A cell suspension obtained after FACS sorting was concentrated at a range of 600-1000 cells/μL. C1 Suspension Reagent was added (all ‘C1’ reagents were from Fluidigm, Inc.) in a ratio of 4 μL to every 7 μL cell suspension as previously described (Zeisel et al., 2015 (link)). 11 μL of the cell suspension mix was loaded on a C1 Single-Cell AutoPrep IFC microfluidic chip designed for 10- to 17-μm cells, and the chip was then processed on a Fluidigm C1 instrument using the ‘mRNA Seq: Cell Load (1772x/1773x)’ script (30 min at 4°C). The plate was then transferred to an automated microscope (Nikon TE2000E), where a brightfield and RFP or EGFP fluorescence image (20 × magnification) was acquired for each capture site using μManager (http://micro-manager.org/ (2)), which took < 15 min. Quality of cells and control for doublets was performed after each experiment as described in (Zeisel et al., 2015 (link)).
C1 single cell autoprep ifc microfluidic chip
Manufactured by Standard BioTools
The C1 Single-Cell AutoPrep IFC microfluidic chip is a lab equipment product designed for the isolation and preparation of single cells. It functions as a microfluidic chip that enables the automated processing of individual cells for various downstream applications.
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3 protocols using c1 single cell autoprep ifc microfluidic chip
1
Single-cell RNA-seq Protocol Using C1 System
2
Single-Cell RNA-seq of Neural Stem Cells
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Sai2 NES cells passage 29, AF22 NES cells passage 20 and Ctrl‐7 NES cells passage 12 were harvested as neural stem cells at day 0 and Sai2 DIFF passage 27, AF22 DIFF passage 16 and 23, Ctrl‐7 DIFF passage 12 as differentiated neurons at day 28 of differentiation.
The cells were dissociated as described under neural stem cell culture. The dissociated cells were passaged through a cell strainer (40 μm, VWR) and diluted to 2,000 cells/ml in NES medium with addition of 5% DNaseI (2,000 U/ml, Qiagen) and 1% BSA (Sigma). Cells were placed on ice until further processed. The cells were loaded according to manufacturers protocol on C1 Single‐Cell AutoPrep IFC microfluidic chip (for cell size 10–17 μm) and processed on a Fluidigm C1 instrument. This chip contains 96 wells for single‐cell capture.
Each chamber of the chip was optically inspected by automated microscope (Nikon TE2000E), empty chambers, cell clumps and dying cells were discarded from further analyze. Lysis of the cells and cDNA production were carried out according to the Linnarsson's laboratory protocol (Islam et al.,2014 ). For analysis, high‐quality single cells were selected through cutoff to fit between 500 and 5,000 genes/cell.
The cells were dissociated as described under neural stem cell culture. The dissociated cells were passaged through a cell strainer (40 μm, VWR) and diluted to 2,000 cells/ml in NES medium with addition of 5% DNaseI (2,000 U/ml, Qiagen) and 1% BSA (Sigma). Cells were placed on ice until further processed. The cells were loaded according to manufacturers protocol on C1 Single‐Cell AutoPrep IFC microfluidic chip (for cell size 10–17 μm) and processed on a Fluidigm C1 instrument. This chip contains 96 wells for single‐cell capture.
Each chamber of the chip was optically inspected by automated microscope (Nikon TE2000E), empty chambers, cell clumps and dying cells were discarded from further analyze. Lysis of the cells and cDNA production were carried out according to the Linnarsson's laboratory protocol (Islam et al.,
3
Single-cell mRNA sequencing using C1 system
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Cell suspensions in a concentration of 600-1000 cells/μL was used. C1 Suspension Reagent was added (all 'C1' reagents were from Fluidigm, Inc.) in a ratio of 4μL to every 7μL cell suspension. 11μL of the cell suspension mix was loaded on a C1 Single-Cell AutoPrep IFC microfluidic chip designed for 10- to 17μm cells, and the chip was then processed on a Fluidigm C1 instrument using the 'mRNA Seq: Cell Load (1772x/1773x)' script (30 min at 4°C). The plate was then transferred to an automated microscope (Nikon TE2000E), and a bright-field image (20× magnification) was acquired for each capture site using μManager (http://micro-manager.org/ (2)), which took <15 minutes. Quality of cells, control for doublets and processing of C1 chips were performed in the Eukaryotic Single Cell Genomics facility at SciLife Lab, as described in Zeisel et al. (2015) (link) and Marques et al. (2016) (link).
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