ATAC-seq of E10.5 WT and Chd4-CMko mouse hearts

Purpose: The goal of this study is to identify the differential cardiac chromatin accessibility between WT and cardiomyocyte conditional knockout (Chd4-CMko) hearts at E10.5 using ATAC-seq. Methods: Three hearts at E10.5 were pooled per genotype per replicate, and were then dissociated into single cells. 40,000 viable cells were taken for were lysed to isolate nuclei, which were treated with Tn5 transposase (Nextera DNA Sample Prep Kit, Illumina) to isolate DNA. Fragmented DNA was then amplified using bar-coded PCR primers and libraries were seuqenced. Results: 15736 differential peaks (2-fold change) were identified between E10.5 WT and Chd4-CMko hearts. Overall design: Each embryonic mouse heart was dissected in cold PBS and individually cryopreserved in 200 mL of DMEM medium with 10% FBS and 10% DMSO (vol/vol) using a Mr. Frosty isopropyl alcohol chamber. Hearts from each genotype were thawed in a 37?-water bath and pooled (three pooled E10.5 hearts per genotype per biological replicate, four biological replicates for each genotype). Samples were pelleted at 1000g for 3 min at 4?. The medium was aspirated, and the hearts were washed with cold PBS. Hearts were then dissociated in 200 mL of 0.05% Trypsin/EDTA at 37? for 10 min, with gentle agitation every 3 min. Dissociation was stopped by adding 400 mL of DMEM/10% FBS (vol/vol), and the samples were passed through a 100-micron cell strainer to remove connective tissue. Cells were quantified using Trypan blue, and 40,000 viable cells were taken for downstream procedures. Dissociated embryonic cardiac cells were lysed to isolate nuclei, which were treated with Tn5 transposase (Nextera DNA Sample Prep Kit, Illumina) to isolate DNA Fragmented DNA was then amplified using bar-coded PCR primers and libraries were pooled. Pooled libraries were then sequenced with the NovaSeq 6000 platform. Reads were aligned to the mm10 reference genomes using BWA-MEM and peaks were called using HOMERv4.10.3. Annotation and analysis were performed using ChIP-Seeker v1.20.0.

研究目的：本研究利用ATAC测序（ATAC-seq），旨在鉴定E10.5时期野生型（WT）与心肌细胞条件性敲除（Chd4-CMko）小鼠心脏之间的心脏染色质差异可及性。
实验方法：每个生物学重复按基因型分别混合3枚E10.5时期的胚胎心脏，随后将其解离为单细胞悬液。取40000个活细胞进行裂解以分离细胞核，使用Tn5转座酶结合Illumina公司Nextera DNA样本制备试剂盒处理细胞核，以分离DNA片段。随后使用带条形码的PCR引物扩增断裂DNA，并构建测序文库并进行测序。
实验结果：在E10.5时期的野生型与Chd4-CMko小鼠心脏之间，共鉴定出15736个差异峰（2倍变化）。
整体实验设计：将每枚胚胎小鼠心脏置于预冷磷酸盐缓冲液（PBS）中解剖，随后使用Mr. Frosty异丙醇冻存盒，将其单独冻存于200 mL含10%胎牛血清（FBS）与10%二甲基亚砜（DMSO，体积比）的DMEM培养基中。将同基因型的心脏置于37℃水浴中解冻，按基因型混合（每个生物学重复混合3枚E10.5时期的胚胎心脏，每个基因型设置4个生物学重复）。样本以1000g转速在4℃下离心3分钟，弃去上清培养基，使用预冷PBS洗涤心脏组织。随后将心脏置于200 mL 0.05%胰蛋白酶-EDTA溶液中，37℃下温和振荡孵育10分钟，每3分钟轻轻摇晃一次。加入400 mL DMEM/10% FBS（体积比）终止解离反应，将样本通过100微米细胞筛以去除结缔组织。使用台盼蓝对细胞进行计数，取40000个活细胞用于后续实验步骤。解离后的胚胎心脏细胞经裂解分离细胞核，再次使用Tn5转座酶结合Illumina公司Nextera DNA样本制备试剂盒处理细胞核以分离DNA片段。随后使用带条形码的PCR引物扩增断裂DNA，并合并测序文库。合并后的文库使用NovaSeq 6000平台进行测序。使用BWA-MEM工具将测序reads比对至mm10参考基因组，使用HOMERv4.10.3软件进行峰识别。使用ChIP-Seeker v1.20.0软件完成基因组注释与数据分析。