Singleron matrix automated single cell processing system

Single-cell suspensions at 1 × 10⁵ cells/mL in concentration in PBS (HyClone, Shanghai, China) were prepared and loaded onto microfluidic devices and scRNA-seq libraries were constructed according to Singleron GEXSCOPE^® protocol by GEXSCOPE^® Single-Cell RNA Library Kit (Singleron Biotechnologies) and Singleron Matrix^® Automated single-cell processing system (Singleron Biotechnologies). Individual libraries were diluted to 4 ng/μL and pooled for sequencing. Pools were sequenced on Illumina HiSeq X (Illumina, San Diego, CA, USA) with 150 bp paired end reads.

The collected pT_CD8+CD28- and pT_CD8+CD28+ were separately stored in the sCelLive^TM Tissue Preservation Solution (Singleron Biotechnologies, Nanjing, China) and then transported to the Singleron lab on ice as soon as possible. The sample was stained with trypan blue (Sigma, Shanghai, China) and microscopically evaluated for cell viability. Single-cell suspensions at 1 × 10⁵ cells/mL in PBS (HyClone, Shanghai, China) were prepared and loaded onto microfluidic devices and scRNA-seq libraries were constructed according to the GEXSCOPE^® protocol using the GEXSCOPE^® Single-Cell RNA Library Kit (Singleron Biotechnologies) and Singleron Matrix^® Automated single-cell processing system (Singleron Biotechnologies). Individual libraries were diluted to 4 ng/µL and pooled for sequencing. Pools were sequenced on Illumina novaseq6000 with 150-bp paired-end reads.

The collected cells were centrifuged, and then the cell pellets were resuspended in PBS (HyClone, Marlborough, MA, USA). GEXSCOPE Red Blood Cell Lysis Buffer (Singleron Biotechnologies, Nanjing, China) was added to the cell suspension to remove the red blood cells. Then, the mixture was centrifuged and resuspended in PBS. Trypan blue exclusion test was used to evaluate the cell viability [23 (link)]. We counted the cells with a TC20 automated cell counter (Bio-Rad, Hercules, CA, USA), and prepared the cell suspension with a final concentration of 1 × 10⁵ cells/mL.
Single-cell suspensions were loaded onto microfluidic devices. ScRNA-seq libraries were constructed on the basis of the Singleron GEXSCOPE^® protocol by the GEXSCOPE^® Single-Cell RNA Library Kit (Singleron Biotechnologies, Nanjing, China) and Singleron Matrix^® Automated Single-Cell Processing System (Singleron Biotechnologies, Köln, Germany). After being diluted to 4nM, individual libraries were pooled for sequencing. Libraries were then sequenced on an Illumina HiSeq X with 150 bp paired-end reads (Illumina, San Diego, CA, USA).