The cell lines RH4, RHJT, RH36 (Peter Houghton, Greehey Children’s Cancer Research Institute, San Antonio, TX), RH5 (Susan Ragsdale, St. Jude Children's Hospital, Memphis, TN), RH30, RD as well as HEK293T cells (ATCC, LGC Promochem) and SMS-CTR were cultured in DMEM (Sigma-Aldrich), supplemented with 100 U/mL penicillin/streptomycin, 2 mmol/L l-glutamine, and 10% FBS (Life Technologies) in 5% CO2 at 37°C. Cell lines were controlled for mycoplasm contamination using the LookOut Mycoplasma PCR Detection Kit (Sigma-Aldrich) and were tested negative. All RMS cell lines were authenticated by short tandem repeat analysis (STR) profiling in 2014/2015 and positively matched72.
Lentivirus production and transduction
HEK293T cells were seeded in T-25 flasks and transfected the next day with 2,8μg of both the lentiviral envelope (capsid) and packaging plasmid, pCMV-VSV-G and psPAX2, respectively (Addgene) together with 7.4μg of the lentiCRISPR plasmid of interest (Table S5A) using the calcium phosphate technique. Medium was changed 24h after transfection and lentiviral supernatant was collected 3 days after transfection, filtered through 0.45μm filter syringes and concentrated using Amicon Ultra tubes (100kDa, Merck). Lentivirus aliquots were either stored at -80°C or directly used to transduce target cells. Target cells were plated in 24-well plates and transduced the day after with virus in culture medium supplemented with 8μg/ml of Polybrene (TR-1003-G, Merck).
Cas9 expressing RH4 cells were generated by transduction of wildtype cells with lentiviral vector coding Cas9 and mNeonGreen (Addgene) followed by sorting. Activity of Cas9 in these cells was tested using Cas9 activity reporter with BFP (Addgene). The lentiviral vectors coding individual sgRNAs (Table S5A) were generated by cloning single guide sequences using the In-Fusion cloning system (Clontech, 638909) into the sg_shuttle_RFP657 vector (Addgene). After transduction of Cas9 expressing cells, efficiency of sgRNA delivery was assessed by flow cytometry. Knockout of individual target genes was validated by western blot anaylsis at indicated timepoints.
CRISPR competition assay
Cas9 expressing RH4 cells (GFP positive) were plated in 24-well format and transduced the next day with sgRNA carrying lentiviruses (with RFP reporter). 2 days after transduction, cells were mixed with same amount of untransduced cells. Part of the resulting mixture was directly used for baseline flow cytometry measurements, while the rest was kept in culture. For flow cytometry analysis, cells were fixed with 0.5% Paraformaldehyde/1xPBS and washed twice in 1xPBS. After resuspension of cell pellets in 1xPBS, samples were analyzed using the BD LSR Fortessa instrument. GFP and RFP signals were acquired to assess percentages of transduced cell populations. Wildtype, untransduced as well as Cas9 expressing RH4 cells were used to set gates and/or compensation respectively. Data were analyzed with FlowJoV10 software. Dead cells and doublets were excluded by manual gating. Measurements were repeated after 7 and 12 days post transduction to follow the development of populations carrying either control or gene targeting sgRNAs (Table S5A). Finally, percentages of RFP positive cell populations were normalized to baseline measurements obtained at day 2.
Total RNA was extracted using the Qiagen RNeasy Plus Mini Kit (Qiagen) and reverse-transcribed using oligo (dT) primers and Omniscript reverse transcriptase (Qiagen). qRT-PCR was performed for gene specific TaqMan assays indicated in Table S5C using TaqMan gene expression master mix (all Life Technologies). Data were analyzed with the SDS 2.4 software. Cycle threshold (CT) values were compared to GAPDH. Relative expression levels were calculated using the ΔΔCT method based on 3 technical replicates. Outliers found in technical replicates (SD >0.5) were removed from the analysis. Mean and upper and lower limit values were calculated for the indicated amounts of biological replicates.
RNA-seq and GSEA
Biological triplicate samples of Cas9 expressing RH4 transduced with indicated sgRNAs were collected 7 days after transduction for RNA isolation. Total RNAs were extracted by RNeasy Plus Mini Kit (Qiagen). Paired-end mRNA libraries were prepared using Truseq Stranded Total RNA Library Prep Kit (Illumina) and sequenced on a Novaseq system as 2x150 base reads by Atlas Biolab GmbH (Berlin, Germany).
