"SMART-Seq Ultra Low Input: Single cell sequencing (단일세포 염기서열 분석) 논문리스트 보기"
* 더 높은 효율을 보유한 업그레이드 제품, SMARTer Ultra Low Input RNA Kit for Sequencing - v3 사용을 추천합니다.
SMARTer Ultra Low RNA Kit for Illumina® Sequencing-HV은 매우 소량의 total RNA(10pg-10 ng)나 소량의 세포(1-1000 cells)를 이용하여 곧바로 transcriptome 분석(RNA-seq)에 이용할 수 있는 library를 제작 할 수 있는 cDNA합성이 가능하다. 합성된 cDNA는 Nextera® DNA sample preparation kit (using our modified protocol) 또는 ThruPLEX® DNA-seq Kit (Code R400523)를 이용하여 Illumina's Genome Analyzer™, HiSeq®, 또는 MiSeq® instrument에 적용 할 수 있는 library를 제작 할 수 있다.
SMARTer Ultra Low RNA Kit for Illumina® Sequencing-HV은 oligo dT priming 방법을 이용하여 high quality RNA (RIN >8)나 intact cells을 이용한다. 기존의 SMARTer Ultra Low RNA Kit for Illumina Sequencing(maximum input volume=1 μl) 에서 input volume이 1-9 ul로 modify된 프로토콜을 이용한다. 변형된 input volume은 희석된 샘플이나 large volume에 들어있는 sorted cell을 이용할 때 유용하다.
그림1. Overview of SMARTer Ultra Low RNA Kit sample preparation for Illumina sequencing
특징
- Sample prep made easy - Use cells directly or total RNA. Single-tube protocol works directly on cells to preserve sample integrity
- Unparalleled sensitivity - Start with as little as 1 cell or 10 pg of total RNA (input range: 1-1,000 cells or 10 pg to 10 ng of total RNA)
- Integration with Illumina sequencing - Compatible with Illumina's NGS platforms
- Excellent RNA-seq data - Whole transcriptome information with full length gene analysis, minimal 5' or 3' bias, less than 2% rRNA reads, broadest coverage of exons, and ERCC correlation when compared to other technologies.
그림2. Electropherogram of amplified SMARTer cDNA. Various amounts of Universal Human Reference Total RNA (UHR) and Human Brain Reference RNA were used as input for SMARTer cDNA synthesis.The cDNA samples were then analyzed for purity and yield on an Agilent 2100 Bioanalyzer. Shown are Bioanalyzer trace overlays of cDNA amplified from 1 ng (red line), 0.1 ng (dark blue line), 0.05 ng (green line), and 0.01 ng (light blue line) of total RNA and a no template control (NTC; pink line). The main peak indicates the purity and yield of cDNA between 0.4 and 9 kb with the highest point at ~2 kb. There was no amplification in the negative control (pink line). Although the amount of input RNA can vary over quite a large range (e.g., 1 ng to 0.01 ng), comparable cDNA output can be obtained by adjusting the number of PCR cycles.
그림 3. Gene expression data obtained from very low amounts of RNA correlate well with data obtained by qPCR. Scatter plots were used to compare differential expression data obtained by sequencing with the SMARTer Ultra Low RNA Kit (1 ng total RNA) and quantitative PCR (qPCR) data available for Universal Human Reference Total RNA (UHR) and Human Brain Reference RNA through the MicroArray Quality Control (MAQC) project. The differential expression of ~700 genes showed correlation values of 0.94, demonstrating that the sequencing results are consistent with orthogonal gene expression technologies.
그림 4. Comparison of transcript coverage with different amounts of input RNA. Shown are overlaid plots comparing the average read coverage from libraries made with 1 ng to 0.01 ng of mouse brain total RNA. The x-axis represents gene length normalized to 100%, where 0 is the 5'-end of each transcript and 100 is the 3'-end. The y-axis indicates the average coverage for a set of 724 genes that are moderately to highly expressed in brain tissue. The results are very consistent through the range of input RNA used, with full-length coverage of the transcripts reflecting no systematic 5'- or 3'-bias.
구성품 (자세한 내용은 CoA를 참조하세요)
* SMARTer Ultra Low Input RNA for Illumina Sequencing Components - HV
* Advantage 2 PCR Kit (Cat. No. 639207)
