ATP was induced in five Prg4-CreERT2;R26-tdTomato mice at 8 weeks of age, followed by tamoxifen injection 3 and 4 days after ATP, and were then euthanized 1 week after the ATP. The Achilles tendons were harvested and minced in ice-cold PBS. The harvested tissues were digested in RPMI 1640 medium (#30264-85, Nacalai Tesque Inc.) containing 0.3% type 1 collagenase (#CLS1, Worthington, Columbus, OH) and 0.4% dispase II (#04942078001, Roche, Basel, Switzerland) at 37°C for 45 min with constant agitation. Cells were filtered through 100-, 70-, and 40-μm strainers, centrifuged, counted, and resuspended at a concentration of 1000 cells/μl. Cell viability was assessed using the trypan blue exclusion method with a LUNA-FL (Logos Biosystems, Anyang, South Korea) automated counter, and samples with viability greater than 80% were processed for further sequencing. Single-cell libraries were prepared using the Chromium Controller (10x Genomics, Pleasanton, CA) according to the protocol of Chromium Next GEM Single Cell 3′ Reagent Kits (v3.1 10x Genomics). Libraries were prepared using Chromium Controller (10x Genomics). Alignment, quantification, and aggregation of sample count matrices were performed using the 10x Genomics Cell Ranger pipeline (v.5.0.0), retaining an average of 61,308 reads per cell mapped read depth normalization.
Chromium controller
Manufactured by 10x Genomics
Sourced in United States
The Chromium Controller is a laboratory instrument developed by 10x Genomics. Its core function is to automate the processing and preparation of samples for analysis using 10x Genomics' proprietary Chromium technology. The Chromium Controller precisely partitions individual cells or nuclei into nanoliter-scale gel beads, enabling high-throughput single-cell analysis.
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Hiseq 4000, by Illumina (34 mentions)
Novaseq, by Illumina (19 mentions)
Hiseq 2500, by Illumina (19 mentions)
Chromium single cell 3 reagent kit v2, by 10x Genomics (16 mentions)
Novaseq 6000 system, by Illumina (16 mentions)
Nextseq 500, by Illumina (16 mentions)
Chromium single cell 3 reagent kit, by 10x Genomics (16 mentions)
Chromium single cell 3 reagent kit v3, by 10x Genomics (15 mentions)
Agilent bioanalyzer, by Agilent Technologies (13 mentions)
C1000 touch thermal cycler, by Bio-Rad (11 mentions)
Chromium single cell 3 reagent kits v2, by 10x Genomics (11 mentions)
Chromium i7 multiplex kit, by 10x Genomics (11 mentions)
2100 bioanalyzer, by Agilent Technologies (11 mentions)
Nextseq 550, by Illumina (10 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (10 mentions)
High sensitivity dna kit, by Agilent Technologies (9 mentions)
Cell ranger, by 10x Genomics (9 mentions)
Bioanalyzer, by Agilent Technologies (9 mentions)
Chromium next gem chip g, by 10x Genomics (8 mentions)
506 protocols using chromium controller
1
Single-cell Transcriptome Analysis of Achilles Tendon Cells
2
Single-cell transcriptomics of mouse colonic cells
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Colonic epithelial and mesenchymal cells (Fig 1 ) were isolated from three female C57BL/6 mice as described above and mixed at a ratio of 1:1. Using the Single Cell 3’ V3 assay, 16,000 cells were loaded on a Chromium Controller (10x Genomics, USA). Colonic mesenchymal Pdgfra-positive cells (Fig 3 ) were isolated as described above from three female PdgfraH2BeGFP mice. Using the Single Cell 3’ V2 assay, 16,000 cells were loaded on a Chromium Controller (10x Genomics). In both cases reverse transcription, cDNA synthesis/amplification and library preparation were carried out in accordance with the manufacturer’s recommendations by the Functional Genomics Center Zürich (FGCZ). scRNA libraries were sequenced on a NovaSeq 6000 instrument (Illumina, Switzerland).
