Geneace sybr qpcr mix

Total RNA (200–300 ng) was isolated using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and subjected to reverse transcription with random hexamer and oligo dT primers using PrimeScript RT Reagent kit (TAKARA BIO, Japan). qPCR was performed using GeneAce SYBR qPCR Mix (NIPPON GENE, Japan) and Thermal Cycler Dice Real Time System (TAKARA BIO, Japan). The value of gene expression was calculated by delta-delta Ct method and normalized for that of GAPDH mRNA. Primer sequences used for qPCR are listed in Supplementary Table S2.

A ChIP assay was performed using the wild-type and bam⁸⁶ mutant ovaries as described (Baxley et al., 2011 (link)). Immunoprecipitation was performed using 1 µg of antibody. As a control, normal mouse IgG (Jackson ImmunoResearch Laboratories) was used. Anti-H3K36me3, anti-H3K4me3 (Kimura et al., 2008 (link)), and anti-RNA polymerase II (8WG16; Covance) antibodies were used for the ChIP assays. Input DNA, mock-precipitated DNA, and DNA from the ChIP assays were analyzed by PCR. Quantitative PCR analyses were performed using GeneAce SYBR qPCR Mix (Nippon Gene). The sequences of the primers used for the ChIP assays are listed in supplementary material Table S1.

For monoculture or 1:1 mixed culture of RasV12 cells with MDCK or MDCK ADAMDEC1-shRNA1 cells, total 2 × 10⁷ cells were seeded on collagen-coated 145-mm dishes. Total RNAs were extracted from isolated cells with TRIzol™ (Thermo Fisher Scientific), purified using RNeasy Mini Kit (QIAGEN), and reverse transcribed using QuantiTect Reverse Transcription Kit (QIAGEN). GeneAce SYBR qPCR Mix (NIPPON GENE) was used to perform qPCR with the StepOnePlus™ system (Thermo Fisher Scientific). In brief, PCR amplification of the targeted fragments was performed with 45 cycles of denaturation at 95 °C (30 s), annealing and extension at 60 °C (30 s). The primer sequences were as fellows. ADAMDEC1: 5′-TGCCGTTCACATTTTGAGAGATAC-3′ and 5′-CCTCACAGCAAGGATTGGAAC-3′; GAPDH: 5′-ATTCTATCCACGGCAAATCC-3′ and 5′-GGACTCCACAACATACTCAG-3′; β-actin: 5′-GGCACCCAGCACAATGAAG-3′ and 5′- ACAGTGAGGCCAGGATGGAG-3′. For data analysis, relative quantification analysis was performed using the comparative CT (2^−∆∆CT) method. For each sample, mRNA levels of ADAMDEC1 were normalized to the GAPDH mRNA or β-actin mRNA. The details of calculation processes are demonstrated in Table S2. We have measured the amplification efficiency of each qPCR primer and confirmed that the amplification efficiency is comparable between the three primers (90–100%).