RNA and Transcription DNA RNA PROTEIN. RNA and Transcription.
A DEVICE FOR SEPARAT ING DNA AND RNA IN 250 CELLS IN … · 2016-04-11 · INTRODUCTION Sequencing...
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A DEVICE FOR SEPARATING DNA AND RNA IN 250 CELLS IN PREPARATION FOR NEXT GENERATION SEQUENCING Gordon D. Hoople* §1 , Andrew Richards §1 , Kun Zhang § , Albert P. Pisano § *University of California, Berkeley, United States § University of California, San Diego, United States 1 Equal contributors to this work ABSTRACT This manuscript reports on a new device that separates DNA and RNA from 250 cells into separate wells. This physical separation is the necessary first step towards sequencing both the genome and tran- scriptome from the same group of cells. A polyacrylamide gel with regions of different densities has been fabricated such that during electrophoresis genomic DNA is stopped in one well while RNA travels through the gel to a separate well. This is the first report of separating DNA and RNA from 250 cells in preparation for next generation sequencing. KEYWORDS: Electrophoresis, Next Generation Sequencing, Polyacrylamide, DNA, RNA INTRODUCTION Sequencing both DNA and RNA from the same cell populations will allow geneticists to examine how variations in DNA impact RNA expression. Current genomic sequencing tools require a minimum of 1 ng (~150 cells) of starting material [1], therefore amplification is required before sequencing small cell populations. Unfortunately, low input amplification and sequencing protocols for DNA and RNA, such as MIDAS [2] and Quartz-Seq [3], are incompatible. One solution to this problem is to physically separate DNA and RNA. Once separated, it becomes possible to utilize these protocols in parallel and create se- quencing libraries that will provide information about both the genome and transcriptome from the same cell population. This paper presents a new device that physically separates DNA and RNA from 250 cells into separate wells. A novel fabrication approach for creating a polyacrylamide device with a high density region (40%T 5%C) and a low density region (8%T 3%C) is described. Separation of DNA and RNA is shown at the 1000, 500, and 250 cell level. Other approaches to solve this problem are also under development. Dey et al. have developed a pro- tocol for simultaneously amplifying and sequencing DNA and RNA from the same single cell [4]. Their approach, however, relies on computational exon masking to distinguish DNA from RNA. The major drawback of this approach is that it requires a priori knowledge of the genome that is not always possible. Macaulay et al. have developed G&T-Seq, a meth- od for separating, amplifying, and sequencing DNA and RNA from the same single cell [5]. This approach separates RNA from genomic DNA by capturing RNA on a magnetic bead using a bioti- nylated oligo-dT primer. Their approach is excel- lent for single cells, but for small cell populations a polyacrylamide based solution may prove more scalable. THEORY The device, shown in Figure 1, separates DNA and RNA by taking advantage of the large size dif- ference between chromosomal DNA and RNA. Chromosomal DNA strands are on the order of 10 7 Figure 1. A device for separating DNA and RNA. The device consists of polyacrylamide gels with two differ- ent densities fabricated on a glass slide. 1957 978-0-9798064-8-3/μTAS 2015/$20©15CBMS-0001 19 th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 25-29, 2015, Gyeongju, KOREA
Transcript of A DEVICE FOR SEPARAT ING DNA AND RNA IN 250 CELLS IN … · 2016-04-11 · INTRODUCTION Sequencing...
A DEVICE FOR SEPARATING DNA AND RNA IN 250 CELLS IN PREPARATION FOR NEXT GENERATION SEQUENCING Gordon D. Hoople*§1, Andrew Richards§1, Kun Zhang§, Albert P. Pisano§
*University of California, Berkeley, United States §University of California, San Diego, United States
1Equal contributors to this work ABSTRACT
This manuscript reports on a new device that separates DNA and RNA from 250 cells into separate wells. This physical separation is the necessary first step towards sequencing both the genome and tran-scriptome from the same group of cells. A polyacrylamide gel with regions of different densities has been fabricated such that during electrophoresis genomic DNA is stopped in one well while RNA travels through the gel to a separate well. This is the first report of separating DNA and RNA from 250 cells in preparation for next generation sequencing. KEYWORDS: Electrophoresis, Next Generation Sequencing, Polyacrylamide, DNA, RNA
INTRODUCTION
Sequencing both DNA and RNA from the same cell populations will allow geneticists to examine how variations in DNA impact RNA expression. Current genomic sequencing tools require a minimum of 1 ng (~150 cells) of starting material [1], therefore amplification is required before sequencing small cell populations. Unfortunately, low input amplification and sequencing protocols for DNA and RNA, such as MIDAS [2] and Quartz-Seq [3], are incompatible. One solution to this problem is to physically separate DNA and RNA. Once separated, it becomes possible to utilize these protocols in parallel and create se-quencing libraries that will provide information about both the genome and transcriptome from the same cell population.
