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Single-cell, single-nuclei, and spatial sequencing

TRANSCRIPTOME PROFILING
WITH HIGH RESOLUTION

Introduction

Single-cell RNA sequencing (scRNA-seq) has emerged as a transformative technology that allows researchers to investigate the heterogeneity and dynamics of gene expression profiles at the individual cell level. By capturing the transcriptomes of thousands to millions of cells in a high-throughput manner, scRNA-seq enables the identification of rare cell populations, characterization of cell states and lineages, and discovery of novel cell types.

Similarly, spatial transcriptomics is a revolutionary technique that enables the simultaneous visualization and quantification of gene expression patterns within intact tissue sections, providing valuable insights into the spatial organization of cells and their molecular profiles.

Watchmaker offers a number of protein, enzyme, and NGS library prep solutions that address key needs and pain points of scRNA-seq, single-nuclei, and spatial workflows.

Critical steps for ensuring data quality

Preserve RNA integrity

  • Sensitive and robust cDNA synthesis is critical for generating high-quality data, as this step determines which transcripts are ultimately detected.
  • RNA degradation is a huge concern prior to and during cDNA synthesis due to the presence of environmental and cellular RNases.
  • Including an RNase Inhibitor - particularly one that is of high-purity and maintains activity at the elevated temperatures needed for reverse transcription - greatly improves yields and sensitivity.

Figure 1A. Prevent RNA degradation through ... MORE

Figure 1B. Prevent RNA degradation ... MORE

Preserve RNA integrity

Figure 1A. Prevent RNA degradation through the addition of RNase inhibitor. The addition of RNase inhibitor to first strand synthesis (FSS) reactions improves cDNA yield even when no RNase A is added to the reaction - highlighting risks of RNase contamination during sample handling. Addition of 5 ng RNase A results in a >10-fold decrease in cDNA yield when no RNase inhibitor is present. Assay performance is rescued by the addition of RNase inhibitor, where all commercial enzymes evaluated perform equivalently. RNase Inhibitors sourced from a variety of vendors were added to oligo-dT-primed FSS reactions with StellarScript HT+ Reverse Transcriptase. Reactions ran for 30 min at 42°C using 10 ng total liver RNA.

Figure 1B. Prevent RNA degradation at elevated temperatures through the addition of RNase Inhibitor. The addition of RNase Inhibitor to high-temperature (55°C) first strand synthesis (FSS) reactions improves cDNA yield even when no RNase A is added to the reaction - highlighting risks of RNase contamination during sample handling. Addition of 5 ng RNase A results in a >10-fold decrease in cDNA yield when no RNase Inhibitor is present. Assay performance is rescued by the addition of RNase Inhibitor. RNase Inhibitor was added to oligo-dT-primed FSS reactions with StellarScript HT+ Reverse Transcriptase. Reactions ran for 30 min at 55°C using 10 ng total liver RNA.

Efficient cDNA amplification with minimal bias

  • cDNA amplification is often required to generate a sufficient amount of material for downstream NGS library preparation.
  • Selecting an amplification solution that generates high yields with a minimal number of cycles, as well as is low-bias (both with respect to GC-content and length) is critical to maintaining an accurate representation of transcript abundance.
  • Equinox® Master Mix for cDNA amplification delivers in all of these areas and is available in a 4X high-concentration format for dilute samples.
Highly efficient library amplification. Human whole genome libraries (10 ng per reaction) were amplified in triplicate with the Equinox Library Amplification Kit, KAPA HiFi HotStart ReadyMix, and NEBNext Ultra II Q5 Master Mix for 0, 2, 4, 6, 8, 10 or 12 cycles. Yields were determined by qPCR-based library quantification at the 2-cycle intervals.Highly efficient library amplification. Human whole genome libraries (10 ng per reaction) were amplified in triplicate with the Equinox Library Amplification Kit, KAPA HiFi HotStart ReadyMix, and NEBNext Ultra II Q5 Master Mix for 0, 2, 4, 6, 8, 10 or 12 cycles. Yields were determined by qPCR-based library quantification at the 2-cycle intervals.

