GSM7431052: M18, Tail Vertebrae, scRNAseq; Homo sapiens; RNA-Seq
Femur, Thoracic and Tail vertebrae of xenograft NSG mice were crushed with PBS to recovered cells from bone marrow. human T-ALL cells were purified after labeling with anti-human CD45 PE conjugated antibody followed by immuno magnetic selection with anti-Phycoerythrin (PE) microbeads (Miltenyi Biotech). Purifications were analyzed by flow cytometry then resuspended in PBS supplemented with 0.04% BSA. Leukemic cells are counted with trypan blue then suspended at 1 x 106 cells/mL in PBS 0.04% BSA. 20 000 cells per condition were loaded in Chromium Next GEM Chip G. Single-cell libraries were generated using Chromium Next GEM Single Cell 3' Reagent Kits (v3.1): GEM, Library and Gel Bead Kit v3.1 (PN-1000128), Chip G Single Cell kit (PN-1000127) and Dual Index kit TT Set A (PN-1000215) (10x Genomics), and following the Chromium Next GEM Single Cell 3' Reagent Kits (v3.1) User Guide (manual part no. CG0000204 Rev D). Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics). The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. Amplified cDNA and libraries were verified on Bioanalyzer 2100 (Agilent Technologies) using High Sensitivity DNA kit (Agilent Technologies). During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample dual index were added.