RNA Sequencing from Single Cell

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RNA sequencing from single cell

Find out how to go from a single to NGS ready library in ~5 hrs

https://www.qiagen.com/us/resources/knowledge-area/single-cell-resource/repli-g-rna-sequencing/


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Complete cell-to-library solution

PCR-free workflow

Primary sample isolation

Single cell isolation

NGS library constructionSample

• Single eukaryotic cell• Picogram leves of purified RNA

• Whole genome NGS library◦ Illumina-compatible◦ Transcript discovery◦ Gene expression ◦ Differential expression

InsightNGS run Data analysis Interpretation

QIAseq FX Single Cell RNA Library Kit

Single cell genomics by QIAGEN, 2016

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For single cell RNA sequencing

Ideally suited for• Sensitive transcript discovery and differential gene

expression analysis from single eukaryotic cells• High frequency detection of sequence polymorphisms• Analyzing both mRNA and long non-coding RNAs in a

single dataset• Studying intercellular heterogeneity• RNA-seq from limited amounts of difficult-to-obtain samples• Researching infectious diseases

QIAseq FX Single Cell

RNA Library Kit



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QIAseq FX Single Cell RNA Library Kit: the contents

Both kits contain:• Cell lysis and gDNA degradation reagents

• Reverse transcription buffers, primers and enzymes

• Enzymes and buffers for cDNA amplification and fragmentation

• Single step NGS library prep

• Single-use, disposable Illumina adapters in 96-well format

• Multiple reagent aliquots to reduce contamination risk and freeze-thaw cycles

What’s not included:• AMPure XP beads for library purification

• PCR reagents for library amplification are not needed as the entire workflow is PCR-free

• qPCR reagents for library quantification: recommended for accurate flow-cell loading

Cat No./ID: 180733QIAseq FX Single Cell RNA Library Kit (24)

Cat No./ID: 180735QIAseq FX Single Cell RNA Library Kit (96)


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Discover QIAseq FX Single Cell RNA Library Kit

• More diverse libraries◦ WTA technology with fewer dropouts and less length-bias ◦ Highly efficient library preparation◦ PCR-free workflow eliminates PCR duplicates

• Highest sequence fidelity◦ High-fidelity WTA minimizes spurious sequence errors; ideal for viral RNA sequencing

• Maximized transcript discovery◦ More transcripts with the same sequencing depth ◦ Sequence both mRNAs and lncRNAs using the same protocol

• Robust & streamlined workflow◦ Everything needed in one package, single use adaptors cut down on contamination possibilities◦ 5.5 hours workflow from single cell to library without any additional kits

• Enables bio-banking◦ Amplified cDNA can be stored for follow-up studies or confirmatory testing.

MDA* instead of

PCR

Innovative QIAseq FX technology

Complete cell-to-library

solution

*MDA = multiple displacement amplification


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QIAseq FX Single Cell RNA Library Kit

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NGS library preparation for single cell RNA sequencing of eukaryotic cells. Ideally suited for differential gene expression, sequence polymorphism analysis.

• Cell-to-Library PCR-free workflow, robust & streamlined workflow. In 5.5 hours hours from single cell to library, no additional kits

• Sensitive transcript discovery - WTA technology with high accuracy

• No PCR duplicates - Highly diverse libraries

Maximized transcript discovery

mRNA & lncRNA in same datasetHigh diversity libraries

Reads / cell

100 pg RNA

Single cellsSingle cells


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QIAseq FX Single Cell RNA Library Kit: streamlined workflow

Cell Lysis• Start with single eukaryotic cells or small amounts

(pg or ng) of high quality, purified RNA

• Starting with 7 µl cell material in PBS (included)

• Prepare lysis buffer, mix with cells, incubate for 8 minutes

• Cool to 4°C

• Add gDNA degradation reagent, incubate for 10 min

Cell lysis15 min

WTA3 h 45 min

FX library preparation

70 min

Purification20 min

ILLUMINA sequencing

5h 30 min with ~1 h hands-on time


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Whole Transcriptome Amplification• Prepare RT mastermix, mix with lysed cells, incubate 1 h

• Prepare cDNA ligation mix, incubate for 30 min

• Prepare cDNA amplification mix, mix with unamplified cDNA, incubate for 2 h

◦ Amplified cDNA can be used directly or frozen until needed

◦ There will be an excess of amplified cDNA, which can be stored for later use or follow-up studies (e.g. confirming deletions detected with NGS via PCR or sanger sequencing).

◦ Library preparation accepts a wide range of inputs. So quantification of the amplified cDNA is generally not necessary.

