M6A modification segregates the stemness program from proliferation in ISCs [ATAC-seq]
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE186916 | GEO | 2023/04/13
REPOSITORIES: GEO
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Project description:Purpose: A method for identifying genome-wide DNA binding sites for Fosb. Methods: Alive cells were sorted from retro-Fosb-OE(over-expression) organoids. The samples were incubated with anti-Fosb antibody (Abcam, ab184938). Purified DNA was subjected to Tru-seq library construction using NEBNext Ultra II DNA Library Prep Kit and sequenced as paired-end with Illumina Novaseq 6000. HISAT2 was used to align the sequences to the mouse genome and generate bam files. bamCoverage (CPM normalized and extended reads) was used to generate bigwig files from bam files. MACS2 was used for peak calling and to generate bed files from aligned reads. HOMER annotatePeaks.pl was used to annotate the peaks. Conclusions: Target genes of Fosb through ChIP assay were consistent with predicted target genes. Thus, we concluded that Fosb, which is a key TF could regulate most of ISC signature genes to maintain Lgr5+ ISCs.
2023-04-13 | GSE186915 | GEO
Project description:Stroma extracts were isolated from 2-week-old WT plants and incubated with either BSF-specific antibodies or with the pre-immune serum. IgGs were captured with SiMAG-Protein G beads (Chemicell) and recovered RNA was used for generation of libraries with the ScriptSeq v2 RNA-seq Library Preparation Kit (Epicentre). Primary reads were aligned to the Arabidopsis chloroplast genome (accession number NC_000932.1) using CLC Genomics Workbench. BAM files were extracted and sorted in Galaxy . Sorted BAM files were converted into RPKM-normalized bigwig files and displayed in IGB. The differential enrichment of BSF/control of the two replicates was displayed across the entire chloroplast genome to identify the RNA targets of BSF.
2019-04-06 | GSE129411 | GEO
Project description:Purpose: To find m6A modified genes which were affected in Mettl3-KO (Mettl3fl/fl; Lgr5-EGFP-IRES-CreERT2; Villin-CreERT2) Intestinal stem cells, we used a method combining RNA-protein immunoprecipitation with high-throughput sequencing to achieve RNA methylation, exactly m6A, analysis at the full transcriptome level. Methods: Intestinal stem cells (Lgr5-high cells) were sorted by flow cytometry in WT and Mettl3-KO intestinal epithelium at 4 dpt. mRNA was purified using Dynabeads mRNA Purification kit (Invitrogen, 61006). Then, mRNA was fragmented into ~100-nucleotide-long fragments by RNA Fragmentation Reagents (Ambion, AM8740) at 94 ℃ for 45s. After that, mRNA were immunoprecipitation with m6A antibody (NEB, NO.E1610S) to enrich the m6A-marked mRNA followed by RNA extraction by RNA clean&concentrator-5 (ZYMO RESEARCH, 1013). The cDNA libraries were generated using SMARTER Stranded Total RNA-seq kit v2. In each sample, reads were aligned to the mouse genome in Ensembl (release 95) with HISAT2. m6A peaks in each sample were identified using exomePeak (v2.17.0) R/Bioconductor package with default parameters. Conclusions: We found that 2074 transcripts showed a reduced m6A modification in Mettl3-KO Lgr5-high ISCs at 4 dpt. The most of targets contained the common m6A motif “RRACH (GGACU)” in the protein-coding region and 3’UTR, especially enriched near the stop codon.
2023-04-13 | GSE186914 | GEO
Project description:Purpose: One goal of this Bulk RNA-seq is to compare genome-wide transcriptome profiles in WT and Mettl3-KO mice intestinal stem cells at indicated time point.The other goal is to compare expression of stemness genes in WT, Mettl3-KO, Mettl3 KO and AKF-OE or AKFP-OE organoids. Methods: For mice, intestinal crypts were isolated and digested with Tryple, Lgr5-high or Lgr5-low cells were sorted by flow cytometry. For organoids, organoids were digested with Tryple and alive cells were sorted by flow cytometry. Total RNA was extracted with RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. Bulk RNA-seq was performed using the Illumina Hiseq X. RNA-seq reads were aligned to the mouse genome available in Ensembl (release 95) with HISAT2. Conclusions: Mettl3 deletion results in reduced expression of most of the stem cell signature genes in Mettl3-KO ISCs, including Lgr5, Olfm4, Ascl2, and Smoc2, but increased gene expression related to cell death, p53, MAPK, YAP and regeneration pathway. AKF and AKFP-overexpression could rescue the expression level of some ISC signature genes compared with Mettl3 detion organoids.