RNA-seq reads were mapped to the human genome build hg19 by STAR (https://github.com/alexdobin/STAR) and quantified as Transcription Per Million (TPM) or Fragments Per Kilobase Million (FPKM) using RSEM (https://deweylab.github.io/RSEM/). Gene set enrichment was assessed using GSEA software (https://www.gsea-msigdb.org/gsea/) and visualized in R (https://github.com/GryderArt/VisualizeRNAseq).
Plasmids for expression of N- and C-terminal BirA-Flag fusion constructs73 were a kind gift of Philip Knobel (Laboratory for Applied Radiobiology, University Zurich). BirA-Flag/PAX3-FOXO1 fusion constructs were generated by amplification of a prevalidated PAX3-FOXO1 cDNA, using primers including restriction sites for AscI (forward) and NotI (reverse), and cloned into N- or C-terminal Bira-Flag backbone vectors. Transient transfection of BirA-Flag/PAX3-FOXO1 fusion constructs or BirA-Flag alone into HEK293T cells was conducted using PEI reagent. Expression as well as subcellular localization of proteins were confirmed by western blot or Immunofluorescence respectively. For Streptavidin Immunoprecipitations, 7.5 Mio. HEK293T cells were plated in a 15cm plate. The next day, cells were transfected with 12.6µg of plasmid DNA in presence or absence of 50µM Biotin. Biotin stock solution (20mM) was obtained by dissolving 100mg of powder (IBA, 2-1016-002) in 2.04ml of NH4OH 28-30% (Sigma Aldrich, ref# 221228),18ml of 1M HCl was added to neutralize the solution (pH~7.5) and sored at 4°C. 24h after transfection, cells were harvested by scraping in 1xPBS. After washing once with 1xPBS, cell pellets were resuspended in 1.5ml Lysis buffer (Table S5F) supplemented with 250U of Benzonase (Novagen, 70664). Lysates were incubated for 1h at 4°C under rotation. After brief sonication to disrupt visible aggregates, centrifugation was performed at 16000g for 30min at 4°C. Cleared input samples were incubated together with 75ul Dynabeads MyOne Streptavidin T1 (Thermo Fisher, 65601) per plate for 2h at 4°C under rotation. For subsequent western blot analysis immunoprecipitates were washed three times with Lysis buffer and eluted from the beads in 1X NuPAGE LDS sample buffer (Thermo Fisher, LuBioScience) at 70°C. For downstream proteomic experiments, beads were washed once in lysis buffer followed by two washing steps with 50mM ammonium bicarbonate. Beads were resuspended in 150µl of 50mM ammonium bicarbonate, snap-frozen and stored at-80°C. For on-bead digestion, 8M urea/100mM Tris-HCl pH8.2 was added to a final concentration of 2M urea. Reduction and Alkylation were carried out using 2mM TCEP and 10mM Chloracetamide for 1h at 30°C under agitation in the dark. The solutions were diluted with Tris-HCl pH8.2 in a 1/1 ratio and digestion was performed with 1µg trypsin per sample overnight at 30°C under agitation in the dark. The next day, supernatant was taken from the beads and pooled with two washing steps with 100ul 10%ACN/Tris-HCl (final concentration of 3%ACN) and acidified to 0.5% TFA. Sample cleanup was performed using Sep-Pack C18 columns and completely dried using speed vac centrifugation. Samples were dissolved in LC-MS solution (3% ACN; 0.1% FA) for further analysis.
Mass Spectrometry: Dissolved samples were injected by an Easy-nLC 1000 system (Thermo Scientific) and separated on an EasySpray-column (75 µm x 500 mm) packed with C18 material (PepMap, C18, 100 Å, 2 µm, Thermo Scientific). The column was equilibrated with 100% solvent A (0.1% formic acid (FA) in water). Peptides were eluted using the following gradient of solvent B (0.1% FA in ACN): 5-25% B in, 60 min; 25-35% B in 10 min; 35-99% B in 5 min at a flow rate of 0.3 µl/min. All precursor signals were recorded in the Orbitrap using quadrupole transmission in the mass range of 300-1500 m/z. Spectra were recorded with a resolution of 120 000 at 200 m/z, a target value of 5E5 and the maximum cycle time was set to 3 seconds. Data dependent MS/MS were recorded in the linear ion trap using quadrupole isolation with a window of 1.6 Da and HCD fragmentation with 30% fragmentation energy. The ion trap was operated in rapid scan mode with a target value of 8E3 and a maximum injection time of 80 ms. Precursor signals were selected for fragmentation with a charge state from +2 to +7 and a signal intensity of at least 5E3. A dynamic exclusion list was used for 25 seconds. After data collection peak lists were generated using FCC74 and Proteome Discoverer 2.1 (Thermo Scientific).
