For MCF10CA single-cell RNA sequencing experiments, spheroids were dissociated as described above and resuspended in DMEM/F12 medium. Capture, full-length cDNA synthesis and amplification was performed on the C1 Single-Cell Auto Prep system for mRNA Seq (Fluidigm) using the IFC for up to 96 cells (medium size 10–17 µm). Cells at a concentration of 350 cells/µl were mixed with C1 Cell Suspension Reagent (Fluidigm) at a ratio of 4:1 immediately before loading on the IFC. Single-cell capture was assessed with an inverted brightfield microscope. Workflow and reagents for single-cell RNA extraction, reverse transcription (RT) and mRNA amplification (18 cycles) were used as described in the SMARTer Ultra Low RNA Kit (for Fluidigm C1). Sequencing libraries were generated with the Nextera XT kit (Illumina) according to an adapted Fluidigm protocol. Concentration and quality of cDNA and sequencing libraries was assessed by a fluorometer (Qubit) and by electrophoresis (Agilent Bioanalyzer high sensitivity DNA chips). Libraries of up to 24 cells were pooled and sequenced as 1 × 50-bp reads on an Illumina HiSeq 2000 machine.
Smarter ultra low rna kit
Manufactured by Standard BioTools
The SMARTer Ultra Low RNA Kit is a laboratory tool designed for the conversion of ultra-low input RNA samples into high-quality cDNA libraries for various downstream applications, such as next-generation sequencing. The kit utilizes a proprietary SMART (Switching Mechanism at the 5' end of the RNA Template) technology to efficiently generate full-length cDNA from minimal RNA input amounts.
Automatically generated - may contain errors
Lab products found in correlation
C1 cell suspension reagent, by Standard BioTools (1 mentions)
Nextera xt kit, by Illumina (1 mentions)
Hiseq 2000 machine, by Illumina (1 mentions)
Agilent bioanalyzer dna high sensitivity chip, by Agilent Technologies (1 mentions)
Ercc spike in, by Thermo Fisher Scientific (1 mentions)
Fragment analyzer, by Agilent Technologies (1 mentions)
Picogreen, by Thermo Fisher Scientific (1 mentions)
Quant it dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
C1 single cell auto prep system, by Standard BioTools (1 mentions)
Nextseq 500, by Illumina (1 mentions)
3 protocols using smarter ultra low rna kit
1
Single-cell RNA sequencing of MCF10CA spheroids
2
Single-cell RNA-seq: Fluidigm C1 protocol
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These steps were performed as described (Camp et al., 2015 (link); Treutlein et al., 2014 (link)). Depending on the size distribution of the cells, cells were loaded at a concentration of 250–500 cells per μl onto small (5–10 μm) or medium (10–17 μm) integrated fluidic circuits (IFCs, Fluidigm). Lysis, reverse transcription and amplification were performed on the Fluidigm C1 platform using the SMARTer Ultra Low RNA Kit for the Fluidigm C1 system. External RNA Control Consortium (ERCC) spike-ins (Ambion) were added to the lysis mix at a dilution of 1:80,000. Resulting cDNA was quantified and checked for its size distribution using a capillary gel electrophoresis system (Fragment Analyzer, Advanced Analytical, 1–6000 bps High Sensitivity).
3
Single-Cell Transcriptome Profiling of Cell Types
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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.
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