Vehicle treated cells were stained with cell tracker as described above. An equal number of stained (vehicle treated) and non-stained (nutlin-3 treated) cells were mixed and diluted to 300 000 cells/ml. Suspension buffer was added to the cells in a 3:2 ratio and 6 μl of this mix of was loaded on a primed C1 Single-Cell Auto Prep Array for mRNA Seq (Fluidigm, 100-6041) designed for medium-sized cells (10–17 μm). Single cell polyA[+] RNA sequencing on the C1 was performed using the SMART-Seq v4 Ultra Low Input RNA Kit for the Fluidigm C1 System (SMART-Seq v4, polyA[+] RNA, Takara, 635026) according to manufacturer's instructions. One microliter of the ERCC spike-in mix was diluted in 999 μl loading buffer to get a 1/1000 dilution of the ERCC spikes. One microliter of this dilution was added to the 20 μl lysis mix. The quality of the cDNA was checked for 11 random single cells on the Fragment Analyzer. The concentration of the cells was measured using the quantifluor dsDNA kit (Promega, E2670) and glomax (Promega) according to manufacturer's instructions. The samples were 1/5 diluted in C1 harvest reagent (Fluidigm). Next, library prep was performed using the Nextera XT library prep kit (Illumina, FC-131-1096) according to manufacturer's instructions, followed by quality control on the Fragment Analyzer.
C1 harvest reagent
Manufactured by Standard BioTools
The C1 Harvest Reagent is a laboratory reagent used for the processing and preparation of samples in various scientific applications. The core function of this product is to facilitate the harvesting and isolation of cells or other biological materials from a sample, enabling further analysis or experimentation.
Automatically generated - may contain errors
Lab products found in correlation
C1 single cell auto prep array for mrna seq, by Standard BioTools (1 mentions)
Nextera xt library prep kit, by Illumina (1 mentions)
Quantifluor dsdna kit, by Promega (1 mentions)
Glomax, by Promega (1 mentions)
Dithiothreitol (dtt), by GE Healthcare (1 mentions)
C1 dna seq cell wash buffer, by Standard BioTools (1 mentions)
C1 chip, by Standard BioTools (1 mentions)
Dtt mix, by GE Healthcare (1 mentions)
Picogreen dsdna quantification assay, by Thermo Fisher Scientific (1 mentions)
Infinite 200 pro plate reader, by Tecan (1 mentions)
Nextera xt sample preparation kit, by Illumina (1 mentions)
3 protocols using c1 harvest reagent
1
Single-Cell RNA Sequencing of Mixed Cell Populations
2
Single-Cell Whole Genome Amplification
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The medium-sized (10 to 17 µm) C1 chip (Fluidigm) was primed with C1 Harvest Reagent, Preloading Reagent, Blocking Reagent and C1 DNA Seq Cell Wash Buffer (Fluidigm) for 10 min before it was loaded with the dissociated single cells. The DTT Mix was prepared by the addition of DTT, Sample and Reaction Buffers (GE Healthcare). The Lysis Mix contained C1 DNA Seq Lysis Buffer and DTT (Fluidigm), while the Reaction-Enzyme Mix consisted of C1 DNA Seq Reaction Mix (Fluidigm), DTT Mix and Enzyme Mix (GE Healthcare). The Lysis Mix, Reaction-Enzyme Mix and C1 DNA Seq Stop Buffer were loaded on the C1 chip followed by the on-chip whole genome amplification experiment. The amplified DNA was harvested from the C1 chip and transferred into 96-well PCR plate. The DNA was quantified using PicoGreen dsDNA quantification assay (Thermo Fisher) on the Infinite 200Pro plate reader (Tecan).
3
Nextera XT Library Preparation
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The library preparation was carried out using the Nextera XT Sample Preparation kit (Illumina) with minor modifications and one-quarter of the recommended volume (7 ) . Briefly, the cDNA and gDNA amplicons were diluted to the range of 0.1 to 0.3 ng/L with C1 Harvest Reagent (Fluidigm) followed by tagmentation at 55 °C for 10 min. The tagmentation was inactivated by addition of NT buffer. The Nextera PCR Master Mix and XT indices were added
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