Proteomic Data Analysis: The acquired raw MS data were processed by MaxQuant (version 18.104.22.168), followed by protein identification using the integrated Andromeda search engine75. Spectra were searched against a Uniprot human reference proteome (taxonomy 9606, canonical version from 2016-12-09), concatenated to its reversed decoyed fasta database and common protein contaminants. Carbamidomethylation of cysteine was set as fixed modification, while methionine oxidation and N-terminal protein acetylation were set as variable. Enzyme specificity was set to trypsin/P allowing a minimal peptide length of 7 amino acids and a maximum of two missed-cleavages. MaxQuant Orbitrap default search settings were used. The maximum false discovery rate (FDR) was set to 0.01 for peptides and 0.05 for proteins. Label free quantification was enabled and a 2 minutes window for match between runs was applied. In the MaxQuant experimental design template, each file is kept separate in the experimental design to obtain individual quantitative values. Protein fold changes were computed based on Intensity values reported in the proteinGroups.txt file. A set of functions implemented in the R package SRMService76 was used to filter for proteins with 2 or more peptides allowing for a maximum of 4 missing values, and to normalize the data with a modified robust z-score transformation and to compute p-values and fold changes using the limma package77. If all measurements of a protein are missing in one of the conditions, a pseudo fold change was computed, replacing the missing group average by the mean of 10% smallest protein intensities in that condition.
Total cell extracts were obtained with RIPA buffer (Table S5F). Protein concentration was measured with Pierce BCA protein Assay Kit (Thermo Fisher Scientific). Proteins were separated using 4%–12% Bis-Tris SDS-PAGE gels (Thermo Fisher Scientific, LuBioScience) and transferred to nitrocellulose membranes (GE Healthcare). After blocking with 5% milk powder in TBS/0.1% Tween, membranes were incubated with primary antibodies overnight at 4°C. After washing in TBS/0.1% Tween, membranes were incubated with HRP-linked IgG antibodies for 1h at room temperature. Proteins were detected by chemiluminescence using ECL detection reagent or SuperSignal West Femto Maximum Sensitivity Substrate (both Thermo Fisher Scientific) after washing in TBS/0.1% Tween. All antibodies used for Western Blot are indicated in Table S5B.
Cells were lysed in 2ml Lysis buffer (Table S5F) per 15-cm dish. Lysates were incubated for 2h at 4°C, with antibody directed against the protein of interest coupled to Dynabeads Protein G (Thermo Fisher Scientific, 10003D) or empty beads as negative control. Antibodies used for CoIPs are indicated in Table S5B. Benzonase (Novagen, 70664) was added to the lysate during this incubation when indicated. After washing 4 times with lysis buffer, proteins were eluted with 1x NuPAGE LDS sample buffer (Thermo Fisher Scientific) at 70°C and analyzed by Western blotting using EasyBlot reagents (Genetex, GTX425858 and GTX221666-01).
Cas9 expressing RH4 cells were transduced with indicated sgRNA as described above. Cells were plated onto chamber slides and immunofluorescence was performed after 7 days post transduction. For experiments with BirA constructs, immunofluorescence was carried out the day after transfection as described above. After washing with 1xPBS, cells were fixed with 4% Formalin followed by washing and quenching with 0.1M Glycine/PBS. Cells were washed 3 more times with 1xPBS and permeabilized with 0.1% Triton X-100/PBS and blocked using 4% horse serum in 0.1% Triton X-100/PBS. Incubation with primary antibody dissolved in 4% horse serum in 0.1% Triton X-100/PBS was done overnight in a humid chamber. The next day, secondary antibodies were added in 4% horse serum/PBS for 1h. Antibodies used for Immunofluorescence are indicated in Table S5B. After washing 3 times with 1xPBS, slides were embedded and counterstained with Vectashield/DAPI solution (Vector Laboratories, H-1200) and sealed with nail polish.
