Single-cell suspensions (1 × 105 cells/mL) in PBS 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 Biotechnologies) [25 (link)]. Individual libraries were diluted to 4 nM and pooled for sequencing. Finally, pools were sequenced on Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) with 150-bp paired end reads.
Matrix single cell processing system
Manufactured by Singleron Biotechnologies
Sourced in China, United States, Germany
The Matrix® Single Cell Processing System is a lab equipment product designed for the isolation, processing, and preparation of single cells. It enables the handling and analysis of individual cells from complex samples. The system provides a platform for various single-cell applications, including but not limited to genomic, transcriptomic, and proteomic studies.
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Lab products found in correlation
Gexscope single cell rna library kit, by Singleron Biotechnologies (9 mentions)
Novaseq 6000, by Illumina (8 mentions)
Scellive tissue preservation solution, by Singleron Biotechnologies (1 mentions)
Scellive tissue dissociation solution, by Singleron Biotechnologies (1 mentions)
Python tissue dissociation system, by Singleron Biotechnologies (1 mentions)
Hiseq x platform, by Illumina (1 mentions)
Single cell rna library kit, by Singleron Biotechnologies (1 mentions)
Novaseq 6000 sequencing platform, by Illumina (1 mentions)
11 protocols using matrix single cell processing system
1
Single-Cell RNA Sequencing Protocol
2
Single-cell RNA Sequencing from Microwell Chip
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Single-cell suspensions (2×105 cells/mL) with PBS (HyClone) were loaded onto microwell chip using the Singleron Matrix® Single Cell Processing System. Barcoding Beads are subsequently collected from the microwell chip, followed by reverse transcription of the mRNA captured by the Barcoding Beads and to obtain cDNA, and PCR amplification. The amplified cDNA is then fragmented and ligated with sequencing adapters. The scRNA-seq libraries were constructed according to the protocol of the GEXSCOPE® Single Cell RNA Library Kits (Singleron) (23 (link)). Individual libraries were diluted to 4 nM, pooled, and sequenced on Illumina novaseq 6000 with 150 bp paired end reads.
3
Single-Cell RNA Sequencing Workflow
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After discarding the supernatant, the remaining sample was re-suspended with PBS (HyClone) into 2×105 cells/mL cell suspension, which was then added into the microwell chip using Singleron Matrix® Single Cell Processing System. Using the same system, Barcoding Beads with barcode labeling were added to the microwell chip. After cytolysis, the polyT structure at the barcode end of the bead surface was coupled with the mRNA tail polyA sequence to capture mRNA. After the complete combination, the magnetic beads were harvested for cDNA reverse transcription and PCR amplification. The obtained cDNA through amplification was fragmented and connected with the sequencing adaptor. The scRNA-seq library was constructed by using GEXSCOPE® Single Cell RNA Library Kits (Singleron) according to the manufacturer’s protocol (14 (link)). The single library was diluted to 4 nM, pooled, and sequenced with Illumina novaseq 6000, and the paired-end read was 150 bp.
4
Single-Cell RNA-Seq Library Preparation
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Single-cell suspensions (2 × 105 cells/mL) with PBS (HyClone) were placed onto a microwell chip using the Singleron Matrix® Single Cell Processing System. Reverse transcription of the mRNA captured by the Barcoding Beads is followed by PCR amplification of the cDNA generated from the Barcoding Bead collection. Sequencing adapters are then ligated to the fragmented cDNA. GEXSCOPE® Single Cell RNA Library Kits (Singleron) were used to construct the scRNA-seq libraries (Dura et al., 2019 (link)). Illumina novaseq 6,000 was used to sequence 150 bp paired end reads from individual libraries diluted to 4 nM, pooled, and processed.
5
Single-cell RNA Sequencing of Spleen Tissue
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The fresh spleen tissues were stored in the sCelLive™ Tissue Preservation Solution (Singleron, China) on ice after the surgery within 30 min, and then digested with 3 mL sCelLive™ Tissue Dissociation Solution (Singleron) by PythoN™ Tissue Dissociation System (Singleron) at 37 °C for 15 min. The cell suspension was collected and filtered through a 40-micron sterile strainer. Afterwards, the red blood cell lysis buffer (RCLB, Singleron) was added and incubated at room temperature for 5-8 min. The mixture was then centrifuged to remove supernatant and suspended softly with PBS.
Single-cell suspensions (2 × 105 cells/mL) with PBS were loaded onto a microwell chip using the Matrix® Single Cell Processing System (Singleron). Barcoding beads are subsequently collected from the microwell chip, followed by reverse transcription of the mRNA captured by the beads to obtain cDNA for PCR amplification. The amplified cDNA is then fragmented and ligated with sequencing adapters. The scRNA-seq libraries were constructed according to the protocol of the Single Cell RNA Library Kits (Singleron). Individual libraries were diluted to 4 nM, pooled, and sequenced on an Illumina Novaseq 6000 (Illumina, USA) instrument with a 150 bp paired-end format. Full identified sequencing data are available in the gene expression omnibus (GEO) under accession number GSE234756.
