Enriched hCD33+ cells from liver and spleen of hNSG-SGM3 mice were resuspended in PBS containing 0.04% BSA, the cell numbers were counted on the Contess II automated cell counter (Thermo Fisher Scientific), and ∼12,000 cells were loaded per channel on Chromium microfluidic chips (10x Genomics). Single-cell capture, barcoding, and library preparation were performed using the 10x Chromium version 2 chemistry according to the manufacturer’s protocol (10x Genomics). The quality of cDNA and libraries was checked on an Agilent 4200 TapeStation, quantified by KAPA quantitative PCR, and sequenced on a HiSeq 4000 (Illumina) to an average depth of 50,000 reads per cell. We quantified gene expression counts from raw sequencing data using Cell Ranger v2.2 with GRCh38. Datasets from liver and spleen of two independent experiments were normalized using Harmony (Korsunsky et al., 2019 (link)).
Chromium microfluidic chips
Manufactured by 10x Genomics
Sourced in United States
The Chromium microfluidic chips are a core component of the Chromium system developed by 10x Genomics. The chips enable the encapsulation of individual cells or nuclei into nanoliter-scale droplets, allowing for high-throughput single-cell analysis.
Automatically generated - may contain errors
Lab products found in correlation
Chromium controller, by 10x Genomics (5 mentions)
Novaseq 6000 platform, by Illumina (2 mentions)
Chromium next gem single cell 5 library gel bead kit v1, by 10x Genomics (2 mentions)
Hiseq 4000, by Illumina (1 mentions)
Agilent 4200 tapestation, by Agilent Technologies (1 mentions)
Novaseq, by Illumina (1 mentions)
Chromium single cell 3 reagent kit v2, by 10x Genomics (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Genomics chromium platform, by 10x Genomics (1 mentions)
Chromium single cell 3 reagent kit v3, by 10x Genomics (1 mentions)
6 protocols using chromium microfluidic chips
1
Single-cell RNA-seq of Human Immune Cells
2
Single-Cell RNA Sequencing of Intracranial Fusiform Aneurysm
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Cell suspension from one intracranial fusiform aneurysm sample (AN-08) underwent single-cell RNA sequencing. This process was conducted using the 10X Genomics single-cell 3' library platform. The intracranial fusiform aneurysm cell suspension was loaded into Chromium microfluidic chips with 3' v3 chemistry and barcoded using a 10 × Chromium Controller (10X Genomics). All subsequent steps adhered to the standard manufacturer's protocols. Sequencing was performed with Illumina (NovaSeq) according to the manufacturer's instructions.
3
Single-cell RNA-seq with 10X Chromium
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The single-cell suspension was loaded into Chromium microfluidic chips with 3’ v2 chemistry and barcoded with a 10× Chromium Controller (10X Genomics, US). RNA from the barcoded cells was subsequently reverse-transcribed and sequencing libraries were constructed with reagents from a Chromium Single Cell 3’ v2 reagent kit (10X Genomics, US) according to the manufacturer’s instructions. Sequencing was performed with Illumina (US) according to the manufacturer’s instructions.
4
Single-Cell Transcriptome and TCR Profiling from Frozen Mononuclear Cells
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Mononuclear cells were isolated from whole blood and bone marrow by centrifugation and resuspended with freezing medium. The cells were then frozen in a freezing container in a − 80 °C freezer. On the date of experiment, the cells were thawed using a water bath at 37 °C and loaded into Chromium microfluidic chips and barcoded within a 10X Chromium Controller (10X Genomics, US). For transcriptome, procedures were performed with reagents: Chromium Next GEM Single Cell 5' Library & Gel Bead Kit v1.1 (10X Genomics, Cat. No. 1000165). TCR enrichment was carried out using the Chromium Single Cell V(D)J Enrichment Kit, Human T Cell (10X Genomics, Cat. No. 1000005) for αβ transcripts, or customer primers for γδ TCR transcripts [9 (link)]. All the libraries were sequenced in a PE150 mode (Pair-End for 150 bp read) on the NovaSeq 6000 platform (Illumina, US).
5
Single-nucleus RNA sequencing of Gulf and non-Gulf samples
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Single-nucleus RNA sequencing (snRNA-seq) was carried out using the 10x Genomics Chromium™ platform. Samples from the GOM and NOM groups were mixed separately and then prepared into nuclear suspensions following the 10x Genomics Cell Preparation Guide [18] . The nuclear suspension was loaded into Chromium microfluidic chips with 3' v3 chemistry and barcoded with a 10x Chromium Controller (10x Genomics, CA, USA). RNA from the barcoded nuclei was subsequently reverse-transcribed, and sequencing libraries were constructed with reagents from a Chromium Single Cell 3' v3 reagent kit (10x Genomics, CA, USA) according to the manufacturer's instructions. Sequencing was performed with an Illumina HiSeq 2000 according to the manufacturer's instructions. Raw read sequences produced by the Illumina pipeline in FASTQ format were preprocessed through Trimmomatic software, in which reads with low-quality, adapters, and fewer than 26 bases were discarded. Raw reads were demultiplexed and mapped to the reference genome by the 10x Genomics Cell Ranger pipeline using default parameters [18] . Cellranger and Seurat were applied for data filtration, expression matrix construction, and single-nucleus analyses [19] (link), [20] (link). In total, 5000 variable genes and 40 principal components (resolution = 0.5) were used for clustering.
6
Single-cell RNA-seq and TCR profiling
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Mononuclear cells were isolated from whole blood and bone marrow by centrifugation and resuspended with freezing medium. The cells were then frozen in a freezing container in a -80°C freezer. On the date of experiment, the cells were thawed using a water bath at 37°C and loaded into Chromium microfluidic chips and barcoded within a 10X Chromium Controller (10X Genomics, US). For transcriptome, procedures were performed with reagents: Chromium Next GEM Single Cell 5' Library & Gel Bead Kit v1.1 (10X Genomics, Cat. No. 1000165). TCR enrichment was carried out using the Chromium Single Cell V(D)J Enrichment Kit, Human T Cell (10X Genomics, Cat. No. 1000005) for αβ transcripts, or customer primers for γδ TCR transcripts9. All the libraries were sequenced in a PE150 mode (Pair-End for 150bp read) on the NovaSeq 6000 platform (Illumina, US).
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