NextSeq 500 paired end sequencing; GSM1970642: WT male 4 cell, repeat 1 [Xist KO]; Mus musculus; RNA-Seq

WT male 4 cell, repeat 1 [Xist KO]

Purpose: The goals of this study are to establish a dynamic roadmap of imprinted X chromosome inactivation and the role of Xist by elucidation of the transcriptome of Xist KO embryos during mouse preimplantation development. Methods: mRNA profiles of the preimplantation embryos WT and KO for Xist were elucidated by RNA-seq at various stages. Trophoblasts isolated from blastocyst outgrowths were also included. The sequence reads for samples where gender could be determined and that passed quality filters were analyzed at the level of autosomes, X chromosome as well as single genes. Results: Female embryos fail to silence the X chromosome at late preimplantation development. General autosomal gene expression is not affected in embryos lacking Xist. Conclusions: Xist is crucial for iXCI. In preimplantation embryos, the main in vivo function of Xist is to regulate iXCI in females. Overall design: Global mRNA profiles of single preimplantation embryos were generated by deep sequencing.

Transcriptome of mouse preimplantation development [Xist KO]

Submitted by Gene Expression Omnibus on 12-OCT-2016

Whole embryos were dissected at the indicated timepoints and the correct stage was verified under the binocular. Single embryos/trophoblast cells were placed in 10ul TCL Buffer (Qiagen) supplemented with 1% BME, and snap frozen. RNA libraries were prepared for sequencing using standard Illumina protocols. The embryo, trophoblast or mock samples were distributed on two 96-well plates and thawed at RT for 10 minutes prior to RNA purification using Ampure RNA beads (Beckmann-Coulter). RNA samples were re-suspended in solution containing 3' RT primer and dNTPs. Reverse transcription was performed with SSII (Life Technologies), whose terminal transferase activity allows incorporation of a PCR binding site at the 3' end of the cDNA using a template-switching oligonucleotide (custom synthesized from Exiqon) as a template. Subsequently, cDNA was amplified using 12 cycles of PCR, followed by tagmentation with Nextera kit (Illumina). Final libraries were amplified by 12 cycles of PCR and sequenced on a NextSeq 500.

Transcriptome of mouse preimplantation development [Xist KO]

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