This paper presents a new device that physically separates DNA and RNA from 250 cells into separate wells. A novel fabrication approach for creating a polyacrylamide device with a high density region (40%T 5%C) and a low density region (8%T 3%C) is described. Separation of DNA and RNA is shown at the 1000, 500, and 250 cell level.
Other approaches to solve this problem are also under development. Dey et al. have developed a pro-tocol for simultaneously amplifying and sequencing DNA and RNA from the same single cell [4]. Their approach, however, relies on computational exon masking to distinguish DNA from RNA. The major drawback of this approach is that it requires a priori knowledge of the genome that is not always possible. Macaulay et al. have developed G&T-Seq, a meth-od for separating, amplifying, and sequencing DNA and RNA from the same single cell [5]. This approach separates RNA from genomic DNA by capturing RNA on a magnetic bead using a bioti-nylated oligo-dT primer. Their approach is excel-lent for single cells, but for small cell populations a polyacrylamide based solution may prove more scalable.
THEORY
The device, shown in Figure 1, separates DNA and RNA by taking advantage of the large size dif-ference between chromosomal DNA and RNA. Chromosomal DNA strands are on the order of 107
Figure 1. A device for separating DNA and RNA. The device consists of polyacrylamide gels with two differ-ent densities fabricated on a glass slide.
1957978-0-9798064-8-3/µTAS 2015/$20©15CBMS-0001 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 25-29, 2015, Gyeongju, KOREA
base pairs long, while RNA is on the order of 103 nucleotides in length. This large discrepan-cy in size can be exploited to physically sepa-rate these molecules using polyacrylamide. The device consists of two distinct regions: a high density (40%T, 5%C) and a low density (8%T, 3%C) polyacrylamide gel (PAG). These gels are bonded to a microscope slide for conven-ient imaging, but could be designed for any form factor.
Cells are lysed in tube and the lysate pipet-ted into the loading wells. Once an electric field is applied, the DNA and RNA experience an electrophoretic force and begin to migrate. The DNA molecules do not migrate far. They
are too large to enter the low density gel and so remain trapped in the loading well. The shorter RNA transcripts, on the other hand, are able to enter the low density region of the gel. These RNA transcripts eventually pass through the low density gel to the capture well where they become trapped by the high density gel. EXPERIMENTAL
Fabricating the polyacrylamide device is accomplished through a combination of photolithography and molding and is summarized in Figure 2. The process begins by treating a glass slide with 3-(Trimethoxysilyl) propyl methacrylate (Sigma Aldrich M6514). This silane treatment is necessary for the creation of a covalent bond between the PAG and glass slide. Next a 250µm SU-8 mold is created that de-fines the negative space of the high density PAG region. The mold is treated with Glass Free (National Diagnostics EC-621) to improve separation of the mold from PAG.
The treated glass slide is positioned 300µm above the SU-8 mold using a latex spacer. The mold cavi-ty is filled with a polyacrylamide monomer solution (40%T, 5%C) containing the photo-initiator VA-086 (1% w/V). The entire assembly is exposed to UV light (13 mW/cm2) for 45 seconds, resulting in rapid polymerization of the high density PAG region. (The approach for curing PAG using UV light has been adapted from Duncombe and Herr [6].) Once polymerized, the glass slide and high density PAG are sepa-rated from the SU-8 mold.
