Post GEM–RT Cleanup and cDNA Amplification v1

The Chromium Single Cell Gene Expression Solution upgrades short read sequencers to deliver a scalable microfluidic platform for 3’ digital gene expression by profiling 500-10,000 individual cells per sample. A pool of ~3,500,000 10x Barcodes are sampled separately to index each cell’s transcriptome. It is done by partitioning thousands of cells (or nuclei) into nanoliter-scale Gel Beads-in-emulsion (GEMs), where all generated cDNA share a common 10x Barcode. Dual Indexed libraries are generated and sequenced from the cDNA and 10x Barcodes are used to associate individual reads back to the individual partitions. After the GEMs Generation and Barcoding, GEMs are broken and pooled fractions are recovered. Silane magnetic beads are used to purify the first-strand cDNA from the post GEM-RT reaction mixture, which includes leftover biochemical reagents and primers. Barcoded, full-length cDNA is amplified via PCR to generate sufficient mass for library construction. This protocol details the post GEM-RT cleanup and cDNA amplification, cleanup - SPRIselect and quantification.