GSM9012260: SCS_DOX_NS_LA; Homo sapiens; RNA-Seq
The C1 integrated fluidics circuits (IFCs) for mRNA seq (small-cell, 5–10 μm) was primed according to the standard Fluidigm mRNA-seq protocol using C1 harvest reagent, C1 preloading reagent, C1 blocking reagent, and Fluidigm cell wash buffer. Priming was performed immediately prior to sample preparation. HCT-116 cells were trypsinised and resuspended in serum-free media at a concentration of 333,000 cells/mL. The standard suspension ratio of 3:2 was used (60 µL of cell suspension was added to 40 µL of Fluidigm suspension reagent) to achieve the final cell mix for IFC loading. After mixing several time the cell preparation, 6 µL of the cell mix was loaded. Following cell loading, light microscopy using EVOS microscope, was used in order to assess each of the 96 capture sites individually and the outcome of loading was recorded in terms of the number of cells present in each capture site and whether debris were present. This information was used as a quality control step to ensure that only confirmed single cell samples were taken forward. Finally, the IFC was placed into the C1 the script “mRNA Seq: Cell Load (1771x/1772x/1773x)” was run. Following the loading, buffer mixes for lysis, reverse transcription and PCR were pipetted into the IFC. The IFC was then placed into the C1 system and the script “mRNA Seq: RT & Amp (1771x/1772x/1773x)” was run overnight. Samples were harvested from the IFC by transferring 3.5 µL of product to a 96 well plate and diluted with 10 µL C1 DNA dilution reagent. The samples corresponding to the confirmed single cell captures were highlighted according to the arrangement on the harvest plate described by Fluidigm. Samples were stored at 4ºC short term and -20ºC long term prior to subsequent quantification and library preparation. Library preparation was performed using the modified Illumina Nextera XT DNA library preparation protocol provided by Fluidigm. The cDNA concentration was quantified using the PicoGreen Assay (Quant-IT PicoGreen dsDNA Assay Kit, Thermo Fisher Scientific, P11496) using 2 µL per 96 samples processed into 2 replicates in a 384-well plate giving a final well volume of 60 µL for reading in a fluorometer. The data was then processed using the Microsoft Excel worksheet, Single-Cell mRNA Seq PicoGreen Template (Fluidigm, PN 100-6260), to quantify the library. Samples were transferred to a new 96-well plate in order to obtain a sample within the range 0.1-0.3 ng/µL by diluting with C1 Harvest Reagent. Only samples with at least 0.1 ng/µL were taken forward for library preparation. Sequencing libraries were prepared using Nextera XT DNA Library (Illumina) with a modified protocol provided by Fluidigm.