Single-cell suspensions (2 × 105 cells/mL) with PBS were loaded onto a microwell chip using the Matrix® Single Cell Processing System (Singleron). Barcoding beads are subsequently collected from the microwell chip, followed by reverse transcription of the mRNA captured by the beads to obtain cDNA for PCR amplification. The amplified cDNA is then fragmented and ligated with sequencing adapters. The scRNA-seq libraries were constructed according to the protocol of the Single Cell RNA Library Kits (Singleron). Individual libraries were diluted to 4 nM, pooled, and sequenced on an Illumina Novaseq 6000 (Illumina, USA) instrument with a 150 bp paired-end format. Full identified sequencing data are available in the gene expression omnibus (GEO) under accession number GSE234756.
6
Single-cell RNA-seq Library Prep
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Single-cell suspensions (1~3 × 105 cells/ml) in PBS (HyClone, Logan, UT, USA) were loaded onto a microwell chip using the Singleron Matrix® Single Cell Processing System. Briefly, the scRNA-Seq library was constructed using the GEXSCOPE® Single Cell RNA Library Kits (Singleron). The library was lastly sequenced with 150 bp diluted to 4 nM and paired-end reads on the Illumina HiSeq X platform following an established protocol (12 (link)). Sequencing data processing and quality control were performed as described in previous publications (13 (link)).
7
Single-cell RNA-seq library preparation
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Single-cell suspensions (1~3×105 cells/mL) in PBS (HyClone) were loaded onto microwell chip using the Singleron Matrix® Single Cell Processing System. Briefly, the scRNA-seq library was constructed using the GEXSCOPE® Single Cell RNA Library Kits (Singleron). The library was lastly sequenced with 150 bp that was diluted to 4nM and paired-end reads on the IlluminaHiSeq X platform following an established protocol (7 (link)). Sequencing data processing and quality control was performed as described in previous publications (8 (link), 9 (link)).
8
Single-cell RNA-seq Library Generation
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For scRNA-seq, single-cell suspensions in PBS (HyClone) with a concentration of 2 × 105 cells/mL were firstly prepared and then loaded onto microfluidic devices using the Singleron Matrix® Single Cell Processing System, and scRNA-seq libraries were constructed according to the GEXSCOPE® protocol using the GEXSCOPE® Single-Cell RNA Library Kit (Singleron Biotechnologies Köln, Germany). The individual libraries were diluted to 4 nM and pooled for sequencing on the Illumina Novaseq 6000 with 150-bp paired-end reads.
9
Singleron Matrix Single Cell RNA-seq
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The prepared single-cell suspension was stored in PBS at a final cell concentration of 2 × 105 cells/mL and then loaded onto a microwell chip of the Singleron Matrix Single Cell Processing System. Subsequently, barcoded Beads were retrieved from the microwell chip for mRNA capture. After barcoding, reverse transcription of the captured mRNA was performed to generate cDNA, followed by PCR amplification. The amplified cDNA was fragmented and ligated using sequencing adapters. The Single-Cell RNA Library Kit (Singleron, China) was used following the manufacturer’s instructions to construct scRNA-seq libraries. Finally, we diluted the individual libraries to a final concentration of 4 nM and performed sequencing on the Illumina NovaSeq 6000 sequencing platform with 150 bp paired-end reads.
10
Single-cell RNA Sequencing of 3D MSCs
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In this study, 3D MSCs were obtained, and single-cell suspensions were collected. Based upon the Singleron Matrix single-cell processing system, single-cell suspensions of 3D MSCs are loaded onto a microwell chip. Subsequently, the captured mRNA is reverse-transcribed and the cDNA was amplified, fragmented and ligated to sequencing adapters. Constructed by the GEXSCOPE Single Cell RNA Library Kit (Singleron), the individual libraries of 3D MSCs were mixed and sequenced by the Illumina platform (novaseq 6000). With the obtained sequencing data, bioinformatics analysis techniques such as t-SNE, PCA, or clustering methods were applied to analyze the differences between individual cells using Seurat package (25 (link)), which allowed for a more in-depth exploration of the changes in single-cell biology features, development, and functional variations of 3D MSCs. COSine similarity-based marker Gene identification (COSG) package was utilized to calculate the markers of each clusters (26 (link)) and GO, KEGG and Reactome enrichment analyses were done to better understand the heterogeneity of MSCs.
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