Control_RNAseq_rep2


title: CaBl: NPCs_MSL2KO_scATACseq_rep2_d


title: CaBl: NPCs_MSL2KO_scATACseq_rep2_c


title: CaBl: NPCs_MSL2KO_scATACseq_rep1_a


title: CaBl: NPCs_MSL2KO_scATACseq_rep1_b


title: CaBl: NPCs_MSL2KO_scATACseq_rep1_d


title: CaBl: NPCs_MSL2KO_scATACseq_rep2_b


title: CaBl: NPCs_MSL2KO_scATACseq_rep2_a


title: CaBl: NPCs_MSL2KO_scRNAseq_rep2


title: genomic DNA from NC-65

description: normal PBMCs sample


title: CaBl: NPCs_Control_scATACseq_rep2_d


title: genomic DNA from NC-62

description: normal PBMCs sample


title: CaBl: NPCs_Control_scATACseq_rep2_c


title: genomic DNA from NC-61

description: normal PBMCs sample


title: Wholebrain_Female18_Control_RNAseq_rep1


title: CaBl: NPCs_Control_scATACseq_rep2_b


title: CaBl: NPCs_MSL2KO_scATACseq_rep1_c


title: genomic DNA from NC-59

description: normal PBMCs sample


title: CaBl: NPCs_Control_scATACseq_rep2_a


title: genomic DNA from NC-58

description: normal PBMCs sample


title: genomic DNA from NC-57

description: normal PBMCs sample


title: genomic DNA from NC-56

description: normal PBMCs sample


title: genomic DNA from NC-55

description: normal PBMCs sample


title: CaBl: NPCs_Control_scATACseq_rep1_c


title: genomic DNA from NC-63

description: normal PBMCs sample


title: CaBl: NPCs_Control_scATACseq_rep1_b


title: genomic DNA from NC-53

description: normal PBMCs sample


title: genomic DNA from NC-50

description: normal PBMCs sample


title: genomic DNA from NC-49

description: normal PBMCs sample


title: genomic DNA from NC-54

description: normal PBMCs sample


title: 9sCa: NPCs_MSL2KO_H3K4me3HiChIP_rep3

value: HiChIP


title: 9sCa: NPCs_MSL2KO_H3K4me3HiChIP_rep1


title: 9sCa: NPCs_MSL2KO_H3K4me3HiChIP_rep2


title: 9sCa: NPCs_Control_H3K4me3HiChIP_rep3


title: 9sCa: NPCs_Control_H3K4me3HiChIP_rep2


title: 9sCa: NPCs_Control_H3K4me3HiChIP_rep1


title: genomic DNA from NC-46

description: normal PBMCs sample


title: 9sCa: NPCs_MSL2KO_scRNAseq


title: CaBl: NPCs_Control_scATACseq_rep1_d


title: genomic DNA from NC-45

description: normal PBMCs sample


title: genomic DNA from NC-44

description: normal PBMCs sample


title: 9sCa: NPCs_Control_scRNAseq


title: CaBl: NPCs_Control_scATACseq_rep1_a


title: 9sCa: NPCs_MSL2KO_scATACseq_d


title: genomic DNA from NC-52

description: normal PBMCs sample


title: genomic DNA from NC-42

description: normal PBMCs sample


title: genomic DNA from NC-41

description: normal PBMCs sample


title: 9sCa: NPCs_MSL2KO_scATACseq_c


title: 9sCa: NPCs_MSL2KO_scATACseq_b


title: 9sCa: NPCs_MSL2KO_scATACseq_a


title: 9sCa: NPCs_Control_scATACseq_d


title: Healthy Control 3

type: protein

city: Liverpool

first: John

last: Frew

address: Elizabeth St

department: Dermatology

country: Australia

institute: Liverpool Hospital

data_processing: Analysis was performed using the Olink Inflammation panel (92 analytes) proseek multiplex assay. (Assarsson et al., 2014). All of the samples met the quality control (QC) for the Olink panels. Gene set enrichment (pathway) analysis was performed using Reactome (Croft et al., 2014), False discovery rate cutoff at 0.05 was used to identify significant enrichment.

label: OLINK PEA

molecule: protein

source_name: Serum

extract_protocol: 5mL of serum was drawn from patients at baseline and after 4 weeks of therapy with Fostamatnib 100mg BD and after 12 weeks of Fostamatinib 150mg BD. Samples were centrifuged after collection, and serum was stored at -80 C.

tag: disease

value: Hidradenitis Suppurativa

tag: timeline

value: Baseline


title: genomic DNA from NC-38

description: normal PBMCs sample


title: Healthy Control 2


title: Healthy Control 4


title: Healthy Control 5


title: Healthy Control 1


title: 9sCa: NPCs_Control_scATACseq_b

value: WT


title: genomic DNA from NC-47

description: normal PBMCs sample


title: HS19 Week 12

value: Week 12


title: 9sCa: NPCs_Control_scATACseq_c


title: genomic DNA from NC-37

description: normal PBMCs sample


title: HS20 Week 12


title: 9sCa: NPCs_Control_scATACseq_a


title: genomic DNA from NC-36

description: normal PBMCs sample


title: CaBl: NPCs_MSL2KO1_RNAseq_rep3

description:
NPCs_MSL2KO1_RNAseq.bw
NPCs_MSL2KO1_RNAseq.genome1.bw
NPCs_MSL2KO1_RNAseq.genome2.bw

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 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.
Genome_build: 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.

