Transcriptome profiling with high resolution

Single-cell, single-nuclei, and spatial sequencing
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RNase Inhibitor

High-concentration Equinox Master Mix

Watchmaker DNA Library Prep Kit with Fragmentation

T4 DNA Ligase

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.

QUICK LINKS

RNase Inhibitor

High-concentration Equinox Master Mix

Watchmaker DNA Library Prep Kit with Fragmentation

T4 DNA Ligase

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 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.
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 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.
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.
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.
Efficient cDNA amplification with minimal bias
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.
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.
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).
Simple, efficient cDNA library preparation
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).
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 Product 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

T4 DNA Ligase

Cost-effective, high-purity ligase suitable
for bead manufacturing with immobilized oligos

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Watchmaker Custom Genomic Solutions

RNase Inhibitor

Prevent RNA degradation during RNA isolation and cDNA synthesis
EQUINOX LIBRARY AMPLIFICATION KIT

Equinox Library Amplification 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

Watchmaker StellarScript Reverse Transcriptases Banner

Stellarscript Reverse Transcriptases

A portfolio of M-MLV RTase variants engineered for reduced RNase H activity, increased thermostability, improved processivity, and enhanced inhibitor tolerance
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