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Sample GSM2374075

Query DataSets for GSM2374075

Status

Public on Jul 24, 2018

Title

HE24W_EVT5_sc15

Sample type

SRA

Source name

Extravillous trophoblast of Week 24

Organism

Homo sapiens

Characteristics

cell type: Extravillous trophoblast
cell population: HLA-G+
Stage: Week 24

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

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

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

mRNA with polyA tail

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

Submission date

Nov 03, 2016

Last update date

May 15, 2019

Contact name

Rui WANG

E-mail(s)

fish_cat_wr@sina.cn

Phone

15801166445

Organization name

Peking University