Cell cycle analysis
Cas9 expressing RH4 cells were transduced with indicated sgRNAs as described above. Cells were harvested by trypsinization, washed in 1xPBS, fixed in 70% ice-cold Ethanol and incubated at -20°C for at least 2 hours. Before flow cytometry, cells were washed by PBS and resuspended in 500μl PI solution (Table S5F). Data were processed by FlowJoV10 software using Dean-Jett-Fox model to assign cell cycle phases.
ChIP reactions to determine SWI/SNF subcomplex genomic binding under basal and Entinostat treated conditions were performed according to established protocols42, 78, 79. For detailed compositions of buffers used refer to Table S5F.
ChIP assays for SWI/SNF interference studies were performed using the iDeal ChIP-seq kit for Transcription Factors (Diagenode) according to the manufacturer’s instructions. Briefly, Cas9 expressing RH4 cells were transduced with indicated sgRNAs. Alternatively, cells were treated with 250nM ACBI1 or cis conformation negative control compound (Table S5E) respectively. After expansion of cells at 7 days post transduction/treatment, cells were fixed using a dual step protocol with ChIP Cross-link Gold (Diagenode, C01019027) for 30min followed by 1% formaldehyde for 15 min, harvested and sonicated with the Bioruptor Pico sonication device (Diagenode) for 13 cycles (30sec ON, 30sec OFF). Sonicated lysates were then quantified and 25µg (35µg for MYCN ChIP) of chromatin were spiked-in with Drosophila Chromatin (Active Motif) and incubated overnight at 4°C with 4µg (7µg for MYCN ChIP) of antibody and an antibody against the Drosophila specific histone variant H2Av (Active Motif) (Table S5B). Amounts of spike-in components were calculated according to manufacturers instructions. As these agents are introduced at identical amounts and concentrations during the ChIP reactions, technical variation associated with downstream steps is accounted for.
After DNA purification, library preparation was performed as previously described 79. DNA libraries were prepared using TruSeq ChIP Library Prep Kit (Illumina, IP-202–1012). DNA was size selected with SPRI select reagent kit (to obtain a 250–300 bp long fragments). Then, libraries were multiplexed and sequenced using NextSeq500 High Output Kit v2 (Illumina, FC-404–2005) on an Illumina NextSeq500 machine. All libraries were quantified using a Qubit fluorimeter to measure concentration and sequenced on NextSeq platform with single-end reads.
For ChIP-qPCR experiments, DNA was purified and qPCR reactions were set up using PowerUp SYBR Green Master Mix (ThermoFisher) with loci specific primers (Table S5D) according to the manufacturer’s instructions. Relative amounts of immunoprecipitated DNA compared to INPUT DNA was calculated using the formula; %recovery = 2^[(Ct(input)-log2(X)-Ct(sample)]x100% whereby X accounts for input dilution. For comparison of same antibody ChIPs between different conditions, Ct values obtained with Drosophila specific Pgbs primer set (Actif Motif) were used to correct for technical variation (Ct(sample)corr=Ct(sample)-(Ct(sample)Pgbs-Ct(control)Pgbs). For quantification normalized to control conditions, dCt values = ((Ct(input)-log2(X)-Ct(sample) were used to calculate relative ddCt values = dCt(control)-dCt(sample) and fold changes were generated by computing 2^(-ddCt).
Analysis of ChIP-seq datasets
For ChIP studies, analysis was performed as previously reported27. ChIP-seq data was mapped to hg19 using BWA80. For ChIP-Rx, we additionally mapped spike in reads to dm3 using BWA80, and normalized human reads to million-mapped Drosophila reads (RRPM, reference normalize reads per million)81. Peaks were called using MACS2.0 (https://github.com/taoliu/MACS), with stringency thresholds of p = 0.0000001 and filtered to remove ENCODE blacklisted regions (https://sites.google.com/site/anshulkundaje/projects/blacklists). Peak intersections we identified using bedtools intersect82, and the resulting heatmaps and metagene plots were plotted using deeptools (https://deeptools.readthedocs.io/en/develop/). Genome tracks were visualized in IGV83 (https://www.broadinstitute.org/igv/igvtools). We performed HOMER for motif analysis45 to define enriched TF binding sites within datasets.