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.


title: genomic DNA from NC-43

description: normal PBMCs sample


title: HS18 Week 12


title: genomic DNA from NC-35

description: normal PBMCs sample


title: HS17 Week 12


title: HS16 Week 12


title: CaBl: NPCs_MSL2KO1_RNAseq_rep2


title: genomic DNA from NC-34

description: normal PBMCs sample


title: HS14 Week 12


title: HS13 Week 12


title: genomic DNA from NC-40

description: normal PBMCs sample


title: genomic DNA from NC-32

description: normal PBMCs sample


title: HS12 Week 12


title: genomic DNA from NC-31

description: normal PBMCs sample


title: HS10 Week 12


title: HS11 Week 12


title: genomic DNA from NC-30

description: normal PBMCs sample


title: HS9 Week 12


title: CaBl: NPCs_MSL2KO1_H3_rep1


title: CaBl: NPCs_MSL2KO1_Input_rep1


title: CaBl: NPCs_MSL2KO1_H3_rep2


title: HS3 Week 12


title: HS5 Week 12


title: HS2 Week 12


title: genomic DNA from NC-26

description: normal PBMCs sample


title: HS1 Week 12


title: genomic DNA from NC-24

description: normal PBMCs sample


title: HS20 Week 4

value: Week 4


title: CaBl: NPCs_MSL2KO1_BSseq_rep2

description:
NPCs_MSL2KO1_BSseq.bw
NPCs_MSL2KO1_BSseq.genome1.bw
NPCs_MSL2KO1_BSseq.genome2.bw

value: BSseq


title: genomic DNA from NC-27

description: normal PBMCs sample


title: HS19 Week 4


title: HS18 Week 4


title: CaBl: NPCs_MSL2KO1_BSseq_rep1


title: CaBl: NPCs_MSL2KO1_RNAseq_rep1


title: genomic DNA from NC-21

description: normal PBMCs sample


title: H16 Week 4


title: CaBl: NPCs_Control_RNAseq_rep3

description:
NPCs_Control_RNAseq.bw
NPCs_Control_RNAseq.genome1.bw
NPCs_Control_RNAseq.genome2.bw
NPCs_DEseq_basic_DEresults.tsv
NPCs_DEseq_allelic_DEresults.tsv
NPCs_DEgenes_classification.txt


title: CaBl: NPCs_Control_RNAseq_rep2


title: CaBl: NPCs_Control_RNAseq_rep1


title: HS6 Week 12


title: HS15 Week 4


title: HS14 Week 4


title: HS4 Week 12


title: HS13 Week 4


title: HS12 Week 4


title: HS11 Week 4


title: genomic DNA from NC-19

description: normal PBMCs sample


title: CaBl: NPCs_Control_H3_rep2


title: genomic DNA from NC-17

description: normal PBMCs sample


title: CaBl: NPCs_Control_Input_rep1


title: HS7 Week 4


title: genomic DNA from NC-23

description: normal PBMCs sample


title: HS6 Week 4


title: CaBl: NPCs_Control_H3_rep1


title: HS5 Week 4


title: genomic DNA from NC-16

description: normal PBMCs sample


title: HS8 Week 4


title: genomic DNA from NC-14

description: normal PBMCs sample


title: HS3 Week 4


title: CaBl: NPCs_Control_BSseq_rep3

description:
NPCs_Control_BSseq.bw
NPCs_Control_BSseq.genome1.bw
NPCs_Control_BSseq.genome2.bw
NPCs_diff.peak.BS.RData


title: HS2 Week 4


title: genomic DNA from NC-13

description: normal PBMCs sample


title: CaBl: NPCs_Control_BSseq_rep2


title: HS4 Week 4


title: HS1 Week 4


title: HS17 Week 4


title: genomic DNA from NC-20

description: normal PBMCs sample


title: genomic DNA from NC-9

description: normal PBMCs sample


title: CaBl: NPCs_Control_BSseq_rep1


title: HS20 Week 0 reanalyzed

description: alternative to GSM7495869


title: HS18 Week 0 reanalyzed

description: alternative to GSM7495867


title: CaBl: ESCs_MSL2KO1_RNAseq_rep3

description:
ESCs_DEseq_basic_DEresults.tsv
ESCs_DEseq_allelic_DEresults.tsv
ESCs_DEgenes_classification.txt


