Microarray and RNA-seq Analysis of AML Subpopulations

Microarray gene expression analyses were performed as described in Wang
et al^{40 (link)}. The log2-normalized
data were linearized and used as input for Gene set enrichment analysis (GSEA,
Broadinstitute.org). One gene matrix transposed file was generated containing
the transcripts from up- and down-regulated genes in the comparison of
NKG2DL^neg and NKG2DL^pos blasts (CD34-expressing and
non-expressing AMLs separately), and the transcripts of the LSC signature, the
HSC signature, the progenitor signature and the 17-genes stemness score
published previously^{43 (link),44 (link)}. GSEA computes if the gene set
is enriched in the generated gene expression data. For RNA-sequencing, total RNA
from 50,000 cell was isolated using an Arcturus PicoPure RNA isolation kit (Life
Technologies, Invitrogen). cDNA libraries were generated with 10 ng of total RNA
using the SMARTer Ultra Low RNA kit for Illumina sequencing (Clontech
Laboratories) and sequenced with a HiSeq2000 device (Illumina). Sequences were
aligned to hg19 reference genome using the STAR alignment software.
DESeq2^{45 (link)} was used to
calculate differentially expressed genes between NKG2DL^neg and
NKG2DL^pos samples. GSEA was performed using default settings.