GSM6131524: Idh1R132H/+ #2; Mus musculus; RNA-Seq
To isolate LSK cells, the femur, tibia and humerus bones were dissected from mice with genotypes of interest. The bone marrow cells were isolated by flushing in 5 mL MACS buffer (phosphate-buffered saline without MgCl2 and CaCl2, supplemented with 0.5% bovine serum albumin and 2mM EDTA pH8.0) using syringes with 26 gauge needles. The isolated cells were filtered through a 70 µm nylon mesh. The cells were centrifuged at 300 x g for 5 minutes at 4°C, and resuspended in 1 mL red blood cell lysis buffer (Sigma) followed by incubation for 5 minutes at ambient temperature. 10 mL cold buffer was added, the cells were centrifuged at 300 x g for 5 minutes a 4°C, and resuspended in 1 mL cold buffer prior to counting on a ViCell Counter. The cells were then washed with 5 mL buffer, centrifuged at 300 x g for 5 minutes a 4°C, and process for lineage depletion using the Mouse Lineage Cell Depletion Kit (Miltenyi Biotec # 130-090-858), following the manufacturer's instructions: cells were resuspended at 2.5 x 108 cells/mL in MACS buffer, and 10 µl of biotinylated mouse lineage antibody cocktail was added per 10 million cells, followed by incubation on ice for 15 minutes. 30 µl of MACS buffer and 20 µl of streptavidin-conjugated magnetic microbeads were added per 10 million cells, followed by incubation ice for 15 minutes, and filtering through a 30 µm nylon mesh in 10 mL cold MACS buffer. The cells were centrifuged at 300 x g for 5 minutes at 4°C, resuspended in 1 mL cold MACS buffer, and processed for lineage separation using an AutoMACS Pro instrument (Miltenyi Biotec) using the “Deplete” settings. Lineage-negative cells were collected.  Cells were incubated for 15 minutes on ice with MACS buffer containing 1:100 Fc blocking antibody (Tonbo Biosciences # 70-0161-M001). Cells were stained with the following antibodies in FACS or MACS buffer for 30 minutes on ice: cKIT-PE/Cy7 (BioLegend # 105814; 1:100), SCA1-FITC (BioLegend # 122505, 1:100), STREPTAVIDIN- APC/Cy7 (BioLegend # 405208; 1:50), alongside DAPI (2ug/mL). After staining, cells were washed twice with cold buffer, resuspended, and sorted using a FACSAria II fluorescence-activated cell sorter (BD Biosciences) into MACS buffer at 4°C. Libraries were prepared using the 10X Genomics Chromium Single Cell 3' v2 Chemistry Reagent Kit, as per the parameters recommended by 10X Genomics.