Protoscript 2 cdna first strand kit

Total RNA was extracted from 10 ovaries from female mice between 4 and 6 weeks of age using TRIzol following the manufacturer’s protocol. First-strand cDNA was synthesized using a ProtoScript II cDNA first strand kit (NEB) with 1 µg of total RNA. The full-length and truncated coding sequences of mouse Mad2l1bp were amplified and cloned into the p3xFLAG-CMV-24 vector. cDNA fragment encoding mouse Mad2 was PCR-amplified and inserted into the pcDNA3.1 vector with a Myc tag. Human MAD2L1BP coding sequence were synthesized from Genewiz and cloned into the pcDNA3.1 vector. All construct sequences were verified by Sanger sequencing.

Total RNA was extracted from 10 ovaries from female mice between 4–6 weeks old using Trizol following manufacturer's protocol. First‐strand cDNA was synthesized using ProtoScript II cDNA first strand kit (NEB) with 1 µg of total RNA. The full‐length coding sequence (CDS) for mouse wild‐type Zp1 (NM_009580.2), Zp2 (NM_011775.7), Zp3 (NM_011776.1), and mutant Zp1 (Zp1Mut,M1‐Q248) was amplified by high‐fidelity PCR enzyme (NEB) using mouse ovary cDNA as templates. PCR products were gel purified and in‐frame cloned into p3xFLAG‐myc‐CMV^TM‐24 vector through double enzyme digestion (Not I & Bgl II) for wild‐type Zp1 and mZp1Mut (M1‐Q248). CDS for mouse Zp2 and Zp3 was in‐frame cloned into a modified pcDNA3.1 vector with myc‐6His tag and 6His tag, respectively, via homologous recombination. Primers were listed in Table 3. All final constructs were eventually examined by Sanger sequencing to ensure mutation free.

RNA was reverse transcribed with the ProtoScript II First Strand cDNA Kit (New England Biolabs). Quantitative realtime PCR was performed with a CFX Connect Real-Time PCR Detection system (Bio-Rad Laboratories) and the Luna Universal qPCR Master Mix (New England Biolabs) in technical triplicates with 20 µl reaction volume and 2 µl of a 1:10 dilution of the cDNA template. Gapdh was used as reference gene for mouse qPCR and eef1a1l was used for zebrafish qPCR. For primers used, see electronic supplementary material Information.
For data analysis, the dCq values were calculated by subtracting Cq[_{target gene}] from Cq[_{reference gene}], and ddCq values were calculated by subtracting the averaged dCq[_control] from the simple dCq[_treatment/KO] [78 (link)]. These ddCq values are presented as log2(fold change). p-Values were determined by two-tailed, unpaired t-test, except for the data presented in figure 1f. There we used a one-tailed test because we knew to expect downregulation from the microarray dataset.
In figure 5a, the expression data for grem2b derives from two runs with the same samples (biological replicates) in the same qPCR cycler. For this, an average dCq value was calculated for each biological replicate.