Step 3: GEM Generation & Barcoding

Single Cell Multiome ATAC + GEX GEM-X Epi Multiome Gel Beads include a poly(dT) sequence that enables production of barcoded, full-length cDNA from poly-adenylated mRNA for GEX library and a Spacer sequence that enables barcode attachment to transposed DNA fragments for ATAC library.

GEMs are generated by combining barcoded Gel Beads, transposed nuclei, a Master Mix, and Partitioning Oil on a Chromium Next GEM Chip JGEM-X MO Chip. To achieve single nuclei resolution, the nuclei are delivered at a limiting dilution, such that the majority (~90-99%) of generated GEMs contains no nuclei, while the remainder largely contain a single nucleus.

Upon GEM generation, the Gel Bead is dissolved. Oligonucleotides containing an Illumina® P5 sequence, a 16 nt 10x Barcode (for ATAC), and a Spacer sequence are released. In the same partition, primers containing an Illumina® TruSeq Read 1 (read 1 sequencing primer), 16 nt 10x Barcode (for GEX), 12 nt unique molecular identifier (UMI), and a 30 nt poly(dT) sequence are also released. The primers are mixed with the nuclei lysate containing transposed DNA fragments, mRNA, and Master Mix, that includes reverse transcription (RT) reagents.

Incubation of the GEMs produces 10x Barcoded DNA from the transposed DNA (for ATAC) and 10x Barcoded, full-length cDNA from poly-adenylated mRNA (for GEX). This is followed by a quenching step that stops the reaction.

Inside Individual GEMs