GSM3833423: Venous Angioblasts 77 days post transplant; Homo sapiens; RNA-Seq
Freshly euthanized mice (<1 min post cervical dislocation) were aseptically prepared allowing 3-5ml of collagenase (0.2%w/v in 20% FCS/PBS) to be perfused in situ via a 26G1/2 needle cannulated portal vein.  Perfused, dissected intact liver was transferred to ice cold HANKS buffer (ThermoFisher, 14175-095) in a 10cm culture plate.  Isolated livers were minced (~2-3mm pieces) with a sterile razor blade, washed twice in hanks buffer by 1 minute settling, and further dissociated for 50 minutes rotating in a 10cm plate at 70RPM (37˚C, 5% CO2, 95% Air) in 24ml liver dissociation solution comprising (66%v/v HANKS buffer, 33%v/v RPMI1640, BSA (0.17%w/v, Proliant), collagenase type 1 (0.3125%w/v, Sigma), DNAse (1U/ml).  Following light trituration by 10ml serological pipette, homogenate was further dissociated for 10 minutes.  Total liver homogenate was filtered (100µm) and centrifuged at 50xg for 5 minutes to pellet hepatocytes and cell clumps.  Supernatant (non-parenchymal fraction) was pelleted (350xg, 5 minutes) and resuspended in ammonium chloride (STEMCELL Technologies, 07850) for 10 minutes on ice to lyse RBCs.  RBC-free NPC was washed three times (90% HANKS buffer, 10% RPMI1640, 0.05% BSA), filtered (40µm) and used for surface marker staining including: anti-CD31-FITC (BD Biosciences, Clone WM59, 15:100), rabbit-anti-LYVE1 (Abcam, Polyclonal, 1:1000), anti-CD32-PeCy7 (Biolegend, Clone FUN-2, 3:100), Donkey-anti-rabbit-APC (FAB fragment, Jackson, 0.5:100), FcR blocking human IgG (Miltenyi, 15:100) in RPMI1640 with 0.5% BSA and 1U/ml DNAse at 1x106 cells/ 100µl.  After antibody staining, cells were washed twice in RPMI1640 with 0.5% BSA and stained with DAPI.  FACS was performed to validate that the sample contained expected DAPI- tdRFP+ CD31+ CD32+ LYVE1+ hPSC-derived LSECs.  Once confirmed, DAPI- (live cells) tdRFP+ (hPSC-derived) cells were purified from the mouse liver cells (tdRFP-).  Sorted cells were washed one time to resupended in PBS for immediate scRNA-seq processing. 10X Genomics Single Cell 3' v2 Reagent Kit
GEO Accession
GSM3833423