GSM5903649: K16/2929; Homo sapiens; miRNA-Seq
Mononuclear cells were isolated by Ficoll density gradient (Axis-shield), complemented by CD34 isolation if CD34 expression was <50% (CD34 MicroBead Kit, Milteny). Cell sorting was performed to isolate CD34+/CD38- and CD34+/CD38+ cells from patients and controls, defined as LSC and HSC, and L-blasts and C-blasts, respectively. Sorted cells were spun down (10 min, 3000 rpm, 4°C) and resuspended in 700µl TRIzol for RNA extraction. RNA was extracted using the miRNeasy Mini or Micro Kit (Qiagen) in combination with on-column Dnase I digestion (RNAse-Free DNase set, Qiagen) according to the manufacturer's instructions. RNA concentrations were measured by Nanodrop (ThermoFisher Scientific) or Qubit RNA HS Assay (Invitrogen). Libraries for RNA sequencing were prepared by Biogazelle using a TruSeq small RNA library kit (Illumina) according to the manufacturer's instructions. The resulting small RNA libraries were concentrated via ethanol precipitation and quantified using the Qubit 2.0 Fluorometer before sequencing with a read length of 75 bp on a NextSeq 500 sequencer (Illumina).