RNA-Seq analysis to capture the transcriptome landscape of a single cell

Abstract

We describe here a protocol for digital transcriptome analysis in a single mouse oocyte and blastomere using a deep-sequencing approach. In this method, individual cells are isolated and transferred into lysate buffer by mouth pipette, followed by reverse transcription carried out directly on the whole cell lysate. Free primers are removed by exonuclease I and a poly(A) tail is added to the 3' end of the first-strand cDNAs by terminal deoxynucleotidyl transferase. Single-cell cDNAs are then amplified by 20 + 9 cycles of PCR. The resulting 100-200 ng of amplified cDNAs are used to construct a sequencing library, which can be used for deep sequencing using the SOLiD system. Compared with cDNA microarray techniques, our assay can capture up to 75% more genes expressed in early embryos. This protocol can generate deep-sequencing libraries for 16 single-cell samples within 6 d.

Figure 1. Typical agarose gel electrophoresis for single cell cDNAs after 20 + 9 cycles of PCR and gel purification.

Figure 2. Workflow of the SOLiD System Express Library Preparation for Fragment Libraries and Multiplexed Fragment Libraries.

Figure 3. Electrophoregram of DNA sheared in C4011-10 tubes generated using Bioanalyzer (Agilent Technologies, Inc.)

Figure 4. Standard curve of the cDNAs.

(a) Standard curve. (b) Standard plot. (c) Library plot.

Figure 5. The single ES cells in PBS-BSA drop.

Black arrowheads indicate individual ES cells. Black arrow indicates the tip of micropipette for picking single cells. The inner diameter of the micropipette is about 2 – 3 fold of the diameter of an individual ES cell. Scale bar: 30um.

Figure 6. GAPDH expression in single and ten mouse ES cells measured by real-time PCR.

One or ten ES cells were picked into each tube to amplify cDNAs by 20 cycles of PCR. Then the PCR product was diluted 10 fold (20ul into 200ul) and take 2ul as template for a 20ul real-time PCR reaction.

Figure 7. Pearson coefficient plots for single cell RNA-Seq of one blastomere of a 4-cell stage embryo, two wildtype mature oocytes, two Dicer knockout mature oocytes, and one Ago2 knockout mature oocyte.

Figure 8. Coverage plots of RNA-Seq reads in a single wildtype mature oocyte.