9902 veriti 96 well thermal cycler

Total RNA was isolated from 50 mg plant tissue with a TRI reagent according to the manufacturer’s instructions (T9424, Sigma-Aldrich, St. Louis., MO, USA). Then, 0.5 μg RNA was used for first-strand cDNA synthesis using the EasyScript ^®One-Step gDNA Removal and cDNA Synthesis SuperMix (TRAN, Beijing, CN). Next, cDNA was used as a template to amplify full length cDNA of GhLETM1 (Gh_A13G094600) using the following primers: forward 5′-CACCATGGCTTCAAGAGTGATCTTGCGAA-3′; reverse 5′-CTACGATCTTCCTGCTTCAGCAGTG-3′. In addition, the specific unique fragment of GhLETM1 was amplified using the following primers: forward 5′-CACCGTTGTGTGACTGGCTGGATTTATCT-3′; reverse 5′-AATTCCAACTTTCTCCTCCTTTCTG-3′. RT-PCR was performed using an ABI 9902 (Applied Biosystems, Veriti^® 96-Well Thermal Cycler 9902), and the PCR program was as follows: 95 °C for 6 min, followed by 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s and finally 72 °C for 2 min. Then, amplified full length cDNA and the unique fragments were cloned and sequenced for OE (Overexpression) and RNAi (RNA interference) construction, respectively.

The CDS sequence of the cotton phosphorus transporter family genes was downloaded from the public CottonGen or the NCBI database according to the GeneBank ID. Supplemental Table S1 listed the IDs of phosphorus transporter family genes and primers.
According to the manufacturer’s instructions (T9424, Sigma-Aldrich, USA), total RNA was isolated from 50 mg plant tissues with TRI reagents. For first-strand cDNA synthesis, the EasyScript^® One-Step gDNA Removal and cDNA Synthesis SuperMix (TRAN, Beijing, CN) was used. RT-PCR was performed using an ABI 9902 (Applied Biosystems, Veriti® 96-Well Thermal Cycler 9902). The PCR program was as follows: 95 °C with 6 min, 35 cycles of 95 °C with 30 s, 60 °C with 30 s, 72 °C with 30 s, and a final step of 72 °C with 2 min. RT-qPCR was performed with UltraSYBR Mixture (Low ROX) using an Applied Biosystems 7500 System (Thermo Fisher, USA). The PCR program was as follows: 95 °C with 10 min, 40 cycles of 95 °C with 15 s and 60 °C with 1 min. Melting curve analysis (95 °C with 15 s, 60 °C with 1 min, 95 °C with 15 s and 60 °C with 15 s) was used for determination of the amplified PCR products specificity. The results were standardized with GhUB7 (DQ116411) of G. hirsutum as internal control^{31 (link)}. Quantitative PCR analysis was repeated at least twice in triplicate for each gene. The relative expression level was determined by 2^−∆∆Ct analysis.

The CP genes of Acanthamoeba were cloned from the cDNA of trophozoites using the primers listed in the Additional file 1: Table S1 by polymerase chain reaction (PCR). PCR was performed in a 9902 Veriti 96-well Thermal Cycler (Applied Biosystems, USA) (94 °C for 3 min; 35 cycles of 94 °C for 15 s, 55 °C for 30 s and 72 °C for 1 min; followed by 72 °C for 7 min). The amplified PCR products were purified and ligated into a pMD19-T vector (Takara, Japan), and the nucleotide sequences were obtained by automated sequencing.
A.castellanii mRNA was extracted from trophozoites and cysts using RNeasy^® Plus Mini Kit (Qiagen, Hilden, Germany), and cDNA was synthesised using a PrimeScript^® 1st strand cDNA synthesis kit (Takara, Japan). Quantitative real-time PCR (qRT-PCR) was carried out in a final reaction volume of 20 μL according to the manufacturer’s recommendations on an ABI 7500 Real-time PCR system (Applied Biosystems, USA). Reactions were performed in a 96-well plate with TB Green Premix Ex Taq II (Takara, Japan) to analyse the expression level of AcCPs. The primers for AcCPs genes and the GAPDH internal reference are listed in Additional file 1: Table S2. The amplification cycling conditions were as follows: 30 s at 95 °C and 40 cycles of 5 s at 95 °C and 35 s at 60 °C. Each experiment was performed at least three times.