GSM8353437: sonKD; Mus musculus; RNA-Seq
24 hours post-transfection, cells were washed with 1xPBS and dettached with 0.05% trypsin in EDTA for 2 minutes at 37C, followed by quenching with complete growth media. Proper viability of cells prior to freezing was quantified with propidium iodide/acridine orange staining in a LUNA-FL Dual Fluorescence Cell Counter. 50% of cells were utilized to validate the knock down at the protein level. The other 50% of cells were centrifuged at 200rcf for 3 minutes. The supernatant was discaded and the pellet was resuspended in 1mL freezing media (80% complete media, 10% heat-innactivated FBS, 10% DMSO), and frozen at -80C. Cells were brought to sequencing facility in dry ice. After quick thawing at 37C, cells were transfered to 0.04% BSA in 1xPBS. Staining with 7-AAD was performed and healthy cells were sorted in 0.04% BSA in PBS. Cells were counted to ensure viability after sorting. This step was quickly followed by single-cell RNA barcoding (10x Genomics' Chromium) and sequencing (Illumina NextSeq 500 instrument, Single Cell 3' v3 chemistry), according standard procedures.