概要: The study of early post-implantation human embryo development is limited by the scarcity of embryo samples and ethical restrictions. We developed a human pluripotent stem cell (hPSC)-based microfluidic platform to model several developmental processes during this stage. We applied single-cell mRNA sequencing technique to examine the transcriptomes of posteriorized embryonic-like sacs obtained using the microfluidic platform, as well as of amniotic ectoderm-like cells generated using a Transwell method and human embryonic stem cells (H9 hESCs).
项目整体设计: Cells retrieved from posteriorized embryonic-like sacs were pooled from 6 microfluidic devices, after 48 hours of exogenous BMP4 stimulation. Amniotic ectoderm-like cells were also obtained by treating H9 hESCs with BMP4 for 48 hours using a Transwell method. H9 hESCs maintained using standard tissue culture dishes were also sequenced for comparison.
Posteriorized embryonic-like sacs (48 hours) within the microfluidic devices, amniotic-like cells derived using transwell and H9 cells were dissociated into single cells by incubating in Accutase for 1 hour. Cells from posteriorized embryonic-like sacs were lysed and barcoded with 10x Chromium Controller Instrument (10x Genomics). Amniotic-like cells derived using transwell and H9 cells were mixed at a 2:1 ratio, before lysed and barcoded with 10x Chromium Controller Instrument.
建库方案:
cDNA library was constructed according to the standard 10x Genomics scRNA-seq library construction protocol (Single Cell 3' v2). The libraries were sequenced on Illumina HiSeq 4000 with paired-end sequencing (One sample per flow cell).
测序信息
分子类型:
poly(A)+ RNA
库的片段类型:
PAIRED
库的链类型:
Forward
测序平台:
ILLUMINA
测序仪型号:
Illumina HiSeq 4000
链特异性:
Specific
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文章
Controlled modelling of human epiblast and amnion development using stem cells.
