Transcriptomic analysis of pediatric T-cell acute lymphoblastic leukemia
Transcriptomic analysis of pediatric T-cell acute lymphoblastic leukemia
Leukemic T-cells were obtained from bone marrow samples by isolation of mononuclear cells, using density gradient centrifugation, followed by immunomagnetic separation, with use of Human T Lymphocyte Enrichment Set-DM (Becton Dickinson) in case of samples with blast percentage below 85%. Thymocyte CD4+ CD8+ and CD34+ samples, obtained from children undergoing cardiac surgery (UZ Gent), were used as controls. Immature CD34+ thymocytes were purified based on MACS purification using CD34 microbeads (Miltenyi Biotec) without lineage depletion, while CD4 and CD8 labeling was used to sort the CD4+ CD8+ double-positive subset by a FACSAriaIII (BDBiosciences). The purity of each subset was at least 98%. Total RNA was isolated using miRNeasy Kit (Qiagen). RNA isolates were DNase treated and purified with use of RNA Clean and Concentrator Kit (Zymo Research). Libraries were generated with Illumina TruSeq Stranded mRNA Prep Kit. Modified fragmentation conditions were used (90'C for 2 minutes)