Singleron matrix single cell processing system

Single-cell suspensions (1 × 10⁵ cells/mL) with PBS were loaded into a microfluidic chip 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) [56 (link)]. scRNA-seq libraries were diluted to 4 nM and pooled for sequencing. At last, pools were sequenced on Illumina HiSeq X with 150 bp paired-end reads.

For samples MBC1, MBC2, MBC3, FBC1, and FBC2, 10,000 cells per sample were loaded into a Chromium Single-Cell 3 Chip Kit v2 (10× Genomics, PN-120236) following the established protocols using the Chromium Single Cell 30 Library V2 Kit (10× Genomics, PN-120234). Briefly, reverse transcription, cDNA recovery, cDNA amplification, and library construction were performed using the Single Cell 3’ Library and Gel Bead Kit v2 (10× Genomics, PN-120237) and Chromium i7 Multiplex Kit v2 (10× Genomics, PN-120262) according to the manufacturer’s instructions. For samples MBC4, MBC5, and MBC6, single-cell suspensions (1 × 10⁵ cells/mL) with PBS (HyClone) 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, 5180011)^{61 (link)}. Individual libraries were diluted to 4 nM and pooled for sequencing. Single-cell library sequencing was performed using the Illumina HiSeq X Ten or NovaSeq 6000, with 150 bp paired-end sequencing.

Single-cell suspensions (1 × 10⁵ cells/ml) with PBS (HyClone) 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)⁵⁴ . 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.

Single-cell suspensions (1 × 10⁵ cells/mL) with PBS (HyClone, Logan, UT, USA) were loaded into microfluidic devices using a Singleron Matrix^® Single Cell Processing System (Singleron Biotechnologies). Subsequently, scRNA-seq libraries were constructed according to the protocol of the GEXSCOPE^® Single Cell RNA Library Kit (Singleron Biotechnologies) [57 (link)]. Briefly, a single-cell suspension was loaded onto the microchip to partition single cells into individual wells on the chip. Cell barcoding beads were loaded into the microchip and washed. Afterwards, 100 μL of single-cell lysis buffer was added to the chip to lyse the cells and capture mRNAs at room temperature for 20 min. The beads, together with the captured RNAs, were flushed out of the microchip and used for subsequent reverse transcription, cDNA amplification, and library construction. After size selection and purification, pools were sequenced on an Illumina Novaseq 6000 (San Diego, CA, USA) with 150 bp paired-end reads.

Single-cell suspensions (1 × 10⁵ cells/mL) in PBS (HyClone) 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 Kit (Singleron). Individual libraries were diluted to 4 nM and pooled for sequencing. Finally, pools were sequenced on an Illumina HiSeq X with 150-bp paired-end reads.

The cellular viability of all freshly PBMCs exceeded 90% evaluated by trypan blue (Sigma, USA) microscopically. Single‐cell suspensions (1 × 10⁵ cells mL⁻¹) with PBS (HyClone, USA) were loaded into microfluidic devices using the Singleron Matrix Single Cell Processing System (Singleron, China). Subsequently, the scRNA‐seq libraries were constructed according to the protocol of the GEXSCOPE Single Cell RNA Library Kits (Singleron, China).^[32] Individual libraries were diluted to 4 × 10⁻⁹ m and pooled for sequencing. At last, pools were sequenced on Illumina HiSeq X with 150 bp paired end reads.

Sorted single-cell suspensions (1 × 10⁵ cells/ml) with PBS (HyClone) 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)^{48 (link)}. Individual libraries were diluted to 4 nM and pooled for sequencing. At last, pools were sequenced on Illumina HiSeq X with 150 bp paired end reads.