GSM5590437: Sample 11_L38; Mus musculus; RNA-Seq
Nuclei were isolated from frozen liver samples (~200mg) as previously described (dx.doi.org/10.17504/protocols.io.3fkgjkw). Briefly, livers were diced in EZ Lysis Buffer (Sigma-Aldrich), homogenized using a disposable dounce homogenizer, and incubated on ice for 5 minutes. The homogenate was filtered using a 70 μm cell strainer, transferred to microcentrifuge tube, and centrifuged at 500 x g and 4°C for 5 minutes. The supernatant was removed and fresh EZ lysis buffer was added for an additional 5 minutes on ice following by centrifugation at 500 x g and 4°C for 5 minutes. The nuclei pellet was washed twice in nuclei wash and resuspend buffer (1X PBS, 1% BSA, 0.2 U/μL RNAse inhibitor) with 5 minute incubations on ice. Following the washes, the nuclei pellet was resuspended in nuclei wash and resuspend buffer containing DAPI (10 μg/mL). The resuspended nuclei were filtered with 40 μm strainer and immediately underwent fluorescence activated cell sorting (FACS) using a BD FACSAria IIu (BD Biosciences, San Jose, CA) with 70 μm nozzle. Libraries were prepared using the 10X Genomics Chromium Single Cell 3' v3.1 kit (10X Genomics)