Tet-dependent transcriptional programs during early embryogenesis

To determine the transcriptional effects of lack of Tet proteins during early embryogenesis, we performed single-embryo RNA-sequencing of control and TKO embryos (E6.75; 4 embryos from each group). Genome-wide analyses showed that Tet deficiency promotes the expression of mesoderm-related genes during early embryogenesis in vivo. Control and Tet1/2/3 TKO embryos were collected at E6.75. Single embryos were lysed and converted into double stranded cDNA using SMARTer ultra-low RNA kit for Illumina sequencing (Clontech). 1 ng of cDNA for each sample was used for preparing libraries using Nextera XT DNA sample preparation kit (IIlumina). Libraries were sequenced on Illumina HiSeq 2500 platforms according to the manufacturer's instructions.为探究早期胚胎发育过程中Tet蛋白缺失所介导的转录组学效应，我们针对对照组及三重敲除（TKO）胚胎（胚胎发育至E6.75时期；每组各取4枚胚胎）开展单胚胎RNA测序（RNA-sequencing）实验。全基因组水平分析结果显示，在体内早期胚胎发育过程中，Tet蛋白缺失会促进中胚层相关基因的表达。我们于胚胎发育E6.75时期收集了对照组胚胎与Tet1/2/3三重敲除（TKO）胚胎。将单枚胚胎裂解后，使用SMARTer超低起始量RNA试剂盒（SMARTer ultra-low RNA kit）将其逆转录为双链互补DNA（double stranded cDNA），以用于Illumina测序（Clontech公司出品）。每个样本取1 ng双链cDNA，使用Nextera XT DNA样本制备试剂盒（Nextera XT DNA sample preparation kit，Illumina公司）构建测序文库。按照制造商的操作指南，在Illumina HiSeq 2500测序平台上完成文库测序。