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Transcriptome analysis of human blastocysts with different levels of chromosomal mosaicism after preimplantation genetic testing.

ABSTRACT: An inner cell mass biopsy and the remaining trophectoderm were separately collected from 14 blastocysts with preimplantation genetic testing result into RNAse-free PCR tubes containing 2 µl of 10X reaction buffer (SMART-Seq v4 Ultra-Low Input RNA kit for Sequencing, Takara Bio, USA). cDNA was obtained from mRNA with 3′SMART-Seq CDS primer II (Takara Bio, USA) and PCR-amplified (17 cycles) from 10 pg of RNA. Amplified cDNA was purified using AMPure XP magnetic beads (Illumina, USA). After confirming cDNA integrity on a 2100 Bioanalyzer (Agilent Technologies, USA), 1 ng of cDNA per sample were fragmented, and libraries were constructed using NexteraXT DNA sample preparation (Illumina, USA). Samples were quantified using Qubit dsDNA Quantitation Assay (Thermo Fisher Scientific, USA), and 3 samples were excluded due to poor cDNA quality. An RNA pool was generated with 25 samples using equal concentration (5 nM) of RNA per sample. Sequencing was performed in duplicate in a single run using an Illumina NovaSeq 6000 S1 platform (Illumina, USA) with a 200-nucleotide read length in a paired-end design (100-bp fragments). FastQC was used for checking the quality of the raw sequence data. Fragments that did not meet quality requirements were trimmed using Trimmomatic. Alignment and quantification were performed using the Salmon algorithm (reference genome GRCh38). Raw counts were directly used for differential gene expression analysis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE194306 | GEO | 2022/01/31

REPOSITORIES: GEO

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