Single Cell RNA-seq (scRNA-seq) Library Structure

Collections of library structure and sequence of popular single cell genomic methods (mainly scRNA-seq).

How to use?

Click the following links to veiw the methods. Notes:

1. the default alignment font is Monaco. Courier New font will be used if Monaco is not available.
2. In a dual-index library, how index2 (i5) is sequenced differs from machines to machines. Miseq and Hiseq2000/2500 use the bottom strand as template, which is why the index sequences are the same as the primer sequences in those machines. MiniSeq, NextSeq and Hiseq3000/4000 use the top strand as template, which is why the index sequences are reverse-complementary to the primer sequences in those machines. All methods listed below use Miseq, Hiseq2000/2500 as examples.

• STRT-seq family (including STRT-seq, STRT-seq-C1, STRT-seq-2i)
• Quartz-seq family (including Quartz-seq and Quartz-seq2)
• SMART-seq family (including SMART-seq, SMART-seq2)
• CEL-seq family (including CEL-seq, CEL-seq2)
• Illumina Bio-Rad SureCell 3' WTA for ddSEQ
• 10x Chromium Single Cell 3' V3 Feature Barcoding
• 10x Chromium Single Cell 3' V2 and V3 GE
• 10x Chromium Single Cell 3' V1 GE
• 10x Chromium Single Cell 5' GE
• SCRB-seq / mcSCRB-seq
• Drop-seq / Seq-Well
• Microwell-seq
• sci-ATAC-seq
• sci-RNA-seq
• Drop-ChIP
• MARS-seq
• SPLiT-seq
• inDrop

Technical comparisons

The basic chemistry is very similar, the main differences among those scRNA-seq methods are summarised in the table below. For a detailed discussion, check the text boxes from our review (open access): From Tissues to Cell Types and Back: Single-Cell Gene Expression Analysis of Tissue Architecture

	Single cell isolation/capture	2nd strand synthesis	Full-length cDNA synthesis	Barcode addition	Pooling before library	Library amplification	Gene coverage
10x Chromium Single Cell 3'	Droplet	TSO	Yes	Barcoded RT primers	Yes	PCR	3'
10x Chromium Single Cell 5'	Droplet	TSO	Yes	Barcoded TSO primers	Yes	PCR	5'
CEL-seq/CEL-seq2	FACS	RNase H and DNA pol I	No	Barcoded RT primers	Yes	In vitro transcription	3'
Drop-seq	Droplet	TSO	Yes	Barcoded RT primers	Yes	PCR	3'
MARS-seq	FACS	RNase H and DNA pol I	No	Barcoded RT primers	Yes	In vitro transcription	3'
Microwell-seq	Nanowells	TSO	Yes	Barcoded RT primers	Yes	PCR	3'
Quartz-seq	FACS	PolyA tailing and primer ligation	Yes	Ligation of barcoded Truseq adapters	No	PCR	3'
Quartz-seq2	FACS	PolyA tailing and primer ligation	Yes	Barcoded RT primers	Yes	PCR	3'
SMART-seq/ SMART-seq2	FACS or Fluidigm C1	TSO	Yes	Library PCR with barcoded primers	No	PCR	full-length
SPLiT-seq	Not needed	TSO	Yes	Ligation of barcoded RT primers	Yes	PCR	3'
STRT-seq	FACS	TSO	Yes	Barcoded TSO primers	Yes	PCR	5'
STRT-seq-C1	Fluidigm C1	TSO	Yes	Barcoded Tn5 transposase	No	PCR	5'
STRT-seq-2i	FACS or dilution	TSO	Yes	Barcoded PCR primers and Tn5 transposase	Yes	PCR	5'
Seq-Well	Nanowells	TSO	Yes	Barcoded RT primers	Yes	PCR	3'
Illumina Bio-Rad SureCell 3' WTA	Droplet	RNase H and DNA pol I	No	Barcoded RT primers	Yes	PCR	3'
inDrop	Droplet	RNase H and DNA pol I	No	Barcoded RT primers	Yes	In vitro transcription	3'
SCRB-seq/ mcSCRB-seq	FACS	TSO	Yes	Barcoded RT primers	Yes	PCR	3'
sci-RNA-seq	Not needed	RNase H and DNA pol I	No	Barcoded RT primers and library PCR with barcoded primers	Yes	PCR	3'

Motivation

I was a little bit bombarded with all the single cell methods and got completely lost. To help myself understand all of them and future troubleshooting, I start to perform an on-paper library preparation whenever I see a new single cell method.

Why bother?

Here I borrow from Feyman:

What I cannot create, I do not understand.

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TODO:

• Waiting for new methods ...

Feedback

I would be very happy if you go through them and let me know what you think. If you spot some errors/mistakes, or I've missed some key methods. Feel free to contact me:

Xi Chen
xc1@sanger.ac.uk