Systems and methods for compartmentalizing and analyzing cells

A method for analyzing a biological sample, comprising:

• (a) introducing the biological sample into a fluidic device, wherein:
• the fluidic device comprises a microfluidic device or a nanofluidic device,
• the microfluidic device or the nanofluidic device comprises a chamber, a port, and a channel,
• the chamber, the port, and the channel are interconnected and in fluid communication,
• the channel comprises a cross-sectional dimension from about 0.1 microns (μm) to about 500 μm, and
• the biological sample comprises a plurality of cells;
• (b) inputting the fluidic device into an instrument, wherein:
• the instrument comprises a temperature controller, and
• the temperature controller is in thermal communication with the fluidic device;
• (c) modulating a temperature of the fluidic device using the temperature controller,
• (d) flowing the biological sample through the channel, wherein during the flowing, the instrument applies a positive pressure, a negative pressure, or a combination thereof to the biological sample,
• (e) forming a plurality of compartments, thereby compartmentalizing and physically separating each of at least 105 cells of the plurality of cells, wherein each compartment of the plurality of compartments co-encloses a capture element of a plurality of capture elements and a cell of the at least 105 cells, wherein the capture element comprises single-stranded DNA, and wherein the capture element comprises a unique molecular identifier, a barcode, and a sequencing primer;
• (f) lysing at least a portion of the at least 105 cells compartmentalized in (e), thereby releasing mRNA molecules from the at least the portion of the at least 105 cells compartmentalized in (e);
• (g) capturing at least a subset of the mRNA molecules using at least a subset of the plurality of capture elements, thereby obtaining captured mRNA molecules, wherein the capturing comprises coupling poly(A) sequences of the mRNA molecules or the subset of the mRNA molecules to poly(T) sequences of the plurality of capture elements or the subset of the plurality of capture elements;
• (h) reverse transcribing at least a portion of an mRNA molecule captured in (g) to form a cDNA molecule, wherein:
• the reverse transcribing comprises adding untemplated cytosine nucleotides to the cDNA molecule,
• the method further comprises coupling a template switch oligonucleotide to the untemplated cytosine nucleotides and extending the cDNA molecule using at least a portion of the template switch oligonucleotide as a template,
• wherein the cDNA molecule comprises the unique molecular identifier, the barcode, and the sequencing primer;
• (i) deconstructing a compartment of the plurality of compartments;
• and
• (j) sequencing at least a portion of the cDNA molecule, a derivative of the cDNA molecule, a complement of the cDNA molecule, or a combination thereof, wherein the cDNA molecule, the derivative of the cDNA molecule, the complement of the cDNA molecule, or the combination thereof are transferred to a sequencing device prior to the sequencing, and wherein the sequencing device is separate from the fluidic device.