Gene expression in WT and Camkk2 KO murine hematopoietic stem cells

To determine the function of Camkk2 in HSC we performed a microarray analysis on isolated KSL from WT and Camkk2 null mice. Using a threshold of 0.05 for statistical significance (p-value) and a log fold change of expression with absolute value of at least 0.6, 1831 differentially expressed genes (DEGs) were identified out of a total of 29352 genes with measured expression. When compared to WT KSL, 1289 genes were significantly downregulated and 533 genes were upregulated in Camkk2 null HSC. The genes downregulated in Camkk2 null KSL were strongly enriched in HSC, early progenitors and lymphoid-myeloid-affiliated genes (s-myly) primed in HSC. Of note, HSC-affiliated genes found downregulated in Camkk2 KSL included Hlf, Meis1, Pbx-1 and Prdm5 which are 4 of the 8 genes capable to reprogram committed murine blood cells into HSC. The GSEA analysis also showed that genes affiliated with the late progenitor signature and erythroid genes primed in HSC and GMP specific genes (d-my) were significantly upregulated in Camkk2 null KSL. These findings indicate Camkk2 controls crucial transcriptional programs involved in the mechanism of HSC differentiation and lineage commitment. Bone marrow was removed from bones of WT and Camkk2 null mice (16 mice/genotypes), and four biological replicates for each genotype were generated. Hematopoietic stem and progenitor cells were isolated based on the expression of Lineage (Lin) markers, c-Kit and Sca-1 expression. Lin-Kit+ Sca1- and Lin-Kit+ Sca1+ (KL and KLS cells) were sorted from mice directly into Buffer RLT (Qiagen). RNA isolation, quality control and all the procedures required for microarray analysis were performed at Sequencing and Genome Technologies Shared Resource (Duke University) according to standard operative procedures.