|Public on Sep 17, 2019
|tissue: TE cells
strain: C57BL/6 X F1
development stage: E3.5
genotype: wild type
|For blastocyst, to separate ICM and TE from E3.5 embryos(Liu et al., 2016), blastocysts were broken using micromanipulator and washed three times in HCZB. The zona pellucidae of blastocysts were digested with 0.5% pronase E (Sigma) for 10-15 mins and washed three times again in HCZB. The embryos were then incubated in Ca2+-free CZB for 30 mins in 37 ℃, and the ICM and TE were separated into single cells by gently pipetting using a pipette with a diameter of 40–60 μm. Each sample was obtained about 10 cells from one embryo for amplification according the cell shape. For placenta, total RNA was isolated from mouse placentae using Dynabeads™ mRNA DIRECT™ Purification Kit (Thermo Scientific™).
For blastocyst, the cDNA library were prepared by SPAPK DNA Sample Pre Kit (Enzymatics) for sequencing using an Illumina HiSeq X Ten sequenator. For placenta, RNA-seq library were prepared by VAHTS mRNA-seq V3 Library Prep Kit for Illumina® (Vazyme), and then applied for deep sequencing on Illumina NovaSeq6000 platform.
|HiSeq X Ten
|TE obtained by Intracytoplasmic wt androgenetic haploid ESC injection
|RNA-seq reads were mapped to the mm9 reference genome using Tophat (version 2.1.1). The gene expression levels were calculated by Cufflinks (version 2.1.1). The expression levels for each transcript were quantified as Fragments Per Kilobase of transcript per Million mapped reads (FPKM).
Supplementary_files_format_and_content: Tab-delimited txt files for RNA seq including FPKM values for each gene
|Jun 05, 2019
|Last update date
|Sep 18, 2019
|Chinese Academy of Science
|institute of biochemistry and cell biology
|Jinsong Li's Lab
|320 Yueyang Road
|RNA Seq analyzed the transcriptional difference between the ICSI and ICAHCI embryo in blastocyst and placenta