Singleron python automated tissue dissociation system

Hepa1-6 tumors were treated with PBS, Virus@gel or V-Navo@gel (1 × 10⁷ PFU) intratumorally for 12 days. Then the tumors were dissociated and stored in the sCelLive^TM Tissue Preservation Solution (Singleron Bio Com, Nanjing, China) on ice after the surgery within 30 min. Tumors were digested with 2 mL sCelLive^TM Tissue Dissociation Solution (Singleron) by Singleron PythoN® Automated Tissue Dissociation System (Singleron) at 37 °C for 15 min. The solution was then centrifuged at 500 g for 5 mins and suspended softly with PBS. Then the single-cell suspensions (1 × 10⁵ cells/mL) were loaded into microfluidic devices using the Singleron Matrix® Single Cell Processing System (Singleron). Subsequently, the scRNA-seq libraries were constructed according to the protocol of the GEXSCOPE® Single Cell RNA Library Kits (Singleron)^{46 (link)}. Individual libraries were diluted to 4 nM and pooled for sequencing. At last, pools were sequenced on Illumina novaseq6000 with 150 bp paired end reads.

Three unilaterally decapsulated testes were dissected from 3 adult WT and 3 adult KO mice. The fresh testicular tissues were stored in the sCelLiveTM Tissue Preservation Solution (Singleron Biotechnologies) on ice within 30 min after the surgery. The tissues were washed with Hanks’ Balanced Salt Solution thrice and then digested with 2 ml sCelLiveTM Tissue Dissociation Solution (Singleron Biotechnologies) using the Singleron PythoN™ Automated Tissue Dissociation System (Singleron Biotechnologies) at 37°C for 15 min. The tissue lysate was centrifuged at 500 × g for 5 min, and the pellet was suspended gently with PBS. The samples were stained with trypan blue (T8154; Sigma), and the cellular viability was evaluated microscopically. Apoptotic or dead cells were discarded.

The fresh tissues were stored in the sCelLiveTM Tissue Preservation Solution (Singleron Bio Com, Nanjing, China) on ice after the surgery within 30 min. The specimens were washed with Hanks Balanced Salt Solution (HBSS) 3 times and then digested with 2 ml sCelLiveTM Tissue Dissociation Solution (Singleron) by Singleron PythoN™ Automated Tissue Dissociation System (Singleron) at 37 °C for 15 min. Afterward, the GEXSCOPE® red blood cell lysis buffer (Singleron, 2 ml) was added, and cells were incubated at 25 °C for another 10 mins to remove red blood cells. The solution was then centrifuged at 500 × g for 5 min and suspended softly with PBS. Finally, the samples were stained with trypan blue (Sigma, USA) and the cellular viability was evaluated microscopically.