GSM5737868: Wild-type Mouse Blastocyst 4; Mus musculus; RNA-Seq
In total, 19 early (E3.25) stage blastocysts (8 Upf2-/-, 11 Upf2+/+) were subjected to mRNA sequencing. As a means of normalization, ERCC Spike-In RNAs (Life Technologies, Cat.no. 4456740) were added to the samples. The ERCC stock provided by the manufacturer was diluted 1:25,000 and 0.25 µl of the dilution was added to each embryo sample before cell lysis and RNA-seq library preparation, and ERCC reads were analyzed downstream in parallel with reads from the embryo transcriptome. RNA isolation and cDNA synthesis were carried out using the SMARTer® Ultra® Low Input RNA Kit for Sequencing – v4 (Clontech, Cat.no. 634888). Genotyping of cDNA was performed using custom primers. In brief, since loxP sites surround exons 2 and 3 of the Upf2 gene, the wild-type locus was detected using forward and reverse primers in exons 1 and 2, respectively; the knock-out locus was detected using forward and reverse primers in exons 1 and 4, respectively. Libraries were generated from the resulting cDNA (0.2ng/ul per sample) of wild-type and Upf2-null embryos using the Nextera XT DNA library preparation kit (Illumina, Cat.no. FC-131-1024) as previously described (Mora-Castilla et al., 2016). Indexed sequence libraries were pooled for multiplexing, normalized by MiSeq read number, and paired-end sequencing was performed on a HiSeq 4000.