| Series | GSE44618 |
| Title | Single Cell RNA-Seq |
|---|---|
| Year | 2013 |
| Country | USA |
| Article | Wold BJ,Myers RM,Gertz J,Schroth GP,McCue K,Williams BA,Marinov GK.From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing.Genome research.2014 Mar |
| PMID | 24299736 |
| Bio Project | BioProject: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA190661 |
| Sra | SRA: http://www.ncbi.nlm.nih.gov/sra?term=SRP018838 |
| Overall Desgin | Single GM12878 cells were picked and RNA-seq libraries were generated using the SMART-seq protocol. We also carried out RNA-seq experiments on pools of 10, 30 and 100 cells, on 100pg and 10ng of total RNA, and on pools of 10 cells that were subsequently split into 10 separate sample and processed as if they were single cells in order to assess technical variation in our experiments. |
| Summary | RNA-seq transcriptome measurements are typically performed by isolating RNA from large numbers of cells in culture or tissues. While highly informative, such experiments mask the variability in gene expression patterns that exists between individual cells. To gain insight into the dynamics of gene expression on the level of single-cells, we have carried out the transcriptomes of single-cells from the GM12878 cell line using RNA-seq. |
| Experimental Protocol | Single GM12878 cells were picked from a culture dish using a micropipet. They were deposited into cell lysis solution (Clontech SMARTer Ultra Low RNA kit) and frozen at -80C. Amplified cDNA was then prepared following the manufacturer's protocol. The amplified cDNA was fragmented and tagged using the Nextera Illumina Genomic DNA prep kit, according to the manufacturer's protocol. The fragmented cDNA libraries were then sequenced on the Illumina HiSeq 2000 instrument. |
| Data processing | Libraries were pooled and sequenced on the Illumina HiSeq or GAIIx. Single-end reads of 100bp length were obtained (excluding the barcode sequence). Sequencing reads were demultiplexed based on barcode sequences and aligned against the male or female hg19 version of the human genome (depending on the sex of the cell line or donor) with TopHat 1.4.1 and the GENCODE V13 annotation using the --GTF option and with de novo junction discovery turned on. Only the first read was used for the few libraries that were sequenced as paired end. Libraries were pooled and sequenced on the Illumina HiSeq or GAIIx. Reads of length 100 bp were obtained (excluding the barcode sequence). Sequencing reads were demultiplexed based on barcode sequence. Reads were mapped using TopHat 1.4.1 and the GENCODE V13 annotation (using the --GTF option in TopHat). Quantification was carried out using Cufflinks 2.0.2 |
| Platform | GPL11154 |
| Public On | Public on Nov 21 2013 |