Single-cell RNA sequencing was performed using the Chromium (10X Genomics) instrument. Single cell suspensions were counted using both the Cellometer K2 Fluorescent Viability Cell Counter (Nexcelom) and a hemocytometer, ensuring viability >80%, and adjusted to 1,000 cells/μl. Samples 1 and 3 were run using the Chromium Single Cell 3′ Library & Gel Bead Kit v2 and Sample 2 and 4 was run using the Chromium Single Cell 3′ Library & Gel Bead Kit v3 (10X Genomics). The manufacturer’s protocol was used with a target capture of 10,000 cells for the 3’ gene expression samples. Each sample was processed on an independent Chromium Single Cell A Chip (10X Genomics) and subsequently run on a thermocycler (Eppendorf). 3’ gene expression libraries were sequenced using the NextSeq 500 high output flow cells.
Chromium single cell a chip
Manufactured by 10x Genomics
The Chromium Single Cell A Chip is a microfluidic device designed for single-cell analysis. It is used to encapsulate individual cells in nanoliter-scale gel beads, enabling the capture and barcoding of cellular transcripts for downstream analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nextseq 500 platform, by Illumina (6 mentions)
Chromium single cell 5 library kit, by 10x Genomics (5 mentions)
Thermocycler, by Eppendorf (3 mentions)
Chromium single cell 3 library gel bead kit v2, by 10x Genomics (3 mentions)
Cellometer k2 fluorescent viability cell counter, by Revvity (3 mentions)
Dynabeads myone silane, by Thermo Fisher Scientific (3 mentions)
Fragment analyzer, by Agilent Technologies (3 mentions)
Chromium single cell 3 library gel beads, by 10x Genomics (3 mentions)
Nextseq 500, by Illumina (3 mentions)
Chromium single cell 5 d j reagent kit, by 10x Genomics (3 mentions)
Chromium single cell 3 library gel bead kit v3, by 10x Genomics (2 mentions)
Chromium i7 multiplex kit, by 10x Genomics (2 mentions)
High sensitivity dna assay, by Agilent Technologies (1 mentions)
Nextseq 500 system, by Illumina (1 mentions)
Partitioning oil, by 10x Genomics (1 mentions)
Spriselect reagent, by Beckman Coulter (1 mentions)
Single cell 3 reagent kits v2, by 10x Genomics (1 mentions)
Chromium single cell 5 d j human t cell enrichment kit, by 10x Genomics (1 mentions)
Chromium single cell 5 library gel bead kit v2, by 10x Genomics (1 mentions)
Nextseq 500 550 high output kit v2, by Illumina (1 mentions)
16 protocols using chromium single cell a chip
1
Single-Cell RNA Sequencing Workflow
2
Scalable Single-Cell RNA-Seq Library Prep
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Single cell RNA-seq libraries were generated using Single Cell 3′ Reagent Kits v2 (10xGenomics) according to the manufacturer’s protocol. Briefly, the Chromium Single Cell A Chip (10xGenomics, 1000074) were loaded with cells, reagents (10xGenomics, PN-120237), gel beads (10xGenomics, 1000092), and partitioning oil (10xGenomics, 220088). cDNA was generated using the Gel Bead-In-Emusions (GEMs)-RT incubation protocol, purified with DynaBeads MyOne Silane beads, amplified with PCR, and further purified with SPRI-select reagent (Beckman Coulter, B23318). After fragmentation, end-repair, A-tailing and size purification, cDNA was ligated with adaptor, followed by quantification with SPRI-select reagents. Libraries were amplified using PCR with sample indexes from Chromium i7 Multiplex kit (10xGenomics, PN-120262). Library quality control was performed on the Agilent Tapestation with D1000 screen tapes, and library yield was quantified by Qubit DNA high sensitivity assay. Sequencing was performed on an Illumina Nextseq 500 system operated by the Next Generation Sequencing at GENEWIZ using the HiSeq 2 × 150 bp high output sequencing kit.
