Anti-YTHDF2 RIP-Seq to identify YTHDF2 target mRNAs in hippocampus

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. Overall design: 2 replicates, each containing input and IP samples

为鉴定小鼠海马体中m6A识别蛋白YTHDF2的靶mRNA，我们开展了抗YTHDF2 RNA免疫沉淀（RNA Immunoprecipitation，RIP）联合RNA测序的实验。使用EZ-Magna RIP™ RNA结合蛋白免疫沉淀试剂盒（Millipore），我们从出生后40天（P40）的野生型小鼠海马体中提取RNA，通过兔源多克隆抗YTHDF2抗体（Proteintech）进行富集沉淀，随后在Illumina NovaSeq 6000平台完成测序。将过滤后的测序读段比对至小鼠参考基因组（GRCm38），所用工具为BWA mem（v0.7.12）。随后使用MACS2（v2.1.0）峰识别软件，鉴定相较于背景信号具有富集的IP区域，并通过Homer软件（Heinz等，2010）检测结合基序。利用PeakAnnotator确认与峰区域相关的基因。差异峰分析基于不同实验中峰的富集倍数：当两组间的比值比（odds ratio）大于2时，该峰被判定为差异峰，并通过相同方法鉴定出与这些差异峰相关的基因。最终，生物学重复的抗YTHDF2 RIP-seq实验共鉴定出408条mRNA转录本。本研究提供了在小鼠海马体中与YTHDF2结合的mRNA对应的基因列表。整体实验设计：设置2次生物学重复，每个重复包含输入（Input）样本和免疫沉淀（IP）样本。