GSM9598106: Liver, STD, rep1; Mus musculus; RNA-Seq
Mice were injected intraperitoneally with 5 IU heparin sodium 5 minutes prior to euthanasia. Livers were excised, washed in ice-cold cell-suspension buffer (PBS + 4% FBS), minced into ~1 mm pieces, and subjected to sequential enzymatic digestion at 37°C with dispase (2.4 U/mL; Gibco #17105-041), collagenase type IA (Sigma-Aldrich #C9891), and collagenase type II (Worthington #LS004176), each followed by mechanical dissociation. After each step, suspensions were centrifuged (400g, 5 min, 4°C) and resuspended in the next enzyme solution. Final suspensions were filtered through 40 µm strainers and red blood cells were lysed with 1x RBC Lysis buffer (BioLegend #420302) for 3 min at room temperature. Cell viability and total cell number were assessed prior to fixation and library preparation. Following liver dissociation, cell viability and total cell number were assessed. For each sample, pooled cell suspensions were combined to obtain approximately 10 million cells. Cells were fixed using the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit, 16 rxns (10x Genomics, PN-1000414). Libraries were prepared using the Chromium Fixed RNA Kit, Mouse Transcriptome (10x Genomics, PN-1000496) with the Chromium Next GEM Chip Q Single Cell Kit (16 rxns) according to the manufacturer's instructions.