A very brief intro to major single-cell protocols

1 The Smart-seq2 protocol

1. First, the biological samples of interest are collected, and cells should be disaggregated into a single-cell suspension.
2. Cells are picked from the suspension and embedded individually in a well containing lysis buffer containing a ribonuclease inhibitor that blocks RNA degradation, and primers for the reverse transcription step.
3. Reverse transcription. Single-stranded RNA molecules are converted into more stable double-stranded cDNA molecules.
4. The cDNA molecules are amplified using PCR amplification.
5. Construction of sequencing libraries using tagmentation. A transposase is used to fragment the cDNA molecules and simultaneously ligate adapter sequences.
6. Another cycle of PCR occurs (the enrichment PCR step). Sample barcodes may be added to allow multiplexing; a dual-index strategy is often used, indices referred to as index 1 (i7) and index 2 (i5) in the Figure below.

Figure: The SMART-Seq2 library prep workflow. Image from Picelli et al. (2014).

2 The 10X Genomics Chromium protocol

• The 10X Genomics Chromium protocol is droplet-based, i.e., cells are embedded into nanodroplets within which library preparation steps occur.
• Within each droplet, gel beads along with reagents are encapsulated together with the cell.
• These gel beads contain millions of sequences on their surface. These sequences consist of a sequencing adapter (‘TruSeq Read’), cell barcode (‘10X Barcode’), molecular barcode (‘UMI’), and a Poly(dT) to capture polyA’d mRNAs. As of v3, two additional capture sequences may be used for capturing additional molecules, e.g., guide RNAs.
• The beads are mixed with the cells of our sample, which are then emulated within oil to create nanodroplets. See ‘Getting Started with Single Cell Gene Expression’ video at 10:40 on the 10X Genomics website for a demonstration. These droplets are often called GEMs, for ‘Gel beads in EMulsion’.
• Within these GEMs, the necessary library preparation steps occur, and the produced molecules each contain a cell barcode as well as a Unique Molecular Identifier (UMI), which may then be sequenced.

Figure: The 10X Genomics Chromium protocol. Images from 10X Genomics.

Figure: The 10X Genomics Chromium protocol. Images from 10X Genomics.

Figure: The 10X Genomics Chromium protocol. Images from 10X Genomics.

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