Low-input unstranded mRNA sequencing

Synopsis

Relatively small model organisms like fruit flies have significantly smaller cells compared to mammalian models. Combined with current trend of tissue-specific and/or rare-cell studies, it is necessary to adopt low-input/single-cell preparation kits as standard ones are typically optimized for mamallian samples.

Here, I provide a succinct summary of current low-input RNA-sequencing cDNA preparation kits.

This work is licensed under CC BY-SA 4.0

Basics

Here are a few basic points on RNA sequencing library preparation:

1. Input amounts provided by a manufacturer is typically total RNA.
2. The amount of mRNA per cell is affected heavily by many factors (incl. species, cell type, differentiation status, etc.) As an estimate, a mamalian cell contains 10pg total RNA while a fruit fly cell contains 10x less (10x smaller genome). Refer to QIAGEN and ThermoFisher for more details.
3. Low-input kits typically start with either purified total RNA or resuspended/FAC-sorted cells. If you plan to start with FACS cells, it is best to sort directly into the lysis buffer provided by the kit. 1xPBS with 0.1% (w/v) BSA is typically sufficient as your sample buffer. Droplet volume is machine-dependent, usually a few picoliters per cell.
4. Final purification step of a kit is typically performed with SPRI beads. SPRIselect, Ampure XP, and Cytiva Sera-Mag Select are completely interchangable for size-selective and non-selective purification applications.
5. PCR is typically used for both cDNA amplification and library preparation steps. While cDNA amplification (if used) does not require sample-specific primers, you need to provide indexing primer sets during library preparation. Indexing primer/adapter sets are typically sold separately.
6. Quality control readouts involve size distribution measured by electrophoresis and concentration measured by fluorometers. Tapestation / Fragment Analyzer are popular choices for the former. Qubit / Promega Quantus are interchangable for the latter.
7. Your library might be 3’-biased dependeing on the detailed mechanism used during cDNA generation. Popular methods include reverse transcription with random primers (RT), template-switching reverse transcription with oligo-dT and template-swtiching oligos (TS-RT), and CEL-seq (in vitro transcription for amplification). CEL-seq methods are 3’-biased and is used for transcript counting. CEL-seq is typically used for highly multiplexed single-cell transcript counting while TS-RT methods are used for whole-transcript sequencing.

Comparison table