A METHOD FOR DETECTION OF WHOLE TRANSCRIPTOME IN SINGLE CELLS

Abstract

We provide a method to efficiently analyze coding RNA and non-coding RNA at single cell level in the present disclosure. A tag sequence is first added to the 3′ of RNA molecules in a single cell, and the tag sequence is subsequently used to capture said RNA and prime reverse transcription of the RNA to cDNA. The resulting cDNA can be amplified and analyzed. The tag sequence can be combined with a cell barcode sequence to decode the identity of single cells, so that a plurality of single cells can be analyzed in parallel.

Claims

1. A method for analyzing whole transcriptome, including coding and non-coding RNA, at single cell level, wherein said method comprising: a) add a specific tag sequence on the 3′ of RNA; b) capture the tagged RNA with a primer that recognize the tag sequence; c) reverse transcribe the tagged RNA to cDNA; d) amplify cDNA; e) analyze amplified cDNA.

2. The method of claim 1, wherein the RNA is non-coding RNA.

3. The method of claim 1, wherein the primer sequence comprises a sequence that acts as cell barcode that identifies each single cells; a specific sequence that can be used to prime the reverse transcription of the tagged RNA; and a sequence that can be used for amplification of the cDNA.

4. The method of claim 1, wherein the primer sequence comprise a unique molecular index (UMI) sequence that can be used to quantify cDNA.

5. The method of claim 1, wherein the tag sequence is added by using an enzyme.

6. The method of claim 1, wherein the tag sequence is added chemically.

7. The method of claim 5, wherein the enzyme is a Poly(A) Polymerase, to add a stretch of A to the 3′ of RNA.

8. The method of claim 5, wherein the enzyme is a terminal transferase, to add specific nucleotide sequence to the 3′ of RNA.

9. The method of claim 5, wherein the enzyme is a ligase, to add specific sequence to the 3′ of RNA.

10. The method of claim 1, wherein the analysis method is sequencing.

11. A product or kit that includes reagents needed to enable the process as described in claim 1.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0010] FIG. 1: Schematic diagram of the present disclosure.

[0011] FIG. 2: Schematic diagram of the embodiment of the present disclosure where Poly (A) Polymerase is used to add a polyA tail to the ncRNA.

[0012] FIG. 3 shows the percentage of UMI.

DETAILED DESCRIPTION

[0013] To overcome the drawbacks of the current single cell ncRNA analysis methods, we use a ncRNA-tagging method to add specific tag sequences to the ncRNA. The tag sequence can then be used to capture ncRNA molecules, and/or as priming site for RT reactions and amplification reactions (FIG. 1).

[0014] One embodiment of the present disclosure is to use Poly (A) polymerase to add a polyA tail to the ncRNA. Afterwards, oligo-dT can be used to capture and reverse transcribe both mRNA and ncRNA with newly added polyA tails. The resulting cDNA can be amplified by PCR if a template switching oligo is introduced during the RT process. With unique cell barcodes in conjunction with the oligo-dT sequence, cDNA molecules from the same single cell can be labeled and a group of single cells can be processed in parallel, enabling high-throughput single cell analysis (FIG. 2).

[0015] GEXSCOPE Single Cell RNAseq Library Construction kit (Singleron Biotechnologies) was used to demonstrate the technical feasibility and the utility of the present disclosure in massively parallel single cell ncRNA sequencing. The experiment was conducted according to manufacture's instructions with modifications described below.

[0016] Briefly, single cell suspension of K562 cells was loaded onto the microchip to partition single cells into individual wells on the chip. Four samples were prepared: two were processed with standard GEXSCOPE protocol for single cell mRNA sequencing (“control”), two were processed with modified protocol to get ncRNA reads (“nc”). Cell barcoding magnetic beads were then loaded to the microchip and washed. Each cell-barcoding magnetic bead contains oligos with a unique cell barcode sequence combined with oligo-dT on the surface. Each oligo on the bead also has a unique molecule index sequence (UMI); the number of UMIs detected in the sequence can be used to accurately quantify different RNA molecules. Only one bead can fall into each well on the microchip based on the diameters of the beads and well (about 30 um and 40 um, respectively). Instead of the lysis buffer contained in the GEXS COPE kit, the following reaction mixture was use to lyse cells and add polyA tails to the ncRNA molecules. E. coli Poly(A) Polymerase and 10×E. coli Poly(A) Polymerase Reaction Buffe are both from New England Biolabs (NEB).

TABLE-US-00001 Components Volume/Reaction (ul) 10× E. coli Poly(A) Polymerase Reaction Buffer 10 ATP (10 mM) 10 E. coli Poly(A) Polymerase 5 10% Triton 2 RNA inhibitor 2.5 Dnase/Rnase-Free Water 70.5 Total 100 ul

[0017] 100 ul reaction mixture was loaded into the chip and let incubate on ice for 10 minutes to lyse cells. After the cells are lysed, the microchip was incubated at 37° C. for 30 minutes so that PolyA tails can be added to the 3′ end of RNA. After being cooled down at room temperature for 30 minutes, the magnetic beads, together with captured RNAs, were taken out of the microchip and subject to RT, template switching, cDNA amplification, and sequencing library construction using reagents from the GEXSCOPE kit and following manufacturer's instructions. The resulting single cell RNAseq library was sequenced on Illumina NovaSeq with PE150 mode and analyzed with scopeTools bioinformatics workflow (Singleron Biotechnologies).

[0018] As shown in FIG. 3, the percentage of the UMIs corresponding to ncRNA in total UMIs increased more than 100%, from an average of 1.5% to 3.6%. The significantly increased percentage of ncRNA UMIs proves the principle of the present disclosure. Furthermore, the percentage of rRNA UMIs remains relatively low at (0.9%, 0.6%).

REFERENCES

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