title: genomic DNA from NC-7

description: normal PBMCs sample


title: HS17 Week 0 reanalyzed

description: alternative to GSM7495866


title: HS15 Week 0 reanalyzed

description: alternative to GSM7495864


title: CaBl: NPCs_Control_BSseq_rep4


title: genomic DNA from NC-5

description: normal PBMCs sample


title: HS14 Week 0 reanalyzed

description: alternative to GSM7495863


title: genomic DNA from NC-3

description: normal PBMCs sample


title: CaBl: ESCs_MSL2KO1_RNAseq_rep2


title: HS12 Week 0 reanalyzed

description: alternative to GSM7495861


title: HS11 Week 0 reanalyzed

description: alternative to GSM7495860


title: HS13 Week 0 reanalyzed

description: alternative to GSM7495862


title: CaBl: ESCs_MSL2KO1_RNAseq_rep1


title: CaBl: ESCs_Control_RNAseq_rep3


title: genomic DNA from NC-2

description: normal PBMCs sample


title: genomic DNA from NC-11

description: normal PBMCs sample


title: genomic DNA from NC-1

description: normal PBMCs sample


title: HS7 Week 0 reanalyzed

description: alternative to GSM7495857


title: HS6 Week 0 reanalyzed

description: alternative to GSM7495856


title: HS10 Week 0 reanalyzed

description: alternative to GSM7495859


title: CaBl: ESCs_Control_RNAseq_rep1


title: CaBl: ESCs_Control_RNAseq_rep2


title: HS19 Week 0 reanalyzed

description: alternative to GSM7495868


title: HS9 Week 0 reanalyzed

description: alternative to GSM7495858


title: HS5 Week 0 reanalyzed

description: alternative to GSM7495855


title: HS4 Week 0 reanalyzed

description: alternative to GSM7495854


title: genomic DNA from E-CRC-31

description: CRC PBMCs sample

value: CRC


title: HS16 Week 0 reanalyzed

description: alternative to GSM7495865


title: genomic DNA from E-CRC-32

description: CRC PBMCs sample

value: Male


title: BlCa: NPCs_MSL2KO1_RNAseq_rep3


title: HS3 Week 0 reanalyzed

description: alternative to GSM7495853


title: genomic DNA from E-CRC-30

description: CRC PBMCs sample


title: BlCa: NPCs_MSL2KO1_RNAseq_rep2


title: genomic DNA from E-CRC-29

description: CRC PBMCs sample


title: BlCa: NPCs_MSL2KO1_RNAseq_rep1


title: PITPNC1sh7_2

data_processing:
The quality of the samples was verified using FastQC v0.11.8 software.
FASTQ files were mapped to GRCh38 human assembly using STAR v2.6.1c with default parameters.
featureCounts v1.6.0 software and Gencode v35 release were used to obtain read counts at gene level considering only those genes aligned to exonic regions
Obtained gene counts were normalized using edgeR and voom function of limma
Assembly: GRCh38
Supplementary files format and content: tab-delimited text files include gene counts obtained with featureCounts for each sample.

source_name: lung cancer cells

growth_protocol: A549 cell line was grown in RPMI (10% serum and 1% antibiotics) and transduced with constitutive shRNAs (GFPsh, PITPNC1sh6 and PITPNC1sh7) for 48 h. Cells were selected with 2 ug/ml puromycin for 3 days and then plated overnight before RNA was harvested.

value: PITPNC1sh7-expressing cells


title: genomic DNA from E-CRC-17

description: CRC PBMCs sample


title: HS1 Week 0 reanalyzed

description: alternative to GSM7495851


title: PITPNC1sh7_1


title: PITPNC1sh7_3


title: PITPNC1sh6_3

value: PITPNC1sh6-expressing cells


title: PITPNC1sh6_2


title: BlCa: NPCs_MSL2KO1_KANSL3_rep2

description:
NPCs_MSL2KO1_KANSL3.bw
NPCs_MSL2KO1_KANSL3.genome1.bw
NPCs_MSL2KO1_KANSL3.genome2.bw


