title: Illumina HiSeq 2500 paired end sequencing; Single-cell RNA sequencing of human liver cells
design: Single-cell RNA sequencing of human liver cells
library_construction_protocol: Physiologically normal human liver tissues were collected from the periphery of liver specimens from donors undergoing surgical resection.  Liver tissue was stored at 4C and processed within 3 h post resection. Liver non-parenchymal cells (NPCs) were isolated following a two-step protocol of collagenase/EDTA digestion. Briefly, hepatocytes were removed by centrifuging the whole digestion at 50 g for 5 min (pellet removed  hepatocyte fraction, cleaned on Percoll), and the supernatant removed and spun again at 50 g for 5 min to remove the remaining hepatocytes, the supernatant was then pelleted at 300 g for 8 min and used as NPC fraction for downstream sequencing library preparation. Cell viability of the isolated NPCs was estimated by trypan blue exclusion.  Samples of 67-89% viable cells from each of the liver samples were subjected to single-cell RNA sequencing using the 10x Genomics Chromium Single Cell 3 Library & Gel Bead Kit v2 following the manufacturers instructions. Approximately 4000 cells were targeted for each sample recovery.