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| Status |
Public on Jun 22, 2019 |
| Title |
I_E8.25_embryo_No.2_1 |
| Sample type |
SRA |
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| Source name |
heart
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| Organism |
Mus musculus |
| Characteristics |
embryonic stage: E8.25
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| Growth protocol |
Mice were housed under a 12-12-hour light-dark cycle and allowed standard laboratory take care.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Single-cell RNA-seq libraries were prepared as described previsouly but with some modifications of Smart-seq2 protocol(Li et.al, Cell Stem Cell, 2017). In short, cardiac cells sorted by FACS or just trypsin digestion are picked into sc-RNA lysis buffer by mouth pipette. Then reverse transcription was performed using oligo(dT) primer which carrying a 8-nt specific barcode and a 8-nt random sequence as unique molecular identifiers (UMIs).
The second stand was synthesized using TSO oligo. After 17 cycle of PCR reaction, all synthesized cDNA was pooled together. Additional 5 cycles of PCR reaction was performed using a biotinylated primer introducing a library specific index. Then cDNA was sheared by Covaris S2 and gain average 400bp fragments and 3’ terminal fragments of the cDNA were captured by Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher). Last, RNA-seq library was constructed by NEBNext Ultra DNA Library Prep Kit for Illumina(E7370) following the manufacture’s guide. All the Single-cell RNA-seq library are subjected to 150 bp paired-end sequencing on an Illumina High seq X ten platform.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
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| Description |
polyA RNA
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| Data processing |
Basecalls performed using CASAVA version 1.8
We used cell-specific barcode sequences attached in read 2 to separate raw reads, and then we trimmed reads by removing the template switch oligo (TSO) and polyA tail sequence.
The clean reads were aligned to the mouse genome (mm10 from UCSC) with TopHat (version 2.0.12). We used HTseq to count the unique mapped reads that were grouped by cell-specific barcode
We calculated the numbers of UMIs of each gene divided by all UMIs and then multiplied by 1,000,000, which were used to quantify the gene expression level as TPM (transcript per million)
Genome_build: mm10
Supplementary_files_format_and_content: txt files which include the TPM values of RefSeq genes.
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| Submission date |
Jan 08, 2018 |
| Last update date |
Jun 22, 2019 |
| Contact name |
Haiqing Xiong |
| E-mail(s) |
haiqingxiong@pku.edu.cn
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| Organization name |
Peking University
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| Street address |
5 Yiheyuan Road, Haidian District
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| City |
Beijing |
| State/province |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
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| Platform ID |
GPL21273 |
| Series (2) |
| GSE108879 |
Single-cell transcriptomic analysis reveals lineage hierarcies and communications for second heart lineage deployment [RNA-seq] |
| GSE108963 |
Single-cell transcriptomic analysis reveals lineage hierarcies and communications for second heart lineage deployment |
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| Relations |
| BioSample |
SAMN08326859 |
| SRA |
SRX3541026 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2915352_I_E8.25_1_gene_expression.txt.gz |
1.5 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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