3
Single-Cell Transcriptomics of Synchronized Malaria Parasites
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For 40 h.p.i. time-point, tightly synchronous parasites were enriched using 63% Percol, washed twice with incomplete RPMI medium, and processed immediately on the 10X Chromium controller (10X Genomics, Pleasanton, CA). For 16 h.p.i. time point parasites were stained with Mitotracker Deep Red FM (Life Technologies, #M22426) for FACS analysis and flow sorting, respectively. Briefly, 50 μl of SYBR Green I stained RBCs were analyzed on BD LSR Fortessa Flow Cytometer with High Throughput sampler (BD Biosciences, San Jose, CA, USA) using BD FACS Diva Software v6.2 and 488 laser excitation / 530 emission filter to determine the concentration of SYBR Green I positive cells per microliter. A BD Influx Cell Sorter (BD Biosciences, San Jose, CA, USA) with BD FACS Sortware v1.0.01 software was used to sort ~40,000 Mitotracker Deep Red FM - positive RBC’s using a 70 μm nozzle, a 640 nm laser excitation / 670 nm emission filter and a pressure setting of 30 psi. Post-sorted cell concentration and quality were checked using a Countess® II Automated Cell Counter (Invitrogen) and FLoid™ Cell Imaging Station (ThermoFisher). Finally, labeled cells (i.e., SYBR Green I or Mitotracker Deep Red FM positive cells) were then loaded onto a 10X chip (Chip G) and processed immediately on the 10X Chromium controller (10X Genomics, Pleasanton, CA).
4
Single-cell transcriptomics of synchronized Plasmodium parasites
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For the 40 h.p.i. timepoint, tightly synchronous parasites were enriched using 63% Percoll, washed twice with incomplete Roswell Park Memorial Institute 1640 medium, and processed immediately on the 10X Chromium controller (10X Genomics). For the 16 h.p.i. timepoint, parasites were stained with Mitotracker Deep Red FM (Life Technologies, #M22426 ) for FACS analysis and flow sorting, respectively. Briefly, 50 µl of SYBR Green I stained RBCs were analysed on BD LSR Fortessa Flow Cytometer with High Throughput sampler (BD Biosciences) using BD FACS Diva Software v6.2 and 488 laser excitation/530 emission filter to determine the concentration of SYBR Green I positive cells µl−1. A BD Influx Cell Sorter (BD Biosciences) with BD FACS Sortware v1.0.01 software was used to sort ~40,000 Mitotracker Deep Red FM-positive RBCs using a 70 µm nozzle, a 640 nm laser excitation/670 nm emission filter, and a pressure setting of 30 psi. Post-sorted cell concentration and quality were checked using a Countess II Automated Cell Counter (Invitrogen) and FLoid Cell Imaging Station (Thermo Fisher Scientific). Finally, labelled cells (that is, SYBR Green I or Mitotracker Deep Red FM-positive cells) were then loaded onto a 10X chip (Chip G) and processed immediately on the 10X Chromium controller (10X Genomics).
5
Single-Cell RNA-seq and Spatial Transcriptomics
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The cell suspension was loaded into Chromium microfluidic chips with 5′ v.1.1 chemistry and barcoded with a 10× Chromium Controller (10x Genomics). RNA from the barcoded cells was subsequently reverse-transcribed, and sequencing libraries were constructed with reagents from a Chromium Single Cell 5′ v1.1 Reagent Kit (10x Genomics) according to the manufacturer’s instructions. Sequencing was performed with an Illumina NovaSeq 6000 PE150 system, depending on the experiment and following the manufacturer’s instructions.
Spatial transcriptomic sequencing was performed with a NovaSeq PE150 platform according to the manufacturer’s instructions (Illumina) at an average depth of 300 million read-pairs per sample.
