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Status |
Public on Jul 24, 2018 |
Title |
HE8W_CTB0_sc6 |
Sample type |
SRA |
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Source name |
Cytotrophoblast of Week 8
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Organism |
Homo sapiens |
Characteristics |
cell type: Cytotrophoblast cell population: HLA-G-/CDH1+ Stage: Week 8
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Treatment protocol |
human placenta tissues were harvested from the hospital put into DMEM supplemented with 5% FBS. the tissue was transferred to the lab on ice within 1 h
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Extracted molecule |
total RNA |
Extraction protocol |
Chrion were scraped from human placenta and the remaining villi were collected and minced into small pieces. The tissue were digested with an enzyme cocktail (0.125% trypsin 0.04% Dnase and 0.05% type Ⅳcollagenase) for 8 min twice at 37 ℃. Cell suspension were collected by filtering the ezyme mixture with 70 μm cell strainer and the enzyme reaction were stopped by addition of 5% DMEM. cells were centrifuged at 1200 g at 4 ℃ on a percoll gradient (7 layers: 10%, 20%, 30%, 40%, 50%, 60% and 70%) and cells from 30% - 50% percoll gradient were collected and washed with DMEM. EVT and CTB were sorted with MACS with PE-conjucted HLA-G and CDH1 antibody combined with anti-PE beads,STB were purified with mouth pipette based on their big size from the cell population and the remaning HLA-G and CDH1 double negative cells were stromal cells. For isolation of EVT from the basal plate of the 24 w placenta, basal plate were scaped from the placenta, minced and digested with the method as described above,finally 24 w EVT were purified by MACs with HLA-G antibody. the single cells were picked into 2ul cell lysis buffer using mouse pipet, then the transcriptome was amplified by modified smart-seq2 protocol which added in barcode for each cell. the amplified cDNAs were sheared into 300bp fragments, the 3' end of the cDNAs were enriched to build library with KAPA Hyper Prep Kits for Illumina
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 4000 |
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Description |
mRNA with polyA tail
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Data processing |
For data from Illumina, Illumina CASAVA version 1.8 were used to the basecalling Read 2 was used to obtain the cell barcodes to further split the reads according to the cell IDs (barcode) and the same time recorded the UMI sequences Read 1 was picked in each cell and these raw reads were trimmed to remove TSO or polyA sequence Adaptor contamination and low-quality reads were discarded from the trimmed Read 1 raw data TopHat(version 2.0.14) with default settings were used for sequence alignment and uniquely mapped reads were kept. Genome_build: Reference sequence and transcript annotation files were from UCSC genome assembly hg19 Supplementary_files_format_and_content: TPM (transcripts-per-million mapped reads) data was calculated as the total UMIs for a genes to be divided by the sum of UMIs from each cell and then multiply by 1,000,000
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Submission date |
Nov 03, 2016 |
Last update date |
May 15, 2019 |
Contact name |
Rui WANG |
E-mail(s) |
fish_cat_wr@sina.cn
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Phone |
15801166445
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Organization name |
Peking University
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Department |
Biodynamics Optical Imaging Center (BIOPIC)
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Lab |
Fuchou Tang
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Street address |
No.5 Yiheyuan Road, Haidian District
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City |
Beijing |
ZIP/Postal code |
100871 |
Country |
China |
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Platform ID |
GPL20301 |
Series (1) |
GSE89497 |
Single-cell RNA-seq reveals the diversity of trophoblast subtypes and patterns of differentiation in the human placenta |
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Relations |
BioSample |
SAMN05977670 |
SRA |
SRX2324978 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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