title: genomic DNA from E-CRC-16

description: CRC PBMCs sample


title: genomic DNA from E-CRC-14

description: CRC PBMCs sample


title: BlCa: NPCs_MSL2KO1_KANSL3_rep1


title: PITPNC1sh6_1


title: GFPsh_3

value: GFPsh-transduced A549 cells


title: BlCa: NPCs_MSL2KO1_Input_rep1


title: GFPsh_2


title: GFPsh_1


title: BlCa: NPCs_MSL2KO1_H3_rep2


title: genomic DNA from E-CRC-11

description: CRC PBMCs sample


title: genomic DNA from E-CRC-09

description: CRC PBMCs sample


title: HS2 Week 0 reanalyzed

description: alternative to GSM7495852


title: BlCa: NPCs_MSL2KO1_H3_rep1


title: genomic DNA from E-CRC-07

description: CRC PBMCs sample


title: BlCa: NPCs_MSL2KO1_BSseq_rep3


title: advanced gastric cancer (NvT3)

city: Seoul

first: EOJIN

last: KIM

address: 73, Goryeodae-ro, Seongbuk-gu

department: Department of Pathology

country: South Korea

institute: Korea University College of Medicine

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence(using fastx_trimmer), then mapped to hg19 whole genome using Tophat
FPKM calculation using Cufflink
Assembly: hg19
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

source_name: stomach

extract_protocol:
RNA was harvested using Trizol reagent (Invitrogen). 1.3 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using the NEBNext Ultra II Directional RNA-Seq Kit standard protocols.

value: stomach

tag: cell type

value: adenocarcinoma

value: no


title: normal gastric tissue (NvT3)

value: normal gastric epithelial and stromal cells


title: BlCa: NPCs_MSL2KO1_BSseq_rep2

tag: strain


title: genomic DNA from E-CRC-05

description: CRC PBMCs sample

value: Female

value: PBMCs


title: genomic DNA from CRC-72

description: CRC PBMCs sample


title: GC, 0-8 hr, scRNA-seq (re-analysis)

city: Kweishan, Taoyuan

first: Shu Yuan

last: Yang

address: Wenhua 1st Rd, No. 259

department: Biomedical science

country: Taiwan

institute: Chang Gung University

description:
10x Genomics
scRNA_0-8 hr
scRNA_0-20hr_aggr_matrix.mtx.gz
scRNA_0-20hr_aggr_barcodes.tsv.gz
scRNA_0-20hr_aggr_features.tsv.gz
The reason why scRNA_0-20hr_aggr_barcodes.tsv.gz has barcodes that end in -1 or -2 is we combined our previous 0-8 hr scRNA-seq data using Cell Ranger v6.1.2.  Barcodes ending in -1 correspond to 0-8 hr embryos and -2 to 8-20 hr embryos.

data_processing:
The scRNA-seq fastq files were demultiplexed, barcode processing, gene counting and aggregated along with our previous  0-8 hr. scRNA-seq data (SRX8341666) using Cell Ranger v6.1.2. Its downstream analysis was made using Monocle3 R package.
1. The scATAC-seq fastq files were demultiplexed, barcode processing using Cell Ranger ARC v1.0.0. 2. Low-quality nuclei were filtered out using DropletUtils, scDblFinder and Signac R packages. 3. The scATAC-seq downstream analysis was made using Signac R package.
Assembly: BDGP6.28.102
Supplementary files format and content: scRNA-seq data: Tab-separated values files and an matrix file
Supplementary files format and content: scATAC-seq data: comma-separated values files

organism: Drosophila melanogaster

source_name: germ cell

extract_protocol:
scRNA-seq: 1. Homogenization of 8-12 and 12-20 hr. embryos 2. Collect vas-GFP cells using Fluorescence-activated cell sorting
scATAC-seq: 1. Homogenization of 0-4, 4-8, 8-12, 12-16 and 16-20 hr. embryos 2. Collect vas-GFP cells using Fluorescence-activated cell sorting 3. Nuclei isolation using 10 mM Tris-HCl (pH7.4) conatining 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.01% Tween-20, 1 mM DTT, 0.01% NP-40, 0.001% Digitonin and 1 U/μl RNase inhibitor
The protocols were based on the manufacturer's instructions using Chromium Next GEM Single Cell 3’ GEM, Library & Gel Bead Kit v3.1 for scRNA-seq and Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle for scATAC-seq

value: germ cell

tag: developmental stage

value: 0-8 hr after egg laying

value: vas-GFP


title: normal gastric tissue (NvT2)

tag: treatment


title: advanced gastric cancer (NvT2)


title: BlCa: NPCs_Control_RNAseq_rep3

value: WT

value: RNAseq


title: BlCa: NPCs_MSL2KO1_BSseq_rep1

value: BSseq


title: genomic DNA from CRC-71

description: CRC PBMCs sample

value: CRC


title: normal gastric tissue (NvT1)


title: genomic DNA from CRC-69

description: CRC PBMCs sample

value: Male


title: iPCR_EEP_Tbx3

city: Amsterdam

first: Bas

last: van Steensel

address: Plesmanlaan 121

country: Netherlands

institute: The Netherlands Cancer Institute (NKI)

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

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).

value: mESCs