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## Step 1: GEM Generation & Barcoding |
GEMs are generated by combining barcoded Gel Beads, a Master Mix containing cells, and Partitioning Oil B onto GEM-X 3' Chip. To achieve single cell resolution, cells are delivered at a limiting dilution, such that the majority (~90-99%) of generated GEMs contain no cell, while the remainders largely contain a sing... |
## GEM-X Chip Workflow |
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Immediately following GEM generation, the Gel Bead is dissolved, primers are released, and any copartitioned cell is lysed. Primers containing an Illumina TruSeq Read 1 (read 1 sequencing primer, Read 1T), 16 nt 10x Barcode, 12 nt unique molecular identifier (UMI), and 30 nt poly(dT) sequence are mixed with both the ce... |
Incubation of the GEMs produces barcoded, full-length cDNA from polyadenylated mRNA. |
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## Inside Individual GEMs - Gene Expression Primer |
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## Step 2: Post GEM-RT Cleanup & cDNA Amplification |
After incubation, 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 co... |
## Pooled cDNA Amplification |
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## Step 3: 3' Gene Expression Library Construction |
Enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size. P5 and P7 adaptors, i7 and i5 sample indexes, and TruSeq Read 2 (read 2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina amplificat... |
## Amplified cDNA Processing (dual index) |
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## Sequencing |
A Chromium Single Cell 3' Gene Expression Dual Index library comprises standard Illumina paired-end constructs which begin and end with P5 and P7. The 16 bp 10x Barcode and 12 bp UMI are encoded in Read 1, while Read 2 is used to sequence the cDNA fragment. i7 and i5 index sequences are incorporated as the sample index... |
Illumina sequencer compatibility, sample indices, library loading and pooling for sequencing are summarized in the sequencing chapter. |
## Chromium Single Cell 3' Gene Expression Library |
See Oligonucleotide Sequences on page 76 |
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