Figure 2. Highly efficient library amplification ... MORE

Efficient cDNA amplification with minimal bias

Highly efficient library amplification. Human whole genome libraries (10 ng per reaction) were amplified in triplicate with the Equinox Library Amplification Kit, KAPA HiFi HotStart ReadyMix, and NEBNext Ultra II Q5 Master Mix for 0, 2, 4, 6, 8, 10 or 12 cycles. Yields were determined by qPCR-based library quantification at the 2-cycle intervals.Highly efficient library amplification. Human whole genome libraries (10 ng per reaction) were amplified in triplicate with the Equinox Library Amplification Kit, KAPA HiFi HotStart ReadyMix, and NEBNext Ultra II Q5 Master Mix for 0, 2, 4, 6, 8, 10 or 12 cycles. Yields were determined by qPCR-based library quantification at the 2-cycle intervals.

Figure 2. Highly efficient library amplification. Human whole genome libraries (10 ng per reaction) were amplified in triplicate with the Equinox Library Amplification Kit, KAPA HiFi HotStart ReadyMix, and NEBNext Ultra II Q5 Master Mix for 0, 2, 4, 6, 8, 10 or 12 cycles. Yields were determined by qPCR-based library quantification at the 2-cycle intervals.

Simple, efficient cDNA library preparation

  • Converting amplified cDNA into sequenceable libraries often requires cDNA normalization prior to library prep to ensure similar final library insert sizes across samples.
  • The Watchmaker DNA Library Prep Kit with Fragmentation alleviates this sample prep bottleneck by fragmenting DNA consistently - independent of input amount.
  • Similarly sized libraries can be achieved using the same fragmentation condition for a 5,000-fold input range.
Consistent fragmentation across a 5,000-fold range of DNA input amounts. Libraries were constructed in duplicate from 500, 10, and 0.1 ng of human genomic DNA fragmented for 20 minutes at 30℃. Final library distributions were assessed using a D1000 assay by TapeStation (Agilent).Consistent fragmentation across a 5,000-fold range of DNA input amounts. Libraries were constructed in duplicate from 500, 10, and 0.1 ng of human genomic DNA fragmented for 20 minutes at 30℃. Final library distributions were assessed using a D1000 assay by TapeStation (Agilent).

Figure 3. Consistent fragmentation across ... MORE

Simple, efficient cDNA library preparation

Consistent fragmentation across a 5,000-fold range of DNA input amounts. Libraries were constructed in duplicate from 500, 10, and 0.1 ng of human genomic DNA fragmented for 20 minutes at 30℃. Final library distributions were assessed using a D1000 assay by TapeStation (Agilent).Consistent fragmentation across a 5,000-fold range of DNA input amounts. Libraries were constructed in duplicate from 500, 10, and 0.1 ng of human genomic DNA fragmented for 20 minutes at 30℃. Final library distributions were assessed using a D1000 assay by TapeStation (Agilent).

Figure 3. Consistent fragmentation across a 5,000-fold range of DNA input amounts. Libraries were constructed in duplicate from 500, 10, and 0.1 ng of human genomic DNA fragmented for 20 minutes at 30℃. Final library distributions were assessed using a D1000 assay by TapeStation (Agilent).

Manufacturing and customization considerations for technology developers

  • Custom fills and formats minimize waste and maximize your operational efficiency
  • Tailored packaging and labeling - including private label - designed to your unique specifications
  • All products are manufactured within an ISO 13485:2016-certified Quality Management System
  • White glove support and a dedicated Project Manager make customization seamless
  • Right-sized processes and terms enable fast turnaround times, as well as serve organizations of all sizes
  • Read more about our Custom Genomic Solutions and capabilities

Discover more with Watchmaker

Equinox® Library Amp Kits

An ultra-high-fidelity DNA polymerase in an optimized hot start master mix format, specifically optimized for high-efficiency, low-bias NGS library amplification...

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RNase Inhibitor

Prevent RNA degradation during RNA isolation and cDNA synthesis, even at elevated temperatures. High-purity and cost-effective...

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DNA Library Prep Kits with Fragmentation

A streamlined single-tube chemistry with reduced bias and artifacts. Ideally suited for PCR-free workflows, ultra-low inputs, and low-quality DNA samples including FFPE ...

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For assay developers – make realizing your next product easier

  • Navigate the complexities of productization with an experienced team who’s as committed to your success as you are
  • All aspects of our customization process are designed to serve you with speed, agility, and above all else, a commitment to quality
  • Tailored fill volumes, labeling (including white and private label), and packaging designed to your specifications
  • All products are manufactured within an ISO 13485:2016-certified QMS (download our certificate)