GSM8752429: leukemia cell lines, rockroi; Homo sapiens; RNA-Seq
For the cell line sample,  suspended cells were washed once with PBS and filtered through a 70  µm cell strainer. Cells were resuspended in PBS and counted with a Neubauer chamber. One million cells were resuspended in 1 mL of sample buffer and counted again at the BD Rhapsody Scanner. A ratio of 1:1:1 SUP-B15, K562 and Loucy cells was calculated using the BD Rhapsody Scanner and prepared before loading the sample on the BD Rhapsody Express machine. For the patient sample, frozen bone marrow samples were defrosted, washed twice in PBS and cells were counted with Trypan blue to assess sample quality. If the viability was below 75%, the cells underwent dead cell removal using magnetic bead-based dead cell removal (Dead Cell Removal Kit, Miltenyi Biotec) which was performed following the manufacturer's recommendations. Part of the sample was stained with fluorescent antibodies  for enriching non-leukemic live cells by FACS sorting. Bone marrow sample was stained firstly with the viability dye for 10 minutes on ice, washed once with PBS and stained with the antibody mix for 30 minutes at room temperature in darkness. After staining the cells were washed once in PBS and resuspended in PBS for FACS sorting using the BD FACS Aria III Fusion. Libraries were generated on a BD Rhapsody Express machine. Libraries for both samples were generated with the following changes to the standard BD Rhapsody workflow: 1) addtion of N1 primer to the RPE PCR; 2) addition of ROIseq primers to the pool of randomeres during RPE extension; 3) separate indexing reaction for the samples in which the N1 primer is added; 4) custom sequencing primer