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| Status |
Public on Sep 09, 2019 |
| Title |
CS11_CH_10x |
| Sample type |
SRA |
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| Source name |
human embryonic cells
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Caudal Half
development stage: CS11
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
A modified STRT-seq protocol was applied for single-cell RNA-seq. Briefly, sorted single cells in good condition were picked into lysis by mouth pipetting, and the scRNA-seq libraries were constructed based on STRT-seq with some modifications. cDNAs were synthesized using sample-specific 25-nt oligo-dT primer containing 8-nt barcode (TCAGACGTGTGCTCTTCCGATCT-XXXXXXXX-DDDDDDDD-T25, X representing sample-specific barcode while D standing for unique molecular identifiers (UMI). After reverse transcription and second-strand cDNA synthesis, the cDNAs were amplified by 16 cycles of PCR using ISPCR primer and 3’ Anchor primer. Samples were pooled and purified using Agencourt AMPure XP beads . 4 cycles of PCR were performed to introduce index sequence and subsequently, 400 ng cDNAs were fragmented to around 300 bp by Covaris S2. After being incubated with Dynabeads MyOneTM Streptavidin C1 beads (Thermo Fisher, 65002) for 1 hour at room temperature, cDNA Libraries were generated using KAPA Hyper Prep Kit (Kapa Biosystems, kk8505). After adaptor ligation, the libraries were amplified by 8 cycles of PCR using QP2 primer and short universal primer. The libraries were sequenced on Illumina Hiseq X Ten platform in 150 bp pair-ended manner
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
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| Data processing |
10X Genomics CellRanger version 2.1 were used to the basecalling.
Sequencing data from 10X genomics was processed with CellRanger software package (version 2.1.0) with default mapping arguments. For modified STRT-seq data, raw reads were first split for each cell by specific barcode sequence attached in Read 2. The TSO sequence and polyA tail sequence were trimmed for the corresponding Read 1 after UMI information was aligned to it. Reads with adapter contaminants or low-quality bases (N > 10%) were discarded. Subsequently, we aligned the stripped Read 1 sequences to hg19 human transcriptome (UCSC) using Hisat2 (version 2.10). Uniquely mapped reads were counted by HTSeq package83 and grouped by the cell-specific barcodes. Duplicated transcripts were removed based on the UMI information for each gene. Finally, for each individual cell, the copy number of transcripts of a given gene was the number of the distinct UMIs of that gene.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include UMI values for each cell
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| Submission date |
Jul 31, 2019 |
| Last update date |
Sep 09, 2019 |
| Contact name |
Jian He |
| E-mail(s) |
jianhe.amms@gmail.com
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| Organization name |
the Fifth Medical Center of the PLA General Hospital
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| Street address |
DongDaJie #8
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| City |
Beijing |
| ZIP/Postal code |
100071 |
| Country |
China |
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| Platform ID |
GPL20795 |
| Series (1) |
| GSE135202 |
Tracing the first hematopoietic stem cell generation in human embryo by single-cell RNA sequencing |
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| Relations |
| BioSample |
SAMN12415849 |
| SRA |
SRX6628630 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3993421_CS11_CH_rawdata.txt.gz |
15.5 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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