GSM5267712: Exercise_rep2; Mus musculus; RNA-Seq
For a single-cell transcriptional profile, the liver was perfused with enzymatic solution of Collagenase, Type 1 (1 mg/ml) (Worthington-biochem) and for 10 min at 37 °C in a water bath with continuous agitation. After this treatment, the enzymatic solution was inactivated by adding DMEM-Dulbecco's Modified Eagle Medium (Gibco) with 10% fetal bovine serum (Gibco) and was immediately centrifuged at 400 g for 5 min at 4 °C. The cell suspension was filtered through a 70-μm cell strainer, subjected to discontinuous Percoll gradient separation (Sigma-Aldrich). NPCs were separated from the hepatocytes by differential centrifugation (400 rpm for 5 min). The supernatant was centrifuged again (1500 rpm for 5 min x2) to obtain NPCs. For 10X Genomics sample processing and cDNA library construction, hepatic CD45+ cells were isolated from NPC fraction using CD45 MicroBeads (Miltenyi Biotec). Samples were prepared as outlined in the 10X Genomics Single Cell 3′v3 Reagent Kit user guide. The samples were washed twice in PBS (Life Technologies) + 0.04% BSA (Sigma) and resuspended in PBS + 0.04% BSA. Cell viability was assessed by staining with Trypan Blue (Invitrogen) and scoring on a hemocytometer (Thermo Fisher). Following counting, the appropriate volume for each sample was calculated for a target capture of 6000 cells. Samples below the required cell concentration as defined by the user guide (i.e., <400 cells/μl) were pelleted, resuspended, recounted, and volume adjusted prior to loading onto the 10X Genomics single-cell-A chip. After droplet generation, samples were transferred onto a pre-chilled 96-well plate (Eppendorf), heat-sealed and reverse transcribed using a Veriti 96-well thermal cycler (Thermo Fisher). cDNA was recovered using Recovery Agent provided by the kit followed by a Silane DynaBead clean-up (Thermo Fisher) as outlined in the user guide. Purified cDNA was amplified for 12 cycles before cleaning using SPRIselect beads (Beckman). Samples were diluted 4:1 in elution buffer (Qiagen) and run on a Bioanalyzer (Agilent Technologies) to determine the cDNA concentration. cDNA libraries were prepared as outlined by the Single Cell 3′Reagent Kit v3(10X) user guide with appropriate modifications to the PCR cycles and based on the calculated cDNA concentration, as recommended by 10X Genomics.