Illumina NovaSeq 6000 paired end sequencing; Comparison of different hashing methods on human PBMCs and primary mice tissues
Comparison of different hashing methods on human PBMCs and primary mice tissues
8 weeks old mice (BALB/cJ, C57BL/6N and DBA/2J) were deeply anaesthetized and perfused transcardially with 20 ml of ice-cold phosphate buffered saline (PBS). Mice were decapitated, brains were divided in two hemispheres and collected in 1 mL ice-cold Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco). Brains were cut into small pieces (1-2 mm) after which enzyme mix was added (30 U ml−1 DNAse I (Roche), 10 U ml−1 collagenase type I (Worthington) and 400 U ml−1 collagenase type IV (Worthington) diluted in 1X Hanks' buffered salt solution (Gibco)). The brains were incubated for 20 min at 37 °C, and resuspended with a 1 mL pipet every 10 minutes. The solutions of three hemispheres per condition were pooled and filtered twice over a 100 μm nylon filter and centrifuged. The pellet was resuspended in 5 ml 70% standard isotonic percoll (SIP, GE Healthcare) diluted in 1X Hanks' buffered salt solution and carefully overlaid with 5 ml of 37% SIP, followed by a 5 ml layer of 30% SIP, forming a three-layered density gradient (centrifuged at 800g, 4 °C, 30 min without acceleration/braking). The 70/37% interphase containing immune cells was collected, centrifuged, pooled per condition, and resuspended in fluorescent activated cell sorter (FACS) buffer (2 mM EDTA (Duchefa), 2% heat-inactivated fetal calf serum (Gibco) dissolved in 1X Hanks' buffered salt solution). The duration of the entire dissociation process was 4 h. 10x Genomics CG000390 Rev B protocol 10x Genomics CG000390 Rev B protocol