Rneasy plus micro kit 50

Total RNA was extracted from the ALDH^hiCD44⁺ and ALDH^lowCD44⁺ cells using an RNeasy Plus Micro kit 50 (Qiagen, Hilden, Germany). The specific primer sequences were as follows: Forward, 5′-ACCCAGAAGACTGTGGATGG-3′ and reverse, 5′-CCACCCTGTTGCTGTAGCC-3′ for GAPDH; forward, 5′-CGCCTTTGTGCTTCTGTTCTTC-3′ and reverse, 5′-CCCCACTCATTCTGGTTGTCG-3′ for Notch1; and forward, 5′-CAATCCAACAGTAGCCTTTATCAG-3′ and reverse, 5′-GCCAAGTGGGTGGTATAGAGG-3′ for β-catenin. The conditions used were 40 cycles of 30°C for 10 min, 42°C for 15 min, 9°C for 5 min and 5°C for 5 min, GAPDH was selected as the reference gene and the specificity of the PCR products was analyzed using dissociation curves. The relative RNA expression values of the transcripts were calculated using the cycle threshold (Ct) and were defined using the following equations: Relative RNA expression = 2^−ΔCt if ΔCt = Ct1 − Ct0, where Ct1 is the objective gene and Ct0 is GAPDH.

Total RNA was isolated from 30 pooled oocytes and 20 pooled embryos followed by synthesis of cDNA using the RNeasy Plus Micro Kit (50) (QIAGEN; 74,034). A random 6 mer (Takara Bio Inc.; RR037A) was used as the first strand primer. Armoured RNA control provided from Cells-to-cDNA II Kit (Thermo Fisher Scientific; AM1723) as an external control was spiked into the cell lysate containing total RNA samples before the RT reaction. Half of the cell lysate without the RT enzyme was used as a negative control. cDNA was synthesised using the Superscript III RT First-Strand system (Life Technologies; 18,080,051). Following the RT reaction, the prepared cDNA samples were amplified and analysed using PCR. The primers used are described in Table S4. Amplifications were run in a Thermal Cycler Dice TP600 (Takara Bio Inc.) and Takara Taq Hot start version (Takara Bio Inc.; R007A). The reaction parameters were as follows: one cycle at 98 °C for 2 min and 40 cycles for Armored RNA control or 26 cycles for MuERV-L at 98 °C for 10 s, 58 °C for 30 s, and 72 °C for 25 s. The RT-PCR products were electrophoresed on 2% agarose gels with TBE and visualised using Midori Green Advanced (1:10,000; NIPPON Genetics Co., Ltd; NE-MG04). Three independent experiments were performed for each.

RNA was extracted using TRI reagent®. RNA samples underwent clean-up using the RNeasy Plus Micro Kit (50) (Qiagen, USA) following the manufacturer’s instructions. Samples were prepared for sequencing by the AGRF and 100 bp single end reads were obtained using the Illumina NovaSeq 6000 platform (Illumina, San Diego, USA). Image analysis was conducted in real time by the NovaSeq Control Software (v1.6.0) and Real Time Analysis (v3.4.4) which performs base calling. The Illumina bcl2fastq 2.20.0.422 pipeline was used to generate sequence data.
Data processing and bioinformatics analysis were also performed by the AGRF. The RNA-seq expression analysis workflow included alignment against the human reference genome version hg38 using STAR aligner (v2.5.3a). This was followed by transcript assembly using stringtie tool (v1.3.3) then quantification and Trimmed Mean of M-values (TMM) normalization. Differential expression analysis was performed using edgeR (v3.22.3) and R (v3.5.0).