GSM4310089: Macrophages SPTA; Mus musculus; RNA-Seq
The abdominal cavity of a living, deeply anesthetized mouse was opened, and the portal vein was catheterized for in situ liver perfusion and digestion with 50 ml of 0.06 U/ml collagenase B buffered solution (Roche, 11088815001). Once digested, livers were dissected out and the mouse was sacrificed. The digested livers were then mechanically disaggregated in a petri dish on ice and filtered through a 100 μm pore cell strainer. The cell suspensions were centrifuged twice at 60 x g for 2 min at 4°C, and the pellets were discarded. The supernatants were then centrifuged at 300 x g for 5 min at 4°C to obtain a pellet of nonparenchymal liver cells containing liver macrophages. To select for F4/80+ macrophages from liver cell suspensions, Dynabeads Sheep anti-Rat IgG (Invitrogen, 11035), in combination with purified rat anti-mouse F4/80 antibody (BD Pharmingen, 0.5 mg/ml, 565409) ), and a DynaMag™-2 Magnet (Thermo Fisher Scientific, 12321D) were used according to the manufacturer's protocol (Invitrogen). The purity of the separated cells was tested by detecting F4/80 positive cells by FACS. Nonparenchymal liver cell suspension enriched for F4/80+ macrophages were processed for library preparation according to the 10x Genomics Chromium Single Cell 3' v3 Reagent Kit instruction guide. Briefly, sample volume was adjusted to a target capture of 10,000 cells and loaded onto the 10x Genomics single-cell-A chip. After droplet generation, samples were subjected to reverse transcriptase and the barcoded cDNA was further amplified for 11 cycles (adjusted for a cell recovery of >6000,  as suggested by the 10x Genomic user guide). cDNA quality and concentration were assessed using High-Sensitivity D5000 ScreenTape (Agilent). cDNA strands were then subjected to enzymatic fragmentation, end repair and A-tailing. Adaptors were ligated to the fragmented cDNA, and sample index was added during sample index PCR (set for 13 cycles, as recommended by 10X Genomics user guide to correlate with a cDNA input of 12-150 ng). Library quality and concentration were assessed using High-Sensitivity D5000 ScreenTape (Agilent). Libraries were pooled in equimolar amounts and sequenced using the Illumina NovaSeq 6000 system according to 10X Genomics recommendations: paired-end reads, R1=28 cycles, i7=8 cycles and R2=91 cycles. Sequencing depth was targeted for 50,000 reads per cell in both samples.