GSM6605939: liver single cell; Mus musculus; RNA-Seq
Livers from adult mice were perfused with 0.5 mg/ml collagenase D through the portal vein. After pelleting down the hepatocytes, the cells remaining in the supernatant were equilibrated with OptiPrep to 17% final concentration. After centrifuging at 400g for 15 min, cells band at the interface were collected and red blood lysis buffer were used after wash. Cell viability was at least 95% (Trypan blue exclusion). Cells were filtered through a 40 um sieve, stored in PBS with 2% FCS. FACS antibodies were incubated for 30 min at 4 ℃. Cells were analyzed or sorted with BD flow cytometer using a 100 μm nozzle. Library was performed according to the manufacturer's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriately sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.