RNA-seq of LSC-like and Mature populations from human AML patient samples
RNA-seq of LSC-like and Mature populations from human AML patient samples
AML samples were collected from bone marrow (BM) and/or peripheral blood (PB) aspirations at the University hospital in Heidelberg or Hannover in accordance with the Declaration of Helsinki after obtaining written consent from each patient. The project was approved by the Ethics Committee of the Medical Faculty of Heidelberg (S-169/2017 and S-648/2021) and the local Ethics Review Committee of Hannover Medical School (ethical vote No.7972_BO_K_2018). Mononuclear cells (MNCs) were isolated by density gradient centrifugation using Ficoll Paque Plus (GE Healthcare, cat # GE17-1440-03), and stored in liquid nitrogen until further use. Primary AML cells were stained with cell-surface antibodies. Cells were sorted into populations according to CD11b, CD64, and GPR56 expression within the live lineage-negative gate. Cells from each population were sorted directly into RNA extraction buffer (Thermo Fisher, cat #KIT0214), snap-frozen, and stored at −80°C until RNA extraction. RNA extraction and purification of FACS-sorted cells was done using PicoPure RNA Isolation Kit according to manufacturer's instructions (Thermo Fisher, cat # KIT0214).  Whole transcriptome amplification was performed using a modified Smart-Seq2 protocol (Picelli et al 2014), with 5μl of a modified RT buffer containing 1× SMART First Strand Buffer (Takara Bio Clontech, cat # 639538), 1 mM dithiothreitol (Takara Bio Clontech), 1 μM template switching oligo (IDT), 10 U μl−1 SMARTScribe (Takara Bio Clontech, cat # 639538) and 1 U μl−1 RNasin Plus RNase Inhibitor (Promega, cat # N2615). Tagmentation of cDNA was done using Nextera XT DNA Library Preparation Kit (Illumina, cat # FC-121-1030).