MEFs, ESCs, or reprogramming cells were digested by 0.25% trypsin, and washed twice with PBS. Single cells were captured using the Fluidigm C1 Single-Cell Auto Prep System. 5-10 mm C1 chips for ESCs, iPSCs and late reprogramming cells (D3, D5, D7) and 10-17 mm C1 chips for MEFs and early reprogramming cells (D0, D1, D2) dependent on the cell diameters. A concentration of 2 3 10 5 cells per ml was used for chip loading. After cell capture, chips were observed under the microscope to exclude capturing multiple cells. Cell lysis and cDNA synthesis were performed on-chip with the SMARTer Ultra Low RNA kit for the Fluidigm C1 system and following the manufacturer's instructions. After cDNA harvesting, the concentrations were analysis with PicoGreen (Thermo Fisher) following the protocol of Single-Cell mRNA Seq PicoGreen Template (Fluidigm, PN 100-6160). 0.5 ng amplified products were used for Nextera XT library preparation, and following the protocol of Using the C1 Single-Cell Auto Prep System to Generate mRNA from Single Cells and Libraries for Sequencing (Fluidigm, . For 10x single-cell RNA-seq, we prepared the libraries following the Chromium Single Cell 3 0 Reagent Kits User Guide.The single cell libraries were quantified by Quant-iT dsDNA Assay Kit, high sensitivity (Thermo Fisher) on Qubit 2.0, and then sequenced on illumina NextSeq 500.
C1 single cell auto prep system
Manufactured by Standard BioTools
Sourced in United States
The C1 Single-Cell Auto Prep System is a laboratory instrument designed to automate the process of isolating and preparing individual cells for downstream analysis. The system uses microfluidic technology to capture, lyse, and extract nucleic acids from single cells in a high-throughput manner.
Automatically generated - may contain errors
Lab products found in correlation
Nextera xt, by Illumina (6 mentions)
2100 bioanalyzer, by Agilent Technologies (5 mentions)
Hiseq 2000, by Illumina (4 mentions)
Nextera xt dna sample preparation kit, by Illumina (3 mentions)
Biomark hd system, by Standard BioTools (3 mentions)
Biomark system, by Standard BioTools (3 mentions)
Nextera xt dna sample prep kit, by Illumina (3 mentions)
Nextera xt dna library prep kit, by Illumina (3 mentions)
Open app program, by Standard BioTools (3 mentions)
Nextseq 500, by Illumina (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Golgiplug, by BD (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Rna fish, by Panomics (2 mentions)
Miseq sequencing system, by Illumina (2 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Live dead stain, by Thermo Fisher Scientific (2 mentions)
Smarter chemistry, by Takara Bio (2 mentions)
Biomark hd, by Standard BioTools (2 mentions)
Diamond cd34 isolation kit, by Miltenyi Biotec (2 mentions)
53 protocols using c1 single cell auto prep system
1
Single-Cell Transcriptome Profiling of Cell Types
2
Transcriptomic Profiling of Mouse Embryonic Stem Cells
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Mouse ES-OS25 cells were grown on 0.1% gelatin-coated surfaces in supplemented GMEM-BHK21 containing 10% fetal calf serum (FCS, PAA Laboratories, Gmbh), non-essential amino acids, sodium pyruvate (1 mM), sodium bicarbonate (0.075%), streptomycin (100 units/ml), L-glutamine (292 μg/ml), penicillin G (100 units/ml), 2-mercaptoethanol (0.1 mM), and human recombinant LIF (1000 units/ml, Chemicon)8 (link), 51 (link).
For single-cell sequencing libraries were prepared according to Fluidigm manual “Using the C1 Single-Cell Auto Prep System to Generate mRNA from Single Cells and Libraries for Sequencing”. OS25 cell suspension was loaded on 10–17 micron C1 Single-Cell Auto Prep IFC, Fluidigm; cDNA was synthesized in the chip using Clontech SMARTer Kit, and Illumina sequencing libraries were prepared with Nextera XT Kit and Nextera Index Kit (Illumina). Libraries from 96 cells were pooled and sequenced on four lanes on Illumina HiSeq2000 using 100 bp paired-end protocol.
