GSM8367131: Human liver, normal, AAV8, NL #2 Heps3; Homo sapiens; RNA-Seq
Following NMP, livers were flushed with 10-15 L of ice-cold normal saline through the portal vein and hepatic artery until the efflux was clear, followed by 1 L UW solution. 50-70 g wedges of the right and left liver lobes were cut and immediately immersed in ice-cold UW solution. Sections were manually perfused with UW solution through open vessels before cell release. To release hepatocytes, sections were perfused with calcium- and magnesium-free HBSS containing 20 mM HEPES, penicillin-streptomycin and 1 mM EGTA for 15 minutes at 37°C followed by perfusion with 1 mg/mL collagenase IV in 20 mM HBSS containing HEPES, penicillin-streptomycin and 5 mM CaCl2.H20 for 22 minutes at 37°C. The crude cell preparation was suspended in DMEM containing penicillin-streptomycin and 5% FBS, filtered through sterile gauze, pelleted at 70g and washed twice with the same suspension media. Hepatocytes were further purified by centrifugation through 45% Percoll at 200g for 15 minutes. For isolation of NPCs, a liver section was perfused for 15 minutes with HBSS containing 1mM EGTA as described for hepatocytes, followed by perfusion with 300 mL of pronase (1 mg/mL in DMEM) and then by 1.5 mg/mL collagenase IV in HBSS containing 5 mM CaCl2 for 22 minutes. The crude cell preparation was filtered through sterile gauze, digested further in 200 mL pronase (0.5 mg/mL) for 30 minutes at 37°C with constant shaking, pelleted at 600g, washed twice and subjected to density gradient centrifugation with 9% Accudenz at 1400g for 17 minutes. Hepatic stellate cells were retrieved from the top of the 9% Accudenz gradient and other NPC populations were retrieved from the mid-portion of the gradient. For NL #2, NL #3, NL #4, and SL #3, some cells were isolated via chunk digestion: ~250 mg of liver tissue was minced and incubated in Gibco Liver Perfusion Media at 37°C for 15 minutes with 20 rpm rotation. After washing with PBS, the tissue sample was incubated with collagenase IV (minimum 600 U/mL) in HBSS supplemented with 1% HEPES for 30 minutes at 37°C with 100 rpm rotation. The tissue sample was then triturated using a 10 mL or 25 mL pipette 10-15 times and passed through a 70 µm filter to acquire single cells and red blood cells were lysed with ACK Lysis Buffer. For all samples, viability was assessed using trypan blue and cells were counted with a hemocytometer. For each sample, libraries were prepared using the Nextera XT DNA Library Preparation kit: First, 800-1000 pg of DNA were dual-indexed with N700 and N500 oligonucleotides. SPRI beads were used to purify library products at sequential 0.6x and 1x ratios by volume. Library concentration was determined to be between 100-1000 bp using an Agilent HSD1000 TapeStation. 6 nM dilutions were prepared for each library.