Chamq sybr color qpcr master mix without rox

Two ecological types of S. alfredii were treated by CdCl₂ with different concentrations (HE: 10 and 100 μM, NHE: 10 μM) and times (0, 6, 24 h, 3, and 7 days). The roots, stems, and leaves of each plant were separated and frozen rapidly in liquid nitrogen. The total RNA was extracted using a Spin Column Plant Total RNA Purification Kit (Sangon) and then synthesized to cDNA with a HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) (Vazyme). Real-time quantitative PCR (RT-qPCR) was performed using a ChamQ SYBR Color qPCR Master Mix (Without ROX) (Vazyme), with LightCycler 480 System (Roche, United States). The RT-qPCR protocol was as follows: 95°C for 3 min, 40 cycles of 95°C for 10 s, 60°C for 30 s. The melting-curve analysis was included to verify the specificity of the primer. The mean amplification efficiency was analyzed with the LinReg software (Ruijter et al., 2009 (link)). The specific primers for RT-qPCR were designed according to the SaPCR2 sequence as follows: SaPCR2 forward 5′-GCGGTGGGATGTGGTCTAC-3′ and SaPCR2 reverse 5′-CGATAATCTCGGCTATTTGGC-3′, SaACTIN1 forward 5′-TGTGCTTTCCCTCTATGCC-3′, and reverse: 5′-CGCTCAGCAGTGGTTGTG-3′ (Chao et al., 2010 (link)). The relative expression levels were calculated using 2^−ΔCt method.

Pregnant mare serum gonadotropin (PMSG, 110914564) and human chorionic gonadotropin (hCG, 110911282) were purchased from San-sheng Biotechnology. Hyauronidase (H4272), Cytochalasin B (CB, C6762), HTF medium (MR-070-D), M2 medium (M7167), bovine albumin (BSA, A7030), KSOM medium (MR-101-D) and NP-40 (I3021) were obtained from Sigma-Aldrich. TRIzol (15596–026) was purchased from Invitrogen. Protein A magnetic beads (88845) and mMESSAGE mMACHINE™ T7 Transcription Kit (AM1344) were obtained from Thermo Fisher Scientific. AMPure XP beads (A63881) were purchased from Beckman. RNasin, TruePrep® DNA Library Prep Kit V2 for illumina Kit (TD501) and ChamQ SYBR Color qPCR Master Mix (Without ROX) (Q321) were purchased from Vazyme. RNA Clean Beads (N243-01B), T7 High Yield RNA Transcription Kit (E131) and NLS-Cas9 Nuclease (E365-01A) were purchased from Novoprotein. KAPA HiFi HotStart ReadyMix (KK2601) was obtained from Roche. Primary antibodies were used against: m⁶A modification (ab15320, abcam), Ythdc1 (14392–1-AP, Proteintech), β-Actin (A5441, Sigma-Aldrich). Actinomycin D (ActD, HY-17559) was obtained from MedChemExpress.