Next Generation Sequencing Facilitates Quantitative Analysis of C57BL/6J mice.
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE153993 | GEO | 2020/07/08
REPOSITORIES: GEO
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Project description:To identify the target mRNAs of the m6A reader protein YTHDF2 in mouse hippocampus, we carired out anti YTHDF2 RNA Immunoprecipitation (RIP) followed by RNA-sequencencing. Using EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore), RNA from P40 wild type mouse hippocampus was pulled down by rabbit polyclonal anti-YTHDF2 (proteintech) and then sequenced on Illumina Novaseq 6000. The filtered reads were aligned to the mouse reference genome (GRCm38) using BWA mem (v 0.7.12).Then the MACS2 (version 2.1.0) peak calling software was used to identify regions of IP enrichment over background, followed by the motif detected by Homer (Heinz et al., 2010). Peak related genes are then confirmed by PeakAnnotator. Different peak analysis was based on the fold enrichment of peaks of different experiments. A peak was determined as different peak when the odds ratio between two groups was more than 2. Using the same method, genes associated with different peaks were identified. Finally, Biological replicates of anti-YTHDF2 RIP-Seq identified 408 mRNAs transcripts. This study provides gene lists which shows mRNA binding with YTHDF2 in mouse hippocampus.
2023-01-19 | GSE171791 | GEO
Project description:We present ATAC-seq combined with direction RNA seq data for eight time points across the intraerythorcytic development cycle of Plasmodium falciparum 3D7. ATAC peaks were called by MACS2 using ATAC-seq libraries generated with genomic DNA as a control. The majority of identified ATAC peaks were located in intergenic regions and captured most (95%) of previously identified binding sites of the transcription factor required for the expression of most invasion-related genes PfAP2-I (Santos et al., 2017). The robustness of the ATAC-seq dataset was confirmed with a second biological replicate both in terms of peak location and relative peak accessibility pattern. Peaks were assigned to the closest gene and the majority of peaks showed a clear positive correlation between chromatin accessibility pattern and relative mRNA abundance (Pearson correlation > 0.6). In addition, they were sufficient to drive the stage-specific expression of a reporter gene, demonstrating their functionality. Motif searches restricted to accessible regions showed enrichment of several predicted Plasmodium motifs while also predicting several novel regulatory elements.
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Project description:ATAC-seq was performed for T-cell blasts for the following study groups: healthy controls (HC, n=6) and patients with AIOLOS E82K (n=6) and Q402X (n=1). ATAC-Seq was performed using the Active Motif ATAC-Seq Kit following the manufacturer’s protocol and sequenced using the Illumina NextSeq 2000 with 42-bp paired end reads. Using Basepair bioinformatics tools and pipelines (https://www.basepairtech.com/), reads were aligned using Bowtie2, and peaks were called with MACS2 and annotated using Homer. Differential testing, annotation, and visualization were performed using R (v.4.3.1) and the following Bioconductor R packages (https://bioconductor.org/): DESeq2 (v.1.40.2), DiffBind (v.3.10.1), ChIPQC (v.1.36.1), ChIPseeker (v.1.36.0), clusterProfiler (v.4.8.2), and goseq (v.1.52.0).
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Project description:Purpose: The goal of this study is to identify the role of HN1L protein as a transcription factor or co-factor in regulating TNBC cells. Methods: Due to the unavailability of a ChIP-grade HN1L antibody, we overexpressed FLAG-tagged HN1L in SUM159 cells and performed ChIP using anti-FLAG antibodies. ChIP DNA was prepared into libraries and sequenced by the Epigenomics Core of Weill Cornell Medical College using SR50 lane. Results: ChIP-Seq analysis began with mapping the sequenced reads to the genome. We utilized the Burrows-Wheeler Aligner (BWA) MEM algorithm to align the sequence reads against the human genome GRCh37/hg19 Assembly. We next used the Hypergoemetric Optimization of Motif Enrichment (HOMER) suite of tools to find and annotate peaks, and identify enriched motifs. HN1L showed 2,249 binding peaks from 10k bp upstream to 5k bp downstream of transcription start site (TSS). Conclusions: among the binding peaks 35 genes overlapped with a previously published BCSC gene signature, 10 genes overlapped with the HN1L knockdown gene signature and 8 genes are well established CSC transcription factors. When applying overlapped genes in the STRING10 pathway analysis database , a protein interaction network centered with STAT3 was obtained. STAT3 and FGFR2 peaks were validated by qPCR. Model-based Analysis of ChIP-Seq (MACS) was then used to confirm the peaks found by Hypergeometric Optimization of Motif EnRichment (HOMER) . Besides the binding peaks found in HOMER, another peak was called by MACS within LEPR , and was also validated by qPCR . These findings indicate that HN1L may act as a transcription factor through binding to enhancer regions of STAT3 and other STAT3 regulators.
2017-12-20 | GSE105446 | GEO
Project description:Purpose: Zinc Finger MIZ-Type Containing 1 (Zmiz1) is a member of the PIAS family of protein and function as a transcriptional coactivator of Notch, Androgen Receptor (AR), p53, Estrogen Receptor (ER), and Smad3/4 . Despite Zmiz1 critical role in angiogenesis, its role in lymphatic vasculature is unknown. Here, we use HDLECs cell line to profileepigenetic changes upon Zmiz1 knockdown using siRNA. Methods: ATAC sequencing library was prepared as per manufacturer instruction (Active Motif, 53150). Briefly, intact nuclei were isolated from control and Zmiz1 siRNA transfected HDLECs. Samples were treated with a hyperactive Tn5 transposase which tag the target DNA with sequencing adapters and fragment the DNA simultaneously. Library was then quantified using Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Q32851) and verified using the Bioanalyzer DNA High Sensitivity Assay Kit (Agilent, 5067-4626). Validated samples were sequenced using the NextSeq1000/2000 P2 Reagents (100 Cycles) v3 (Illumina, 20046811) on a Nextseq1000/2000. Resulting sequencing data were analyzed using basepairtech ATAC-Seq pipeline (www.basepairtech.com). Briefly, sequenced reads were aligned to the human (hg19) reference genome using Bowtie2. ATAC-Seq peaks and differentially accessible regions were quantified using MACS2 and DESeq2. Results: ATAC seq peaks analysis using MACS2 identified 576 peaks of which are mostly located in intergenic regions followed by introns. We found significantly reduced open chromatin near Prox1 loci upol loss of Zmiz1. Conclusions: We identify Zmiz1 regulates chromatin accessibility near Prox1 genomic loci in lymphatic endothelial cells
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Project description:We used ATAC-seq (Buenrostro et al, 2013) to identify regions of open chromatin in FACS sorted mouse endothelial cells from E12.5 hearts. Please note that the animals were injected at different dates, which resulted in different effects of the knockout, as indicated in the batch attribute of the samples. This data set is part of the study \Endocardial Tbx20 is essential for mesenchymal and myocardial cell movements required for cardiac septation\. Peaks were called using HOMER (-gsize 1.87e9 -region -tbp 1). Replicate 1: -fdr 1e-8, replicate 2: default parameters. Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ. 2013. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods 10: 12138.
2017-06-02 | E-MTAB-3972 | biostudies-arrayexpress