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What’s New for C1 Applications on Script Hub

View the growing list of community-developed apps for single-cell biology

When we introduced Script Hub™ as an open-source platform for C1™ methods and applications, we invited the single-cell community to join us on a quest. The mission: Develop an array of innovative single-cell applications to uncover biological diversity and discover the functional role of unique cell populations in health and disease.

Scientists from around the world have responded and are sharing their groundbreaking protocols and data with the entire C1 user community. This information is available for everyone to access on Script Hub, and features novel single-cell applications for epigenetics, T cell receptor sequencing, DNA sequencing and more.

Here’s an overview of one of the most-visited areas on Script Hub, where C1 mRNA sequencing methods including full-length mRNA seq and transcript counting deliver unparalleled breadth and value for your single-cell analysis.

Full-length mRNA sequencing

Sequencing of full-length transcripts is crucial for uncovering deep insights into the biology of individual cells. The approach offers single-base resolution of the RNA sequence to yield a highly detailed view of each transcript. Measure individual gene expression and quantify allele or splice-variant expression, map transcriptional start sites, identify alternative splicing patterns and discover novel transcripts or gene fusions.

Several specialized approaches are now available on Script Hub to power your single-cell studies, including:

  • C1 TCR Seq: T cell receptor sequencing leverages the detail of whole transcriptome analysis to simultaneously reconstruct the variable region of the TCR and identify all other genes that regulate each T cell’s function. TCR seq brings together full-length mRNA sequencing with TraCeR, a powerful new bioinformatics tool. For a deeper dive into single-cell TCR seq, read our interview with TraCeR’s developers.
  • C1 SMART Seq®: Amplifying full-length cDNA for mRNA sequencing empowers C1 discovery experiments. SMARTer® and C1 SMART Seq2 allow you to create reverse-transcribed poly(A) RNA and employ specialized template-switch technology to generate full-length cDNA prior to next-generation sequencing.

For a complete list of protocols available for full-length sequencing on the C1 system, see Table 1.

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Full-Length Methods

Script Name

Reverse Transcription Method

Amplification Method

Analysis Method

Developer

Advantages

C1 SMARTer v1

Poly(A), template switch

PCR

Full-length

Fluidigm

Standard protocol on all C1 systems

C1 SMART Seq2

Poly(A), template switch

PCR

Full-length

Oxford

Generate longer cDNA

Full-length mRNA Sequencing (SMART Seq v4)

Poly(A), template switch

PCR

Full-length

Clontech

New RT and DNA polymerase with greater sensitivity than SMARTer v1

TCR Sequence Determination by TraCeR

Poly(A), template switch

PCR

Full-length

Wellcome Trust Sanger Institute

Provides a more comprehensive view of the T cell repertoire

Table 1. Full-length sequencing protocols available on Script Hub
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Transcript counting methods

Determine relative numbers of transcripts expressed by each cell with quantitative transcript counting for single-cell gene expression. It’s a cost-effective way to rapidly survey cellular heterogeneity and identify novel cell populations. There are two common approaches to transcript counting by RNA sequencing:

  • 3′ end counting: This frequently-used method leverages molecular tags on the polyadenylated region of the gene to detect transcripts differing in either the sequence of 3′ terminal exon or the length of the 3′ untranslated region (UTR) of the transcript. It offers the benefit of separating coding RNA from non-coding RNA during reverse transcription.
  • 5′ end counting: This approach uses tags against the 5′ end of the transcript to identify transcription start sites and promoters. Leveraging the resulting information aids in identifying promoter usage or transcription factor binding sites.

For a complete list of C1 protocols available on Script Hub for transcript counting methods, see Table 2.
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Transcript Counting Methods

Script Name

Reverse Transcription Method

Amplification Method

Analysis Method

Developer

Advantages

C1 mRNA Seq HT

Poly(A), template switch

PCR

3'

Fluidigm

On-IFC cell barcoding simplifies sample handling and reduces library prep and sequencing costs.

C1 STRT Seq

Poly(A), template switch

PCR

5'

Karolinska Institute

Detects long non-coding RNAs and non-poly(A) transcripts

C1 CEL Seq

Poly(A)

in vitro transcription

3'

Technion – Israel Institute of Technology

PCR-based amplification alternative

C1 CAGE

Random priming, template switch

PCR

5'

RIKEN Institute

Detects capped RNA transcripts; users can share data with the FANTOM5 consortium.

Table 2. Transcript counting protocols available on Script Hub
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Maximize the power of your single-cell analysis on the C1 system, with access to the largest collection of single-cell genomics applications in the industry. Use these innovative mRNA sequencing protocols on Script Hub together with Open App™ IFCs to advance your single-cell studies. Join us on the quest to simplify the complex and reveal new insights into the biology of life.