GSM5065206: CON3; Mus musculus; RNA-Seq
Liver NPCs were isolated from mice according to a two-step collagenase method. In detail, murine livers were perfused in situ via the inferior vana cava with calcium-free Hank's Balanced Salt Solution (HBSS, Gibco, Cat#14170112) containing 0.2 mg/mL EDTA (Sigma, Cat#E6758), followed by 0.4 mg/mL pronase (Sigma, Cat#P5147-1G) and 0.2% collagenase type II (Worthington, Cat#LS004176) at a perfusion rate of 8 mL/minute. Then livers were surgically removed and cut into small pieces. Tissues were transferred in HBSS containing 0.2% collagenase type II, 0.4 mg/mL pronase and 0.1 mg/mL DNase I (Roche, Cat#10104159001), and then incubated for digestion at 37℃ in a water bath for 20 minutes. DMEM (Mediatech, Cat#10-013-CV) containing 10% serum (FBS, Gibco, Cat#10099-141C) was added at the end of the incubation. Sequentially, hepatocytes removal was achieved by centrifugation for 3 minutes at 50 g. Then cell suspension was filtered using a 40 μm nylon cell strainer (Falcon, Cat#352340). Erythrocytes were lysed by treatment with 3-5 mL ACK lysing buffer (Gibco, Cat#A1049201) for 5 minutes after which PBS (Beyotime Biotec, Cat#C0221A) was added to terminate the lysis. The resulting suspension was subjected to Dead Cell Removal Kit (Miltenyi Biotec, Cat#130090101) to remove dead cells according to the manufacturer's recommendations. The obtained cell pellet was washed twice and suspended in PBS. Cell viability was assessed by Trypan Blue (Gibco, Cat#15250-061). Liver NPCs single cell suspensions were loaded onto the 10x Genomics Chromium chip (10x Genomics; Pleasanton, CA, USA) to generate droplets. Then the obtained Gel Beads-in-emulsion (GEMs) were transferred into a PCR tube strip, followed by reverse transcription using ProFlex PCR System (Thermo Fisher, MA, USA). The resulting cDNA was purified and amplified for 12 cycles before cleanup with SPRIselect beads (Beckman, Cat#B23318). Based on the cDNA concentration determined by Qubit (Thermo Fisher, MA, USA), libraries were prepared using the Chromium Single Cell 3′ Library & Gel Bead Kit v3 (10x Genomics; Pleasanton, CA, USA) according to the manufacturer's instructions.