GSM3580368: BEC_ctr2; Mus musculus; RNA-Seq
Optimal isolation of single biliary epithelial cells was obtained by modifying previously established, two-step liver perfusion protocols(Dorrell et al., 2008; Li et al., 2010). Mice were euthanized, the heart and liver were surgically exposed, and a cannula was immediately inserted through an incision in the right atrium through the suprahepatic vena cava. Mice livers were then perfused by a pre-warmed (37°C) perfusion buffer (50 mM EDTA, 10 mM HEPES in 1X HBSS [Gibco, #14185-052]) at an initial flow rate of 3 ml/min for 2.5 minutes, followed by an increase in flow rate to 4 ml/min for an additional 2.5 minutes. This was then followed by perfusion with a pre-warmed (37°C) Collagenase Type IV (150 U/mL; Invitrogen, #17104019) digestion buffer solution (1.25 mM CaCl2, 4 mM MgCl2, 10 mM HEPES in 1x HBSS) for 8-10 minutes at a flow rate of 4 ml/min. For the duration of the two-step liver perfusion, the infrapatic vena cava was digitally occluded every 30 seconds for 10 seconds. After isolating the liver and surgically removing the gallbladder, the capsule was disrupted and the liver was gently and mechanically agitated to release dissociated single cells. The remaining liver was than subjected to serial digestions in Collagenase Type IV (150 U/mL), Accutase (EMD Millipore), and trypsin (0.25%) for 30 minutes, 30 minutes, and 20 minutes, respectively, at 37°C. Dissociated cells were collected after each step and filtered through a 100 μm cell strainer, washed, and re-suspended in ice-cold resuspension buffer (2% FBS, 1.25 mM CaCl2, 4 mM MgCl2, 10mM HEPES, 5 mM glucose in 1X HBSS). Hepatocytes were pelleted and removed by a 30xg spin and the remaining cells were spun at 300xg to isolate biliary cells. After treatment in red blood cell lysis buffer (eBioscience) on ice for 7 mins, the cells were re-suspended in cold resuspension buffer. For hepatocyte isolation, a similar perfusion protocol as described for BEC isolation was used with adaptation of the digestion step, by using liver digest medium (Thermo Fisher, #17703034) and increasing the digestion time to 16-20 minutes while maintaining identical flow rates increasing the digestion time to 16-20 minutes. The liver was then excised, the liver capsule removed, and the liver gently swirled in resuspension buffer to yield a cell suspension. After filtration through a 100 µm cell strainer, the hepatocytes were selectively pelleted by centrifugation with 30xg for 5 minutes, which removes the non-parenchymal cell fraction. The resuspended hepatocytes were then mixed 1:2.2 with 40% iodixanol (Optiprep, Axis-Shield, #1114542) solution (diluted in resuspension buffer) for a volume of 4.8ml, overlayered with 3 ml 18% iodixanol and 0.5 ml resuspension buffer and spun at 500xg for 25 minutes at 4°C with reduced deceleration. The top cell layer was collected, mixed with resuspension buffer supplemented with 3% BSA and washed once at 30xg for 5 min at 4°C. The cells were then re-suspended in ice-cold resuspension buffer (containing 2% BSA and 9% Optiprep), quantified and assessed for viability by Trypan blue stain. Routinely, viability of over 98% was obtained. Isolated single cells were encapsulated, and libraries were generated using inDrops(Zilionis et al., 2016). Approximately 1500 cells were encapsulated for each sample Illumina TruSeq adapaters For BEC sample raw data - R1: transcript (read with biological content), R2: first 8 nucleotides of cell-specific barcode, R3: Library index; R4: first 8 bases correspond to second half of cell-specific barcode; next 6 bases correspond to the UMI; the rest contains the polyA tail.