For single-cell sequencing libraries were prepared according to Fluidigm manual “Using the C1 Single-Cell Auto Prep System to Generate mRNA from Single Cells and Libraries for Sequencing”. OS25 cell suspension was loaded on 10–17 micron C1 Single-Cell Auto Prep IFC, Fluidigm; cDNA was synthesized in the chip using Clontech SMARTer Kit, and Illumina sequencing libraries were prepared with Nextera XT Kit and Nextera Index Kit (Illumina). Libraries from 96 cells were pooled and sequenced on four lanes on Illumina HiSeq2000 using 100 bp paired-end protocol.
3
Single-Cell RNA Sequencing Protocol
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LC2ad were dissociated to single cells by Accumax treatment (Innovative Cell Technologies). We employed the C1 single-cell auto prep system (Fluidigm) and SMART-Seq v4 Ultra Low Input RNA Kit with oligo DNA, identically to Smart-Seq2. We executed the C1 protocol ‘SMART-Seq v4 Rev B’. We acquired approximately 1 ng of FL-cDNA. As a control, we also conducted cDNA synthesis from bulk cells using a general thermal cycler with the same reaction conditions. Using SeqAmp DNA Polymerase (TAKARA Bio), we conducted a further amplification (1 cycle of 1 min at 96 °C, 5 cycles of 30 s at 95 °C, 65 °C for 30 s, 7 min at 68 °C, 1 cycle of 10 min at 72 °C). We purified the re-amplified cDNA using Agencourt AMPureXP (Beckman Coulter). Approximately 300 ng of FL-cDNA was obtained.
4
Single-Cell RNA-Seq of Etv2-EYFP Embryos
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Etv2-EYFP embryos were harvested from time mated females at E7.25, E7.75 or E8.25 and screened using microscopy for EYFP expression14 (link). Embryos were divided into EYFP-positive and -negative pools for dissociation with TrypLE Express (Gibco by Life Technologies). After dissociation, cells were diluted with 10% foetal bovine serum in DMEM and pelleted at 1,000g. Cells were resuspended in 0.1% propidium iodide and 2% serum in PBS. EYFP-negative embryos were used as a gating control sample. Propidium iodide-negative, EYFP-positive cells were sorted by FACS using a MoFlo XDP (Beckman Coulter) into DMEM plus 10% foetal bovine serum. FACS sorted cells were resuspended at 500 cells μl−1 before loading onto a Fluidigm 10–17 μm integrated fluidics circuit for capture, viability screening, lysis and library amplification on a C1 Single-Cell Auto Prep System (Fluidigm).
5
Single Nuclei RNA-seq of Cardiomyocytes
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Single nuclei were isolated from snap-frozen mouse and human left ventricle and processed by mechanical dissociation at 4000 Hz (4 × 20 s pulses) in Lysonator cartridges (SG Microlab devices) and counterstained with DAPI. CM nuclei were stained with PCM1 antibody (1:500, HPA023374, Sigma), secondary anti-rabbit Alexa 488 (1:500) or Alexa 568 antibody (1:500), and captured individually using C1 Single Cell Auto Prep system (10–17 uM mRNA seq chip, Fluidigm). Automated imaging of captured nuclei was performed on an inverted microscope (Olympus) with 10× objective (Olympus) and CCD camera (Axiocam MR3, Zeiss) to confirm the identity of wells containing only single PCM1+ CM captured. PCM1+ CM nuclear RNA-seq libraries were prepared using Nextera XT DNA sample preparation kit (Illumina). Each sample was sequenced with paired end 2 × 101 bp reads on HiSeq 2500 (Illumina).