3
Single-cell RNA-seq of immune cells
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Single-cell RNA sequencing was performed using the Chromium (10X Genomics) instrument. Single cell suspensions were counted using both the Cellometer K2 Fluorescent Viability Cell Counter (Nexcelom) and a haemocytometer and adjusted to 1000 cells/µl. UMM061, UMM062, UMM063, UMM064, UMM065, UMM066, UMM069, UMM067L, UMM041L, and BSSR0022 were run using the Chromium Single Cell 5’ Library & Gel Bead Kit v2, Chromium Single Cell V(D)J Human T Cell Enrichment Kit, and Chromium Single Cell V(D)J Human B Cell Enrichment Kit, (10X Genomics). UMM059 was run using the Chromium Single Cell 3′ Library & Gel Bead Kit v2 (10X Genomics) which is not compatible with the Chromium Single Cell V(D)J product. The manufacturer’s protocol was used with a target capture of 10,000 cells for the 5’ gene expression samples and a target capture of 5,000 cells for the 3’ gene expression sample (UMM059). Each sample was processed on an independent Chromium Single Cell A Chip (10X Genomics) and subsequently run on a thermocycler (Eppendorf). 3’ and 5’ gene expression libraries were sequenced using the NextSeq 500 150-cycle high-output flow cells. B- and T- cell VDJ libraries were sequenced on a MiSeq instrument.
4
Single-cell transcriptome profiling with 10X Genomics
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Single-cell suspension processed with 10X Single-cell 3’ Gene Expression kit (ver. 2 and ver. 3.1) (10X Genomics, Cat. 120237 and Cat. 1000121), using the Chromium Single Cell A chip (10X Genomics, Cat. 1000009) (ver.2) and Chromium Next GEM Chip G Single Cell kit (10X Genomics, Cat. 1000120) (ver. 3.1) on the 10X Chromium Controller (10X Genomics, Cat. 1000202) according to manufacturer specifications. Library construction performed with 10X Single-cell 3’ Library Construction Kit, utilizing the Chromium i7 Multiplex kit (10X Genomics, Cat. 120262). Hashtag library recovery performed according to Biolegend protocol: https://www.biolegend.com/en-us/protocols/totalseq-a-antibodies-and-cell-hashing-with-10x-single-cell-3-reagent-kit-v3-3-1-protocol . Library quantification performed with Qubit dsDNA HS assay (Thermo Fisher Scientific, Cat. Q32854), fragment profiles checked using the High Sensitivity NGS Fragment Analysis kit (Advanced Analytical Technologies GmbH, Cat. DNF-474-0500) on the 5200 Fragment Analyzer (Advanced Analytical Technologies GmbH, Cat. FSv2-CE2). Libraries sequenced with NextSeq® 500/550 High Output Kit v2.5 (75 cycles) (Illumina, Cat. 20024906) on the NextSeq 500.
5
Single-cell RNA-seq Using 10X Genomics
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scRNA-seq was performed according to the 10X Genomics protocols. Briefly, the prepared single cell suspension was carefully mixed with reverse transcription mix using the Chromium Single Cell 3’ Library & Gel beads chemistry v2 (10x Genomics) and loaded into a Chromium Single Cell A Chip (10x Genomics).
During the encapsulation process in the 10X Genomics Chromium system, the cells were lysed within the droplet and released polyadenylated RNA, which then bound to the barcoded bead that was captured with the cell. Following the guidelines of the 10x Genomics' user manual, the droplets were directly subjected to reverse transcription, the emulsion was broken and cDNA was purified using Dynabeads MyOne Silane (Thermo Fisher Scientific). After the PCR amplification of cDNA with eight cycles, it underwent purification and a quality control check on the Fragment Analyzer (Agilent).
The cDNA was fragmented for five minutes and dA-tailed, followed by an adapter ligation step and an indexing PCR of 10 cycles in order to generate libraries. After quantification, the libraries were sequenced on NextSeq500 platform (illumina) using a high-output flowcell in PE mode (R1: 26 cycles; I1: 8 cycles; R2: 57 cycles).
During the encapsulation process in the 10X Genomics Chromium system, the cells were lysed within the droplet and released polyadenylated RNA, which then bound to the barcoded bead that was captured with the cell. Following the guidelines of the 10x Genomics' user manual, the droplets were directly subjected to reverse transcription, the emulsion was broken and cDNA was purified using Dynabeads MyOne Silane (Thermo Fisher Scientific). After the PCR amplification of cDNA with eight cycles, it underwent purification and a quality control check on the Fragment Analyzer (Agilent).
The cDNA was fragmented for five minutes and dA-tailed, followed by an adapter ligation step and an indexing PCR of 10 cycles in order to generate libraries. After quantification, the libraries were sequenced on NextSeq500 platform (illumina) using a high-output flowcell in PE mode (R1: 26 cycles; I1: 8 cycles; R2: 57 cycles).
