GSM7117463: Olive oil, biol rep 1, MMPsense negative, scRNAseq; Mus musculus; RNA-Seq
Murine livers were collected in RPMI1640 media on ice, minced and digested in 100 U/mL collagenase (Sigma-Aldrich) at 37°C for 1 hour with shaking. Digested tissue was filtered through 70 μm nylon mesh and hepatocytes removed by spinning at 50xg for 3 minutes. The remaining cells were passed through a 25 % Percoll (GE Life Sciences) cushion and erythrocytes were lysed with ammonium-chloride-potassium buffer. Isolated cells were stained with 30 μg/mL Hoechst 33342 (Miltenyi) and Zombie NIR viability dye (diluted 1:250 in PBS; Biolegend) before FACS sorting (Sony SH800) to obtain live, single cells. These were methanol fixed based on the 10x Genomics recommended fixation protocol and stored at -80°C. Library was performed according to the manufacter's instructions for methanol fixed samples (single cell 3' v2 or v3.1 protocols, 10x Genomics). Briefly, hepatic cells in methanol were pelleted, counted, washed and 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 Ilumina 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 apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. For samples that were lipid hashed, library construction was performed as described in PMID 31209384 (McGinnis, Gartner et al., 2019).