Non-catalytic functions of Tet2 are essential to regulate hematopoietic stem and progenitor cell homeostasis

To identify genes that are influenced by the catalytic and non-catalytic functions of Tet2 in hematopoietic stem and progenitor cells (HSPCs), we analyzed the gene expression profiles of Tet2 catalytic mutant (Tet2 Mut), Tet2 knockout (Tet2 KO) and wild-type HSPCs (or LSK, Lin–Sca-1+c-Kit+) and multi-potent progenitor (or MPP, Lin–) cells by RNA-seq. LSK and MPP cells were sorted from pooled bone marrow of two 10-week-old Tet2 Mut, Tet2 KO and wild-type mice by FACS. RNA was extracted using QIAGEN RNAeasy Kit. RNA was amplified and barcoded and used to prepare libraries of two technical replicates, with ERCC spike in controls. As such, there were two replicates for each genotype for each cell type (total of 12 samples). The libraries were subjected to 150bp paired-end sequencing (Illumina Next-seq 500 platform) at the Einstein Epigenomics core following their protocols. This generated 15~40 million read pairs per sample. The reads were trimmed using the TrimGalore (v 0.4.1, https://github.com/FelixKrueger/TrimGalore) to remove adaptors and then mapped to the mouse genome (mm10) by TopHat2 software (v 2.0.13) with default parameters. Details of RNA-seq and data analysis are described in the methods sections of the manuscript.