Cells were cultured in a 384-well format and the day after, treatment with titrated concentrations of indicated compounds (Table S5E) was performed using the HP D300e digital dispenser platform. Their viability was measured by WST-1 assay 72hours after transfection. Cells were incubated with the Cell Proliferation Reagent WST-1 (Roche) for at least 20 min and absorbance was measured in a plate reader at 640nm and 440nm.
Size exclusion chromatography (SEC)/IP-MS experiments
Isolation of nuclear extracts: Nuclear extracts from RH4, RH30, RH4-PAX3-FOXO1-FLAG t(2;13) rhabdomyosarcoma cell models were prepared as previously reported 35, 38, 42, 57, 84. Briefly, 10-20 million cells were washed in PBS, and subsequently trypsinized for dissociation. The trypsin was quenched with DMEM with 10% FBS, pelleted (centrifugation, 40C, 180g), and washed again in PBS. After decanting, cells were resuspended in 1mL Buffer A (Table S5F) and diluted with Buffer A to a final volume of 10 mL. Cells were incubated on ice for 7 minutes and pelleted by centrifugation at 1000g. After decanting, pellets were resuspended in 600 mL Buffer C (Table S5F). To this suspension was added 66.6 mL ammonium sulfate (3M solution, Sigma, Cat# A4418) and rotated at 40C for 30 minutes. The suspensions were pelleted with ultracentrifugation (100,000 rpm) in 1 mL thick-wall polycarbonate tubes (Beckman Coulter Cat# 343778) at 40C for 11 minutes. After pelleting chromatin, the supernatant was transferred to new thick-wall polycarbonate tubes, into which 200 mg ammonium sulfate was suspended. The suspensions were incubated on ice for 20 minutes, ultracentrifuged at 100,000 rpm for 11 minutes, and nuclear fraction was used for further size exclusion chromatography experiments (SEC; Protein Characterization Laboratory, Cancer Research Technology Program, Frederick National Laboratory).
Size exclusion chromatography: One hundred ug of nuclear extract were size fractionated on Acquity I Class UPLC (Waters), using BioSep SEC 300 (7.8x300 mm) size exclusion column (Phenomenex), equilibrated with 150 mM NaPO4 pH 7.2. The proteins we eluded of the column using 150 mM NaPO4 pH 7.2 at 350 ul/minute. Fraction size was estimated by running the Bio Rad gel filtration standard (MW 1,350 to 670,000).
Immunoprecipitation: After size-exclusion chromatography (SEC) for fractionation of nuclear extracts, immunoprecipitation was performed on fractions that stained positive for BAF or PBAF by western blotting. Briefly, 500 mL SEC fractions were diluted 1:1 (v/v) in SEC-IP buffer (Table S5F) and incubated with 3 mg antibody (Table S5B) and rotated gently at 40C for approximately 16 hours. To the mixtures, Protein A Dynabeads (ThermoFisher Cat# 10002D) was added and incubated for an additional 4 hours at 40C with rotation. Immunoprecipitation reactions were washed successively with ice cold SEC-IP buffer, and then twice with LCMS buffer (Table S5F). Beads were resuspended in 30 mL LCMS buffer and used subsequently for LCMS experiments.
On Bead Trypsin Digestion: The beads were resuspended in 25 mM NH4HCO3, pH 8.4 and heated at 95oC for 5 min to denature the proteins. The samples were digested overnight with 2 mg of trypsin at 37°C. The supernatant containing the tryptic digest was collected after centrifugation of the beads, the beads were washed twice with 25 mM NH4HCO3, pH 8.4 and the supernatant and the wash combined for maximum recovery. The peptides were desalted using C18 columns (Thermo Scientific, CA) and lyophilized.
Nanoflow LC and Mass spectrometry: The dried peptides were reconstituted in 0.1%TFA and subjected to nanoflow liquid chromatography (Thermo Easy nLC 1000, Thermo Scientific) coupled to high resolution tandem MS (Q Exactive, HF, Thermo Scientific). MS scans were performed in the Orbitrap analyser at a resolution of 60,000 with an ion accumulation target set at 3e6 over a mass range of 380-1580 m/z, followed by MS/MS analysis at a resolution of 15,000 with an ion accumulation target set at 2e5. MS2 precursor isolation width was set at 1.4 m/z, normalized collision energy at 27, and charge state one and unassigned charge states were excluded.