2023-04-13 | GSE186912 | GEO
Project description:Purpose: The transcription fact Lola is identified as a conponent acting downstream of Hippo signaling to restrict ISC proliferation and regulate midgut homeostasis. To further elucidate the mechanism of Lola regulating ISC proliferation and midgut homeostasis,Chromatin immunoprecipitation assay followed by sequencing (ChIP-seq) was performed to identify genes suppressed directly by Lola. Methods: Chromatin lysates were clarified from homogenized and sonicated S2 cells; protein-DNA complexes were isolated with antibody. Genomic DNA fragments were purified with a DNA purification kit (QIAGEN) and subjected to high throughput sequencing using Illumina HiSeq2500. Process: After the genomic DNA segments were sequenced using Illumina HiSeq2500. After quality control with fastqc (version 0.11.8), we built genome index from Drosophila melanogaster genome(BDGP6)with bowtie2-build. Reads were aligned to Drosophila genome BDGP6 index with bowtie2. Then we converted sam files to bam files with samtools. Peaks were called from the aligned reads using MACS2 callpeak, and peaks annotation using R package ChIPseeker. Conclusion: Thousands of Lola-associated chromatin binding sites were identified in cultured S2 cells. Analysis of the Lola binding profiles revealed that Lola mainly binds to the regions around transcription start sites (TSS) and promoters.
2019-09-07 | GSE136999 | GEO
Project description:Purpose: To monitor the different of ISCs and cell fate alteration upon Mettl3 deletion, we use single cell sequencing to examine the sequence information from individual cells with optimized next-generation sequencing (NGS) technologies, providing a single-cell resolution of cellular differences. Methods:Alive intestinal epithelial cells were sorted from WT and Mettl3-KO mice at 4 dpt. ScRNA-seq libraries were generated using the Chromium Single Cell 3’ Reagent Ki V3 (10X Genomics). The libraries were sequenced as paired-end with Illumina Novaseq 6000. Raw reads were aligned to the GRCm38/mm10 mouse genome, and Cell Ranger (v3.1.0) was used to estimate unique molecular identifiers (UMIs). Raw aligned features were loaded and processed using the Seurat package (v4.0.2). Low-quality cells were filtered if they expressed no more than 200 genes or with more than 20% of mitochondrial genes. Results: After cells with low information content and a high fraction of mitochondrial RNAs were excluded, 7865 cells (4467 WT and 3398 KO) were analyzed. Conclusions: Mettl3 deletion reduced the expression of stem cell signature genes but unchanged proliferation profile in ISCs. Thus, we conclude that Mettl3 deletion leads to loss of stemness but not proliferation, and enhanced regeneration and apoptosis.
2023-04-13 | GSE186913 | GEO
Project description:Tissue homeostasis requires long-term lineage fidelity of somatic stem cells. Whether and how age-related changes in somatic stem cells impact the faithful execution of lineage decisions remains largely unknown. Here, we address this question using genome-wide chromatin accessibility and transcriptome analysis as well as single cell RNA-seq to explore stem cell intrinsic changes in the aging Drosophila intestine. These studies indicate that in stem cells of old flies, promoters of Polycomb (Pc) target genes become differentially accessible, resulting in the increased expression of enteroendocrine (EE) cell specification genes. Consistently, we find age related changes in the composition of the EE progenitor cell population in aging intestines, as well as a significant increase in the proportion of EE-specified ISCs and progenitors in aging flies. We further confirm that Pc-mediated chromatin regulation is a critical determinant of EE cell specification in the Drosophila intestine. Pc is required to maintain expression of stem cell genes while ensuring repression of differentiation and specification genes. Our results identify Pc group proteins as central regulators of lineage identity in the intestinal epithelium and highlight the impact of age-related decline in chromatin regulation on tissue homeostasis.
2021-04-02 | GSE157794 | GEO
Project description:Tissue homeostasis requires long-term lineage fidelity of somatic stem cells. Whether and how age-related changes in somatic stem cells impact the faithful execution of lineage decisions remains largely unknown. Here, we address this question using genome-wide chromatin accessibility and transcriptome analysis as well as single cell RNA-seq to explore stem cell intrinsic changes in the aging Drosophila intestine. These studies indicate that in stem cells of old flies, promoters of Polycomb (Pc) target genes become differentially accessible, resulting in the increased expression of enteroendocrine (EE) cell specification genes. Consistently, we find age related changes in the composition of the EE progenitor cell population in aging intestines, as well as a significant increase in the proportion of EE-specified ISCs and progenitors in aging flies. We further confirm that Pc-mediated chromatin regulation is a critical determinant of EE cell specification in the Drosophila intestine. Pc is required to maintain expression of stem cell genes while ensuring repression of differentiation and specification genes. Our results identify Pc group proteins as central regulators of lineage identity in the intestinal epithelium and highlight the impact of age-related decline in chromatin regulation on tissue homeostasis.
2021-04-02 | GSE157793 | GEO