6
Single-cell RNA-seq of mouse cells
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After digestion, the single-cell suspensions were washed and resuspended in 0.04% BSA in PBS at a concentration of 106 cells/ml. A hemocytometer was used to manually count the cells to determine the concentration of the suspension. Single-cell RNA-sequencing libraries were prepared using the Chromium Single Cell 3’ reagent kit v3 (10x Genomics, Pleasanton, CA) following the manufacturer’s protocol.67 Cells were diluted into the Chromium Single Cell A Chip to yield a recovery of 6,000 single-cell transcriptomes with <5% doublet rate. Libraries were sequenced on the NextSeq 500 (Illumina, San Diego, CA).68 The sequencing data was aligned to the mouse reference genome (mm10) using CellRanger v5.0.0 (10x Genomics).67
7
Single-cell RNA-seq Using 10X Genomics
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scRNA-seq was performed according to the 10X Genomics protocols. Briefly, the prepared single cell suspension was carefully mixed with reverse transcription mix using the Chromium Single Cell 3’ Library & Gel beads chemistry v2 (10x Genomics) and loaded into a Chromium Single Cell A Chip (10x Genomics).
During the encapsulation process in the 10X Genomics Chromium system, the cells were lysed within the droplet and released polyadenylated RNA, which then bound to the barcoded bead that was captured with the cell. Following the guidelines of the 10x Genomics' user manual, the droplets were directly subjected to reverse transcription, the emulsion was broken and cDNA was purified using Dynabeads MyOne Silane (Thermo Fisher Scientific). After the PCR amplification of cDNA with eight cycles, it underwent purification and a quality control check on the Fragment Analyzer (Agilent).
The cDNA was fragmented for five minutes and dA-tailed, followed by an adapter ligation step and an indexing PCR of 10 cycles in order to generate libraries. After quantification, the libraries were sequenced on NextSeq500 platform (illumina) using a high-output flowcell in PE mode (R1: 26 cycles; I1: 8 cycles; R2: 57 cycles).
During the encapsulation process in the 10X Genomics Chromium system, the cells were lysed within the droplet and released polyadenylated RNA, which then bound to the barcoded bead that was captured with the cell. Following the guidelines of the 10x Genomics' user manual, the droplets were directly subjected to reverse transcription, the emulsion was broken and cDNA was purified using Dynabeads MyOne Silane (Thermo Fisher Scientific). After the PCR amplification of cDNA with eight cycles, it underwent purification and a quality control check on the Fragment Analyzer (Agilent).
The cDNA was fragmented for five minutes and dA-tailed, followed by an adapter ligation step and an indexing PCR of 10 cycles in order to generate libraries. After quantification, the libraries were sequenced on NextSeq500 platform (illumina) using a high-output flowcell in PE mode (R1: 26 cycles; I1: 8 cycles; R2: 57 cycles).
8
Single-cell B cell profiling by 10x
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Single-cell sequencing libraries were constructed from the isolated BM PCs following the demonstrated 10x Genomics’ protocol: ‘Direct target enrichment - Chromium Single Cell V(D)J Reagent Kits’ (CG000166). Briefly, single cells were co-encapsulated with gel beads (10x Genomics, 1000006) in droplets using 5 lanes of one Chromium Single Cell A Chip (10x Genomics, 1000009) with a target loading of 13,000 cells per reaction. V(D)J library construction was carried out using the Chromium Single Cell 5′ Library Kit (10x Genomics, 1000006) and the Chromium Single Cell V(D)J Enrichment Kit, Mouse B Cell (10x Genomics, 1000072) according to the manufacturer’s instructions. Final libraries were pooled and sequenced on the Illumina NextSeq 500 platform (mid output, 300 cycles, paired-end reads) using an input concentration of 1.8 p.m. with 5% PhiX.
9
Enriched Single-Cell VDJ Sequencing
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Single-cell sequencing libraries were constructed from the isolated bone marrow plasma cells following the demonstrated 10X Genomics’ protocol: “Direct target enrichment-Chromium Single Cell V(D)J Reagent Kits” (CG000166 REV A). Briefly, single cells were co-encapsulated with gel beads (10X Genomics, 1000006) in droplets using 5 lanes of one Chromium Single Cell A Chip (10X Genomics, 1000009) with a target loading of 13,000 cells per reaction. V(D)J library construction was carried out using the Chromium Single Cell 5′ Library Kit (10X Genomics, 1000006) and the Chromium Single Cell V(D)J Enrichment Kit, Mouse B Cell (10X Genomics, 1000072) according to the manufacturer’s instructions. All of the reverse transcribed complementary DNA was used as input for VDJ library construction. Final libraries were pooled and sequenced on the Illumina NextSEq. 500 platform (mid output, 300 cycles, paired-end reads) using an input concentration of 1.8 pM with 5% PhiX.
10
Single Cell RNA-seq Library Preparation
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