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
For the mice (8 weeks old BALB/cJ) brain cell isolation, the Miltenyi Biotech Neural Tissue Dissociation kit (130-092-628) was used without perfusion according to manufacturer's guideline. 8 weeks old mice (C57BL6/J and C57BL6/N were used for lung and spleen isolation. Single cell suspensions were prepared by digestion in 100µg/mL Liberase TL (Cat 05401020001, Roche, Germany) and 100µg/mL DNase I (11284932001, Roche, Germany) for 30 minutes at 37°C before passing through a 100µm cell strainer. Spleens were rinsed with ice-cold PBS and mashed into small pieces using a plunger of a 10 mL syringe on top of a sterile 100 µm cell strainer mesh in a petri dish containing 2 ml ice-cold RPMI. Red blood cells in the lung and spleen single cell suspensions were lysed using ammonium chloride lysis buffer (10 mM KHCO3, 155 mM NH4Cl, 0,1 mM EDTA in MilliQ water).  Single cell suspensions were washed with 1x dPBS before incubation with antibodies. 10x Genomics CG000390 Rev B protocol 10x Genomics CG000390 Rev B protocol