4.5. RT-PCR, PCR, qPCR and Sanger Sequencing

cDNA synthesis was performed using M-MLV Reverse Transcriptase for primary AML samples and Random Hexamers (Invitrogen, Thermo Fisher, Waltham, MA, USA) or the SuperScript III First-Strand Synthesis System (Invitrogen) for RNA extracted from transduced c-Kit⁺ cells. Polymerase chain reaction (PCR) primers were designed to amplify fragments containing the fusion boundary detected by RNA-seq using Primer3 (http://primer3.ut.ee/, Table S1). Quantitative PCR (qPCR) was performed using Brilliant III Ultra-Fast QPCR Master Mix (Agilent Technologies, Santa Clara, CA, USA) on an Mx3000p qPCR system (Agilent Technologies) and standard cycling set-up (Table S1). TaqMan gene expression for BCL11B mRNA (Hs01102259_m1) was performed on BM cells from AML patients (blasts ≥ 80%, n = 10) and peripheral blood mononuclear cells from healthy controls (n = 3), using GAPDH (Hs02786624_g1) as reference gene, on the Applied Biosystems 7500 Real-Time PCR System (Thermo Fischer Scientific). Gene expression was quantified by the 2^-ΔΔCt method, using the average of healthy controls as reference sample. Long-distance PCR were performed with LA Taq DNA Polymerase (Takara Bio, Shiga, Japan) following manufacturer instructions for human genomic DNA. Fast Start Taq DNA Polymerase (Roche, Basel, Switzerland) was used for standard PCR reactions. Products were purified with the QIAquick PCR purification kit (Qiagen) or conventional agarose gel electrophoresis and extraction of specific bands with the QIAquick Gel Extraction kit (Qiagen). PCR products were sequenced by Sanger Sequencing using an ABI PRISM 3730 automated DNA sequencer (Applied Biosystems) and the Big Dye Terminator DNA sequencing kit (Applied Biosystems, Foster City, CA, USA). Fusion detection was performed using NCBI Blast alignment and BLAT software tool (http://genome.ucsc.edu/cgi-bin/hgBlat?command=start) to reference genome GRCh37/hg19.

BCR and TCR clonality assay was performed as described by the BIOMED-2 study [66].