GSM7081871: NEK2 knockout 5TGM1, scRNAseq; Mus musculus; RNA-Seq
Mouse tibia and femur were harvested. Bone marrow cells were obtained by flushing using a 25-gauge needle with ice-cold PBS buffer. Debrics were removed by passing through a 70 μm nylon cell strainer and cells were collected in a 50 mL Falcon conical tube. Cells were centrifuged at 300g for 5 minutes and cell pellet was resuspended with 500 μL RBC lysis buffer at room temperature for 2 minutes, and neutralize the lysis buffer by adding 5 mL DMEM complete medium. Then cells were centrifuged at 300g for 5 minutes and resuspended with frozen medium (60% DMEM, 30% FBS, 10% DMSO) and stored at -80°C freezer. On the day for scRNA-seq analysis, frozen cells were recovered in 37°C water bath and washed twice with PBS buffer. Dead cells were removed using Dead cell removal kit (130-090-101, Miltenyi Biotec). Samples with viability higher than 95% were processed for 10x genomics scRNA-seq. Approximately 10,000 cells were targeted, and single-cell emulsions were generated with the Chromium Controller (10x Genomics, Pleasanton, CA, USA) using the Chromium Next GEM Chip G (Part Number 1000120, 10x Genomics) with the Chromium NextGEM Single Cell 3' v3.1 kit (Part Number 1000121, 10x Genomics) following the standard protocol. Libraries were assessed for mass concentration using the Qubit 1X dsDNA HS Assay Kit (catalog # Q33231; Invitrogen, Waltham MA, USA) and the Qubit 4 fluorometer (catalog # Q33238; Invitrogen, Waltham MA, USA). Library fragment size was assessed with the High Sensitivity NGS Fragment Analysis Kit (catalog # DNF-474-0500; Agilent, Santa Clara CA, USA) on the Fragment Analyzer System (catalog # M5310AA; Agilent, Santa Clara CA, USA). Libraries were functionally validated with the KAPA Universal Library Quantification Kit (catalog # 07960140001; Roche, Basel CH).