Spatial transcriptomic sequencing was performed with a NovaSeq PE150 platform according to the manufacturer’s instructions (Illumina) at an average depth of 300 million read-pairs per sample.
6
Syngeneic Murine Tumor Single-Cell RNA-seq
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We implanted two mice for each syngeneic model resulting in a total of 12 samples. Each mouse tumor was harvested when the tumor size reached 100 – 200 mm3. Each sample was minced and digested with reagents from Mouse Tumor Dissociation Kit (Miltenyi) according to the manufacturer’s instructions. Cells were resuspended at 2×105 cells/mL in PBS-0.04% BSA. Each sample was processed individually and run in technical duplicates. For each sample (except CT26 and MC-38) one replicate was enriched for CD45 positive cells. Live CD45 positive cells were sorted with BD Aria after staining with FITC-CD45 (Biolegend) and 7-AAD. Single cell suspensions of all samples were resuspended in PBS-0.04% BSA at 5×105 cells/mL and barcoded with a 10× Chromium Controller (10× Genomics). In total, this procedure resulted in 24 samples. RNA from the barcoded cells for each sample was subsequently reverse-transcribed and sequencing libraries were constructed with reagents from a Chromium Single Cell 3′ v2 reagent kit (10× Genomics) according to the manufacturer’s instructions. Sequencing was performed with Illumina HiSeq according to the manufacturer’s instructions (Illumina).
7
PBMC Isolation and GEM Generation
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Resuspend the PBMCs in 1 mL of RPMI 1640 complete medium, avoid heavy bubbles.
Ten microliters of trypan blue (0.4%) (Solarbio, Cat# C0040) is added to 90 μL of cell suspension in a 1.5 mL microcentrifuge tube, and the mixture is incubated at 25°C for 3 min after mixing.
Ten microliters of mixed cells is added to the blood counting chamber, and the cell numbers and viability are detected (
PBMCs are diluted to 0.7–1.2 × 106 cells/mL, and GEM generation is performed for PBMCs on a 10× Chromium Controller (10× Genomics, Cat# PN110203).
8
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.
9
Single-Cell ATAC-Seq with 10x Chromium
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Resuspended cell nuclei were used for transposition and loaded into the Chromium Next GEM Chip H with 10× reagents and barcoded with a 10× Chromium Controller (10x Genomics). DNA fragments from the barcoded cells were subsequently amplified, and sequencing libraries were constructed with reagents from a Chromium NextGEM Single Cell ATAC Reagent kit ver 2 (Cat# CG000496, 10x Genomics) according to the manufacturer's instructions. Libraries were then pooled and loaded on an Illumina NovaSeq 6000 PE50, a 50 bp paired‐end module at Novogene (Beijing, China).
10
Single-Cell RNA Sequencing of Tumor Samples
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Tumor fragments and LCAs were dissociated using TrypLE™ (Gibco). Crude dissociates from parental tumor samples were treated with RBC lysis buffer (Thermo Fisher Scientific) for 5 min. All samples were washed three times by centrifugation at 200 × g for 5 min and resuspended in 10 mL of calcium-free, magnesium-free PBS (Gibco). Cells were strained through a 40 μm filter (Corning), analyzed for viability by trypan blue staining, and counted using an automatic cell counter (Thermo Fisher Scientific). Samples had a viability of > 85% and were diluted to a final concentration of 100 viable cells/mL in PBS with 0.01% BSA (w/v). The single-cell suspension was loaded into Chromium microfluidic chips with 3’ (v2 or v3, depending on project) chemistry and barcoded with a 10 × Chromium Controller (10 X Genomics). RNA from the barcoded cells was subsequently reverse-transcribed, and sequencing libraries were constructed with reagents from a Chromium Single Cell 3’ v2 (v2 or v3, depending on project) reagent kit (10X Genomics) according to the manufacturer’s instructions. Sequencing was performed with Illumina (HiSeq 2000 or NovaSeq, depending on project) according to the manufacturer’s instructions (Illumina).
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