Cell lysis15 min

WTA3 h 45 min


70 min

Purification20 min

ILLUMINA sequencing



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NGS Library Preparation• Prepare FX mastermix, add to diluted WTA

product and incubate for ~15 min. Insert size can be set by user

• Hold at 4°C

• Add adapters from single-use adapter plate

• Prepare ligation mastermix, add to samples and incubate for 15 min to produce library

Cell lysis15 min

WTA3 h 45 min


70 min

Purification20 min

ILLUMINA sequencing



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Library Purification• Remove excess adapters with double-sided

Ampure XP cutoff

◦ No PCR amplification necessary; protocol generates sufficient library without enrichment

◦ Library quantification via qPCR (i.e. QIAseq Library Quant) is highly recommended to ensure accurate clustering on sequencer

Cell lysis15 min

WTA3 h 45 min


70 min

Purification20 min

ILLUMINA sequencing



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Discover more: libraries with higher diversity

The QIAseq FX Single Cell RNA Library Kit detects a greater number of transcripts than competitor C/I at the same sequencing depth. To account for cell-to-cell differences in transcript abundance, libraries were produced from 100 pg of reference RNA from PBMCs. After sequencing, quality control and mapping, annotated transcripts with >1 TPM were quantified from either the full dataset or rarified sub-fractions. Saturation curves are from different sample preparation methods. Each point on the curve was generated by randomly selecting a number of raw reads from each sample library and then using the same alignment pipeline to call genes with mean TPM>1.

Discovery plot


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Discover more: highly diverse libraries from single cells

Libraries were generated from single isolated PBMCs with the QIAseq FX Single Cell RNA Library Kit. After QC and mapping, the number of annotated transcripts with >1 TPM was computed. Data were then rarified repeatedly, where a subset of reads was selected at random, and the number of annotated transcripts with >1 TPM was computed for each sub-sampled set of reads. This was repeated at multiple read depths.

Discovery plot

Reads/cell


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Discover more: detect high numbers of transcripts

Libraries were generated from three individually isolated HeLa cells or from 100 pg of bulk RNA isolated from HeLa cells using the QIAseq FX Single Cell RNA Library Kit. After QC and mapping, the number of annotated transcripts with >1 FPKM was computed.

Number of transcripts


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PCR-free library preparation yields highly diverse datasets

Single cell RNA libraries from PBMCs were generated using the QIAseq FX Single Cell RNA Library Kit or a kit from supplier C/I. Libraries were sequenced on Illumina NextSeq. Plotted are the percentage of duplicates that were obtained from the FastQC report of the sequenced libraries.

Note: While true PCR duplicates cannot be produced with the PCR-free QIAseq protocol, some duplicates will still be counted using this calculation method. This is completely due to the high abundance of transcripts and probability, which dictates that at higher transcript abundance the chance of two reads being counted as a duplicate increases (using this calculation method).

Level of duplicates


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Uncover lncRNAs and mRNAs with a single protocol

Single cell RNA libraries from PBMCs and HeLa Cells were generated using QIAseq FX Single Cell RNA Library kit or a kit from supplier C/I. Plotted are the percentage of reads that map to lncRNAs detected in PBMC and HeLa preparations. QIAseq detects a significantly higher percentage of lncRNAs compared to supplier C/I.

Percentage of mapped reads that are lncRNAs


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High proportion of reads map to protein-coding genes

Single cell libraries were prepared from PBMCs or total RNA from PBMCs using the QIAseq FX Single Cell RNA Library Kit and sequenced on NextSeq. Plotted is the percentage of reads that map to different RNA biotypes.

RNA biotypes


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Consistent, robust cDNA amplification enables bio-banking

Whole transcriptome amplification yield from single cells using the QIAseq FX Single Cell RNA Library Kit or a competing, PCR-based method (Supplier C). Eight replicates from different single cells are shown.

Reproducible cDNA yield


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High reproducibility

Four individual HeLa cells were isolated from the same cell culture and libraries were prepared with either the QIAseq FX Single Cell RNA Library Kit or a competing workflow. After sequencing to equal depth, quality control, alignment and TPKM calculation, pairwise comparisons of the number of common transcripts detected with >1 FPKM divided by sum of all transcripts in both preps were made between all tested cells. The data shown represent mean of 6 pairwise comparisons with SD. This graph demonstrates that the more sensitive transcript detection and the capturing of both lncRNAs and mRNAs does not come at the cost of reproducibility, and that this method is at least as reproducible as other workflows.


Download product profile

https://www.qiagen.com/us/resources/resourcedetail?id=56f6a3f5-64fd-448f-a263-9207137c1367&lang=en

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Single cell genomics by QIAGEN

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For in-depth, molecular analysis of single cells

Single cell WGA or WTA

Single cell isolation

Single cell sequencing

• Easily access single cells

• Affordable

• Gentle on cells

QIAscoutQIAseq

FX Single Cell Library Kits

REPLI-g Single Cell Kits

Single cell miRNA analysis

• Create superior-quality NGS libraries directly from single cells

• Provides best-in-class amplification of genomes or transcriptomes from single cells

• Profile miRNA expression using qPCR

miScript Single Cell qPCR Kit


Visit the single cell resource center

https://www.qiagen.com/us/products/life-science-research/single-cell-resource/

RNA Sequencing from Single Cell

Healthcare

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