GSM6248703: BM sample pre/post allo-SCT; Homo sapiens; RNA-Seq
We prepared BM samples at three time points from identical patients: non-hCR before SCT, CR at engraftment after allo-SCT, and non-hCR at relapse. We purified CD34+ cells from each sample using an indirect CD34 MicroBead Kit (Miltenyi Biotec). Cells (4,000) from each sample were used for single-cell isolation using BD Rhapsody Single-Cell Analysis System (BD Biosciences). Before single-cell isolation, all cells were labeled using a BD Single-Cell Multiplexing Kit and BD AbSeq antibody against TIM-3; six samples derived from two patients were loaded onto a single cartridge cDNA library preparation was performed according to the manufacturer's instructions. For cDNA amplification, we used costumed 435 primer pairs (46 genes associated with stem cell signature on BD Onco-BC Panel Hs, which contains 389 genes). An aliquot of the PCR products was used for assessing concentration and for the quality check of size and integrity using a Qubit (High Sensitivity dsDNA kit, Thermo Fisher Scientific) and the Agilent 4200 TapeStation system (High Sensitivity D1000 screen tape, Agilent), respectively.