| Series | GSE53386 |
| Title | Single-cell RNA-Seq transcriptome analysis of circular RNAs in mouse embryos |
|---|---|
| Year | 2015 |
| Country | China |
| Article | Not set |
| PMID | NA |
| Bio Project | BioProject: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA231896 |
| Sra | SRA: http://www.ncbi.nlm.nih.gov/sra?term=SRP034543 |
| Overall Desgin | 3 Single mouse ES cells, 7 single HEK293T cells, 8 diluted mouse ES total RNA samples and 19 mouse embryo samples are performed using SUPeR-seq to evaluate its ability to detect genes in single RNAs and to cover poly(A)- RNA species. |
| Summary | We reported a new method of single-cell universal poly(A)-independent RNA sequencing (SUPeR-seq) to obtain single cell whole transcriptome analysis that covers both poly(A) plus and poly(A) minus RNA species. We built libraries of single mouse ESC , 10pg, 100pg and 1ng diluted mouse ES total RNA, single HEK293T cell and mouse early embryos before 4-cell stage. We analyzed these data using bioinformatics and find a good coverage of non-polyadenylated RNAs, circular RNA for example. to make a comparison,we also used mouse ES cells and HEK293T cells to build libraries using the protocol publish by F.Tang in 2009(sample name Tang2009_*). We also de novo assembled new genes in mouse ESCs and early embryo samples, further validated known and novel zygotic genes with a-Aminatine treated 2-cell embryos(sample name SUPeR-seq_a-AM_treated_2-cell*). |
| Experimental Protocol | Single cells were picked into the lysis buffer which contained Rnase inhibitor to prevent RNA degration; Cells were picked into the lysis buffer to release all RNAs; then the released RNAs were reverse transcribed with SuperScript III(Invitrogen), then digested the unreacted primers with ExoSAP-IT(USB) and tailed the first strand cDNA with a poly(A) sequence with terminal deoxynucleotidyl transferase (TdT, Invitrogen) , then used poly(T) primers to synthesize the sencond strand cDNA.the cDNAs were then enriched by two rounds of PCR amplifications each with size-selection by 2% agarose gel recovery then the library construction waa conducted following the illumina library preparing protocol. The quality-ensured libraries were used for pair-end deep sequencing on Illumina HiSeq2000 and Illumina HiSeq2500 Sequencers. Single cells were picked into the lysis buffer which contained Rnase inhibitor to prevent RNA degration; Cells were picked into the lysis buffer to release all RNAs; then the released RNAs were reverse transcribed with SuperScript III(Invitrogen), then digested the unreacted primers with ExoSAP-IT(USB) and tailed the first strand cDNA with a poly(A) sequence with terminal deoxynucleotidyl transferase (TdT, Invitrogen) , then used poly(T) primers to synthesize the sencond strand cDNA.the cDNAs were then enriched by two rounds of PCR amplifications each with size-selection by 2% agarose gel recovery.then the library construction waa conducted following the illumina library preparing protocol. The quality-ensured libraries were used for pair-end deep sequencing on Illumina HiSeq Sequencer. |
| Data processing | Illumina CASAVA version 1.8 were used to the basecalling.; Reads were trimmed to remove the adapter sequences and low quality bases.; RNA-seq reads were aligned to the reference genome using BWA software; gene expression level was estimated using comstomized Perl scripts.; Genome_build: mm10 and hg19; Supplementary_files_format_and_content: txt files reflect RPKM of each gene in Refseq. Illumina CASAVA version 1.8 were used to the basecalling.; Reads were trimmed to remove the adapter sequences and low quality bases.; RNA-seq reads were aligned to the mouse refference genome (mm10) using BWA software; gene expression level was estimated using comstomized Perl scripts.; Genome_build: mm10; Supplementary_files_format_and_content: txt files reflect FPKM of each gene in Refseq. Illumina CASAVA version 1.8 were used to the basecalling.; Reads were trimmed to remove the adapter sequences and low quality bases.; RNA-seq reads were aligned to the mouse refference genome (mm10) using BWA software; gene expression level was estimated using comstomized Perl scripts.; Genome_build: mm10 and hg19; Supplementary_files_format_and_content: txt files reflect FPKM of each gene in Refseq. Illumina CASAVA version 1.8 were used to the basecalling.; Reads were trimmed to remove the adapter sequences and low quality bases.; RNA-seq reads were aligned to the mouse refference genome (mm10) using BWA software; gene expression level was estimated using comstomized Perl scripts.; Genome_build: mm10 and hg19; Supplementary_files_format_and_content: txt files reflect RPKM of each gene in Refseq. |
| Platform | GPL11154;GPL13112;GPL16791;GPL17021 |
| Public On | Public on Jun 16 2015 |