Tet enzymes are required for mammalian embryonic hematopoiesis

In this study we have examined the role of Tet enzymes in regulation of embryonic Endothelial cells (ECs) and Hematopoietic stem and progenitor cells (HSPCs). We have performed transcriptomic and methylation analyses of Tet deficient and wild type ECs and HSPCs isolated from E11.5 embryonic aorta-gonad-mesonephros (AGM) to identify differentially expressed genes and differentially methylated regions. Overall design: Endothelial cells (CD31+C-Kit-) and Hematopoietic stem and progenitor cells (CD31+C-Kit+) were isolated by FACS from E11.5 aorta-gonad-mesonephros (AGM) (Tet1/2/3 F/F +/Rosa26-CreER and littemate control Tet1/2/3 F/F +/+, Tamoxifen E7.5) RNA and DNA were extracted from ECs or HSPCs at the same time using QIAGEN All Prep DNA/RNA Micro Kit (QIAGEN, cat. no.80284). Gene expression profiling by RNA-seq: RNA-seq libraries were prepared first by amplifying RNA using SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing (Takara, cat. no.634889) and then libraries were prepared using Nextera XT DNA Library Preparation Kit (illumine, cat.no.15055293). Three technical replicates from three independent batches of cells were used. The libraries were then subjected to 75 bp paired-end sequencing (Illumina NextSeq 500 platform) at the Einstein Epigenomics core. Whole genome bisulfite sequecing (WGBS): WGBS library construction was done using Pico Methyl-Seq TM Library Prep Kit (ZYMO research, cat. no.D5455 for IIIumina-based Sequencing). The libraries were sequenced using Novagene Hiseq 4000 platform. 150 bps paired-end reads were sequenced and data were analyzed as detailed in methods section.