6
Single-Cell Gene Expression Analysis of Inflammatory Monocytes
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A total of 90-target genes, relevant to monocyte function, that included major cytokines and pathway proteins involved in inflammation were selected for pre-amplification in the IFCs using the Fluidigm C1 Single-Cell Auto Prep System according to the manufacturer’s protocol. qPCR-based gene expression assay of the target gene pre-amplified cDNAs were carried out using 96.96 IFCs on the BioMark HD System (Fluidigm) as described in the protocol. Raw data was analyzed using the Fluidigm Real-Time PCR Analysis software (v. 4.1.2) and quality check was performed by inspecting melt curves, amplification curves. A failure score was calculated for each cell as described previously [9 (link), 13 (link)]. Cells with failure score (total CT value) greater than two standard deviations above the mean were excluded from downstream analysis. The limit of detection CT values was set at 28 [10 (link)]; CT values greater than or equal to 28 were considered non-detected and were assigned a value of zero for analysis. Gene expression values were calculated by subtracting the threshold cycle value for each gene for each cell from the number of cycles in the PCR reaction. In this way, higher numbers represent greater gene expression, and lower numbers indicate less expression.
7
Single-Cell RNA-Seq of Mouse Dendritic Cells
8
Single-cell RNA sequencing workflow
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Single-cell capture was performed by C1 single-cell auto prep system (Fluidigm) following the manufacturer’s instructions17 (link). The microfluidics circuit used was the C1™ Single-Cell mRNA-seq IFC, 17–25 µm. All 96 chambers were inspected under an inverted phase contrast microscope; only chambers containing a non-damaged single cell were considered for downstream analysis. For the cell lysis and cDNA synthesis, we used the SMARTer Ultra Low RNA kit for Illumina Sequencing (version 2, Clontech) and a C1 Auto Prep System instrument (Fluidigm) with the original mRNA Seq Prep script provided by the manufacturer (1772×/1773×, Fluidigm). We assessed cDNA quality on 2100 Bioanalyzer (Agilent) with the high sensitivity DNA chips (Agilent) and quantified the cDNA using Qubit dsDNA BR assay kit (Invitrogen). Sequencing libraries were prepared with 0.3 ng of pre-amplified cDNA using Nextera XT DNA kit (Illumina) according to manufacturer’s instructions. Libraries were sequenced on an Illumina HiSeq2000 machine as 100 bp reads single-end.
9
Single-cell gene expression profiling
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HAECs were dissociated as described above, then single cells were separated using a C1 Single-cell AutoPrep system (Fluidigm), followed by quantitative PCR on the Biomark system (Fluidigm) using SsoFast evaGreen Supermix (Biorad) and the primers described in Table S7 .
10
Single-Cell RNA-Seq Using C1 System
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The C1 Single-Cell Auto Prep System (Fluidigm) was used to perform SMARTer (Clontech, Mountain View, CA) cDNA generation and amplification. Prior to loading the single-cell suspension onto the C1 chips, we stained the cells with the LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells (Life Technologies, Carlsbad, CA). After loading, we visualized each microchamber in the C1 chip to identify those chambers that contained a single live (Calcein+/Ethidium homodimer−) cell. We selected two cells from stage 1 (cell A and cell B) and two cells from stage 2 (cell C and cell D), with an average cDNA concentration of 0.4 ng/µL in an approximate output volume of 15 µL (ST1). DNA concentration was quantified using the Qubit dsDNA High Sensitivity Kit (Life Technologies), according to the manufacturer’s instructions. The resulting cDNAs were diluted to a final concentration of 0.1 ng/µL and then converted to Illumina sequencing libraries using the Nextera XT (Illumina) kit using protocols specifically designed for the mosquito HTS (TTP Labtech) (ST2). We generated libraries in three different final reaction volumes (2 µL, 4 µL, and 8 µL) using Axygen (Palo Alto, CA) 384 plates. Dual indexing was performed using the Nextera XT Index Kit v2 Set A (96 Indexes) to enable multiplexing of libraries.
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