The process is then repeated using a new 250µm SU-8 mold to define the loading and capture wells. For the second molding process, the high density PAG serves as the spacer to suspend the glass slide above the SU-8 mold. The mold cavity is flooded with low density PAG solution (8%T, 3%C, 1% w/v VA-086) and cured with UV light (13 mW/cm2) for 20 seconds. The final device, shown previously in Figure 1, can then be separated from the SU-8 mold.
To demonstrate the separation of DNA and RNA, HEK293a cells were lysed in tube at a concentration of 1000 cells/µL and serially diluted to concentrations of 500 cells/µL and 250 cells/µL. 1µL of these so-lutions was then loaded into the various loading wells of the device. An RNA ladder 100-2000 base pairs in length was used as a reference to show RNA mobility through the low density gel. Electrophoretic mi-gration was induced by applying an electric field of 30 V/cm for 20 minutes. After electrophoresis, nucle-ic acids were fluorescently stained and then imaged, appearing in Figure 3 as crisp black bands. RESULTS AND DISCUSSION The results are most easily understood by first examining the two empty wells on the right side of Figure 3. These wells show the background level of fluorescence. This can be compared to the lane loaded with RNA ladder, which shows one black band at the edge of the capture well. The presence of a single black band in this lane suggests that the RNA has passed through the low density gel and been captured only at the edge of the capture well. In comparison, the lanes loaded with cell lysate show nucleic acid has been
Figure 2. The device is fabricated using a combination of molding and photolithography.
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captured in both the loading and capture wells. This suggests that the long genomic DNA has been trapped in the loading well while the shorter RNA transcripts are able to pass through the low density gel to the capture well. This result indicates the viability of separating DNA and RNA in small populations of cells using high and low density polyacrylamide gels.
Figure 3: A fluorescent image of the device shows the separation of genomic DNA and RNA in as low as 250 cells. False color has been added to the regions of low and high density polyacrylamide gel. The DNA and RNA show up as crisp black bands at the edges of the wells. CONCLUSION This manuscript has presented a new device for electrophoretic separations of DNA and RNA in small cell populations. The device takes advantage of the large size difference between chromosomal DNA and RNA. A novel fabrication approach utilizing a combination of molding and photolithography to define regions of high and low density polyacrylamide has been presented. A separation was performed on HEK293a cells that demonstrated the successful separation of DNA and RNA in as little as 250 cells. This separation is the first step in preparing these samples for next generation sequencing. ACKNOWLEDGEMENTS
The authors would like to acknowledge the National Science Foundation and the Korean Ministry of Science, ICT and Future Planning for providing funding for this work. They would also like to thank the members of the Zhang and Pisano labs for their contributions to this project.
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“Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells,” Na-ture Biotechnology, vol. 31, no. 12, pp. 1126–1132, Nov. 2013.
[3] Y. Sasagawa, I. Nikaido, T. Hayashi, H. Danno, K. D. Uno, T. Imai, and H. R. Ueda, “Quartz-Seq: a highly re-producible and sensitive single-cell RNA sequencing method, reveals non-genetic gene-expression heterogenei-ty,” Genome Biol, vol. 14, no. 4, p. R31, 2013.
[4] S. S. Dey, L. Kester, B. Spanjaard, M. Bienko, and A. van Oudenaarden, “Integrated genome and transcriptome sequencing of the same cell,” Nature Biotechnology, pp. 1–7, Jan. 2015.
[5] I. C. Macaulay, W. Haerty, P. Kumar, Y. I. Li, T. X. Hu, M. J. Teng, M. Goolam, N. Saurat, P. Coupland, L. M. Shirley, M. Smith, N. Van der Aa, R. Banerjee, P. D. Ellis, M. A. Quail, H. P. Swerdlow, M. Zernicka-Goetz, F. J. Livesey, C. P. Ponting, and T. Voet, “G&T-seq: parallel sequencing of single-cell genomes and transcrip-tomes,” Nat Meth, vol. 12, no. 6, pp. 519–522, Apr. 2015.
[6] T. A. Duncombe and A. E. Herr, “Photopatterned free-standing polyacrylamide gels for microfluidic protein electrophoresis,” Lab Chip, vol. 13, no. 11, p. 2115, 2013.
CONTACT * Gordon Hoople; [email protected]
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