High throughput analysis of fixed cells

A method for single-cell RNA profiling, comprising:

• loading a plurality of fixed cells into a plurality of microwells;
• loading a plurality of barcoded microcarriers into the plurality of microwells, wherein at least 5% of the plurality of microwells each is loaded with one of the plurality of fixed cells and one of the plurality of barcoded microcarriers, and wherein each of the plurality of barcoded microcarriers comprises:
• a microbead,
• a plurality of molecular barcodes each comprising an identical cell barcode, a unique molecule identifier (UMI) different across the plurality of molecular barcodes, a PCR handle, and a poly-T sequence capable of hybridizing to poly-A tails of messenger ribonucleic acid (mRNA) targets of the plurality of fixed cells, and
• a plurality of releasable linkers each associated with the microbead and linked with one of the plurality of molecular barcodes;
• releasing the molecular barcodes from the microbeads in the microwells, thereby diffusing the molecular barcodes into the fixed cells and hybridizing the molecular barcodes to mRNA targets of the fixed cells;
• reverse transcribing the mRNA hybridized with the molecular barcode to generate barcoded complementary deoxyribonucleic acids (cDNAs);
• pooling the barcoded cDNAs from the plurality of microwells;
• and
• analyzing the barcoded cDNAs or products thereof