Conserved roles for murine mDUX and human DUX4 in activating cleavage stage genes and MERVL/HERVL retrotransposons [RNA-Seq Mouse siRNA]. Mus musculus

To better understand transcriptional regulation during human oogenesis and pre-implantation embryonic development, we defined stage-specific transcription, which revealed cleavage stage as highly distinctive. We present multiple lines of evidence that two cleavage-specific homologs, mouse mDUX and human DUX4, each activate hundreds of cleavage-specific endogenous genes (e.g. ZSCAN4, ZFP352, KDM4E) and retroviral elements (MERVL/HERVL-family). Remarkably, mDux expression converts mouse ESCs into two-cell embryo-like (2C-like) cells by binding to MERVL promoters/enhancers and restoring the chromatin landscape (via ATACseq) to the pattern of mouse two-cell embryos Overall design: We used siRNAs to transiently deplete mESCs of Chaf1a alone and in combination with mouse Dux (mDux). After two consecuitive days of depletion, RNA was collected (2 biological replicates/condition) for sequencing. 125bp libraries were prepped and sequenced in paired-end format. All sequencing was done on the Illumina HiSeq 2500 platform. Chaf1a silencer select siRNA (and negative control siRNA) was purchased from Life Technologies (s77588). mDux siRNAs (si308 and si309) were synthesized as siRNA pools using Giardia Dicer as described below. Briefly, primers were designed to amplify two ~400bp fragments of the endogenous mDux locus from genomic mouse DNA and add T7 handles. mDux (si308) 5'-AACTCCTCCTCCTTGATCAACTG -3', 5'-CTTCTCTCTGTGGCCAAAAGC-3'. mDux (si309) 5'-CTTCTGCAGAGAGTCCCAGAC-3', 5'-GGCAGATCAGGTGTTGTGTC-3'. Purified PCR products were then used as template for in vitro transcription using the MEGAscript® T7 Transcription Kit (ThermoFischer, AM1334). Template DNA was then degraded and the ssRNA was allowed to anneal before dicing. Diced siRNAs were purified using the PureLinkTM Micro-to-Midi Total RNA purification Kit (Invitrogen, 12183-018) with modifications. siRNA concentration was measured with the Qubit® RNA HS Assay Kit (ThermoFisher, Q32852).