Proteomics Data Processing: The raw data was searched against the full human uniprot protein database using the SEQUEST algorithm in the Proteome Discoverer 2.2 software (Thermo Scientific, CA). The precursor ion tolerance was set at 10 ppm and the fragment ions tolerance was set at 0.02 Da along with methionine oxidation included as dynamic modification. Only fully tryptic peptides with up to two mis-cleavages and FDR of 1% using the percolator validator algorithms were accepted.
CRISPR domain screening experiments
For our experiments to define essential domains in rhabdomyosarcoma, we performed pooled CRISPR screens as previously reported27, 85, 86. We targeted the domains of chromatin regulatory complexes in our studies, with specific guide RNAs in pooled experiments (Table S1). Our screens included the human SWI/SNF-like ATPase domains (SMARCA4 ATPase, CHD4 ATPase, SRCAP ATPase, INO80 ATPase, TTF2 ATPase, EP400 ATPase, CHD8 ATPase, CHD2 ATPase, ATRX ATPase, CHD6 ATPase, RAD54L ATPase, HLTF ATPase, CHD1 ATPase, SMARCA2 ATPase, CHD7 ATPase, CHD1L ATPase, CHD5 ATPase, ERCC6 ATPase, CHD3 ATPase, SHPRH ATPase, SMARCAD1 ATPase, RAD54L2 ATPase, CHD9 ATPase, HELLS ATPase, SMARCAL1 ATPase). Additionally, our CRISPR targets included bromodomains incorporated into human chromatin regulatory complexes (BRD4, PBRM, TAF1, CREBBP, KAT2A, TRIM28, SMARCA4, BRD8, BPTF, BRD9, EP300, ZMYND8, BAZ1B, BAZ2A, BRD3, ASH1L, TRIM33, SP140, PhIP, BRDT, SP140L, ATA2B, BRD1, CECR2, BRPF1, SP100, SMARCA2, ATAD2, TRIM24, BRWD1, BRPF3, BRD2, BRWD3, BAZ1A, KAT2B, TRIM66, BAZ2B, KMT2A, ZMYND11). In these experiments, negative and positive control guide RNAs (sgRNAs) were included as internal standards and cloned into lentiviral expression vectors for comparison to the sgRNAs targeting domains in chromatin regulatory complexes. After expression of sgRNAs in RH4, RH30, CTR, or RD cells expression Cas9, harvesting for genomic DNA was carried out 3-days after lentiviral transduction and again at approximately 12 days after transduction. Relative comparison of enrichments for specific targeting sgRNAs and control sgRNAs was carried out by PCR amplification of guide RNAs from genomic DNA, and indexing with custom barcodes as previously reported27, and library amplification for sequencing on the MiSeq platform (Illumina). Relative read counts corresponding to individual sgRNA sequences were normalized to total read depth per sample, and fold enrichments (dependencies) for individual chromatin regulatory domains were determined.
Public datasets analyzed in this study
For this study we used the ENCODE Consortium (https://sites.google.com/site/ anshulkundaje/projects/blacklists) dataset, as well as two ChIP-seq datasets recently reported26, 27 (GSE116344, GSE83725; GEO Omnibus). Additionally, we used gene expression datasets (phs000720; GEO Omnibus and phs001928; dbGAP).
The data sets of RNA-seq and ChIP-seq generated in this study have been deposited in the Gene Expression Omnibus database with accession number GSE162052 (Release date set to Dec. 31, 2021).
The mass spectrometry proteomics data of our BioID experiments supporting the findings in Figure 2A,B have been deposited to the ProteomeXchange Consortium via the PRIDE87 partner repository with the dataset identifier PXD022187. Reviewer account details:
The mass spectrometry proteomics data of SEC-IP-MS experiments supporting the findings in Figure 2F-H have been made publicly available and were deposited to the ProteomeXchange Consortium via the MassIVE partner repository with the dataset identifier MSV000086494.
We have made our code available on github (https://github.com/GryderArt), as open-source software for genomics data analysis (including integration of algorithms such as BCHNV, ROSE2, EDEN, COLTRON). These pipelines are build using Bowtie288, MACS289, DESeq290, and for visualization R-Studio was used (https://www.rstudio.com/products/RStudio/).
Reagents or Resources used in this study
Detailed information about Materials and Reagent resources can be found in Supplementary Table S5