Polyatract mrna isolation system

Cells were cultured at 5% CO2/37°C in a humidified incubator in DMEM (HepG2) or DMEM/F12 (HEK293) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were plated into 12-well plates the day before transfection. Transfection of plasmid DNA was performed using lipofectamine 3,000 (Invitrogen Life Technologies, CA, United States) according to the manufacturer’s instructions. Cells were harvested 48 h after transfection for total RNA preparation or lysed with RIPA buffer for Western blotting. In the CYP2D6 genomic DNA transfection experiments, mRNA was purified from total RNA using the PolyA Tract mRNA isolation system (Promega) to avoid contamination of plasmid DNA. To determine the mRNA stability of the CYP2D6 splice isoforms, HepG2 cells (expressing full-length CYP2D6, CYP2D6∆E3, or CYP2D6∆E6) were treated with actinomycin D (20 μg/mL) for different time periods before harvesting. To test protein stability, CYP2D6, and CYP2D6∆E3 transfected HEK cells were treated with 10 μg/mL cycloheximide for 6 h before harvesting.

Twenty specimens of H. petersii were collected and milked for 2 days by electrical stimulation. Total RNA was prepared from the glands by using TRIzol reagent (Invitrogen). Poly (A) mRNA was purified by using a Poly (A) Tract mRNA isolation system (Promega). The cDNA library was constructed with the Superscript plasmid system cDNA library construction kit (Gibco/BRL). cDNAs were cloned into the pSPORT1 plasmid (Gibco/BRL) and transformed into Escherichia coli DH5α cells. cDNA clones were randomly chosen and sequenced to obtain a reliable representation of the toxin content in the venom gland. Positive clones were identified by using an ABI Prism 377XL DNA sequencer with a universal T7 promoter primer.

Total RNA was isolated from cotton seedlings using a kit (Promega, Madison, WI, United States) according to the manufacturer’s instructions. The PolyATract mRNA Isolation System (Promega) was used to generate polyadenylated mRNA. The cDNA library was prepared as previously described (Wang et al., 2011b (link); Liu et al., 2017 (link)) and propagated on 140 mm plates to obtain about 10⁶ clones. The conserved region of SNAP33 (Heese et al., 2001 (link); Wang et al., 2017 (link)) was used as a probe to screen for positive clones (Liu et al., 2016 (link); Wang et al., 2017 (link)).
The theoretical isoelectric point (pI) and molecular mass were calculated with ProtParam¹. A transmembrane hidden Markov model (TMHMM) analysis of the transmembrane domain was performed using the TMHMM online tool². Multiple amino acid sequence alignment was performed with Clustal Omega³, and the multiple alignment file was shaded with BoxShade⁴. A motif analysis was performed using the National Center for Biotechnology Information (NCBI) conserved domain search program⁵. A phylogenetic tree was constructed with the neighbor-joining method using MEGA 6, with bootstrap values from 1000 replicates indicated at the nodes.