Transcriptome Analysis of Leukemia Stem Cells

Total RNA was extracted from sorted LSCs (lin⁻c-kit⁺Sca1⁻CD16⁺CD34⁺) from two full blown leukemia mice developed from MLL-AF9-transduced Hsf1^fl/flVav-Cre HSPCs and two full blown leukemia mice developed from MLL-AF9-transduced Hsf1^fl/fl HSPCs (duplicate per genotype), respectively. cDNA synthesis and amplification were performed using the SMARTer Ultra Low Input RNA Kit (Clontech) starting with 20 ng of total RNA per sample, following the manufacturer’s instructions. cDNA was fragmented with Q800R sonicator (Qsonica) and used as input for NEBNext Ultra DNA Library Preparation Kit (NEB). Libraries were sequenced on Illumina’s HiSeq2000 in single-read mode with a read length of 50 nucleotides producing 60–70 million reads per sample. Sequence data in fastq format were generated using the CASAVA 1.8.2 processing pipeline from Illumina. Differential expression analysis was performed using the DESeq2 v1.30.1 R package^{97 (link)}. We considered genes differentially expressed between 2 groups of samples when the DESeq2 analysis resulted in an adjusted p-value of <0.01 and the log2-fold change in gene expression greater than or equal to 1.5 or less than or equal to −1.5. Gene set enrichment analysis (GSEA) 3.0 was done according to the previous report using the software downloaded from GSEA website (http://software.broadinstitute.org)^{98 (link)}.