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ChIP-seq analysis
Published in Altuna Akalin, Computational Genomics with R, 2020
After we are convinced that the data is of sufficient quality, we can proceed with the downstream analysis. One of the first steps in the ChIP-seq analysis is peak calling. Peak calling is a statistical procedure, which uses coverage properties of ChIP and Input samples to find regions which are enriched due to protein binding. The procedure requires mapped reads, and outputs a set of regions, which represent the putative binding locations. Each region is usually associated with a significance score which is an indicator of enrichment.
CEBPA mutants down-regulate AML cell susceptibility to NK-mediated lysis by disruption of the expression of NKG2D ligands, which can be restored by LSD1 inhibition
Published in OncoImmunology, 2022
Meng Liu, Mengbao Du, Jian Yu, Zijun Qian, Yang Gao, Wenjue Pan, Xiujie Zhao, Mowang Wang, Huimin Li, Jiaqi Zheng, Qianshuo Huang, Li-Mengmeng Wang, Haowen Xiao
For the chromatin immunoprecipitation (ChIP) assay, 1 × 107 HEK293T cells transfected with wild-type C/EBPα expression vector (p3× FLAG-Myc-CMV- CEBPA-p42) or empty vector were harvested and processed with the SimpleChiP® Enzymatic Chromatin IP Kit (Agarose Beads) (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions. Immunoprecipitation was performed with rabbit anti-FLAG® M2 (14793, Cell Signaling Technology), anti-histone H3 (D2B12) XP® (Cell Signaling Technology, #4620) and IgG control antibodies (Cell Signaling Technology, # 2729). The antibody-bound chromatin was captured with Protein-G sepharose beads, washed, de-cross-linked and precipitated. Libraries were sequenced following the protocol provided by the I NEXTFLEX® ChIP-Seq Library Prep Kit for Illumina® Sequencing (NOVA-5143, Bioo Scientific, Austin, TX) and sequenced on Illumina Xten using the PE 150 method. Sequencing reads were mapped to the reference genome assembly (hg18) using Bwa (version 0.7.15), and peak calling was performed using MACS2 software (version 2.1.1.20160309).
Integrated analysis of mRNA–m6A–protein profiles reveals novel insights into the mechanisms for cadmium-induced urothelial transformation
Published in Biomarkers, 2021
Bin Wu, Xu Jiang, Yapeng Huang, Xiaoling Ying, Haiqing Zhang, Bixia Liu, Zhuo Li, Dengfeng Qi, Weidong Ji, Xingming Cai
Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and data analysis were performed according to the protocol previously reported (Dominissini et al. 2013). Briefly, TRIzol (Invitrogen) was used to extract total RNA. mRNA was purified from total RNA using the Gen Elute mRNA Miniprep Kit (Sigma-Aldrich). Then, mRNA was fragmented using an RNA fragmentation kit (Ambion) and immunoprecipitated with a mixture of Protein A beads (Thermo Fisher) and anti-m6A antibody (Synaptic Systems). By competition with m6A nucleotide solution (Sigma-Aldrich), the immunoprecipitated RNA was washed and eluted. The resulting RNA fragments were used for library construction and sequenced with Illumina Nextseq 500. Read mapping, m6A peak calling, motif search, and analysis of differentially methylated peaks were performed using TopHat2 (Kim et al. 2013) and the exomePeak R/Bioconductor package as previously described (Dominissini et al. 2013).
Characterization of DNA hydroxymethylation profile in cervical cancer
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Jing Wang, Yi Su, Yongju Tian, Yan Ding, Xiuli Wang
DNA libraries were sequenced on an Illumina Nextseq500 platform. The raw sequence reads of input, MeDIP and hMeDIP were trimmed adaptors and filter out low-quality reads using Cutadapt (v1.9.1) and Trimmomatic (v0.35) [17], and checked the quality of clean reads using Fastqc [18]. Next, clean reads were mapped to the human genome (assembly hg38) using the Bowtie 2 (v2.2.6) algorithm [19]. The process of 5hmC and 5mC peak calling (p<.01) were performed by MACS 2 (v2.1.1) [20] and analyzed the different binding domains based on FDR value less than 0.05 and annotated by DiffBind [21]. The peaks on certain genomic loci were visualized by Integrative Genomics Viewer (IGV).