Transcriptaid t7 high yield transcription kit

The HOTTIP plasmid were transcribed and biotin-labeled in vitro with Bio-16-UTP (Life Technologies) using a TranscriptAid T7 High Yield Transcription Kit (Life Technologies). Protein–RNA interactions were carried out using a Pierce Magnetic RNA-Protein Pull-Down Kit (Life Technologies) with the lysates of chondrocytes. Then, the retrieved proteins were detected by western blot analysis or resolved by in-gradient gel electrophoresis followed by mass spectrometry (MS) identification (Xu et al., 2017 (link)).

T7 transcription reactions (60 μl final volume) using the TranscriptAid T7 high yield transcription kit (catalogue #K0441; Thermo Scientific, Thermo Fisher Scientific, USA), were set up as follows: 1.5 μl RNase Inhibitor (60 U; catalogue #M0314L; New England Biolabs), 24 μl NTP mix (final concentration of 10 mM each of ATP, CTP, GTP and UTP), 12 μl 5X TranscriptAid reaction buffer (catalogue #K0441; Thermo Scientific, Thermo Fisher Scientific, USA), 6 μl dithiothreitol (DTT; 0.1 M solution; catalogue #707265ML; Thermo Scientific, Thermo Fisher Scientific, USA), 6 μl TranscriptAid enzyme mix (catalogue #K0441; Thermo Scientific, Thermo Fisher Scientific, USA), 3 μl 2^nd T7 promoter sequence oligonucleotide (1 μM stock solution), 6.3 μl of the ligation product, and 1.2 μl DFHBI-1T (10 μM final concentration). Reactions were mixed gently via pipetting. Three 15 μl aliquots of each reaction were loaded into 384-well plates (catalogue #781096, Greiner Bio-One, Kremsmünster, Austria) and measured using a CLARIOstar plate reader (BMG, Ortenberg, Germany) with the following settings: Excitation 440–15 nm/Emission 510–20 nm, orbital shaking for 5 seconds at 500 rpm before measurement every 5 minutes. Delta (Δ) fluorescence measurements (a.u.) per hour were calculated between 20 and 80 minutes of the reactions.

RNA pull‐down assay was conducted according to the previous reports.
³⁰ (link) Briefly, the LINC01128 overexpression vector was designed by Sangon Biotech to serve as a template to amplify the LINC01128‐corresponding antisense and sense strands using the T7 promoter‐containing primers. Biotinylated LINC01128 was synthesised by the TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific), which was subsequently incubated with streptavidin‐coated magnetic beads. The RNA pull‐down assay was conducted using the Magnetic RNA‐Protein Pull‐Down Kit (Pierce 20164, Thermo Fisher Scientific). Subsequently, the precipitated proteins were employed for silver staining, western blotting and mass spectrometry. The RNA–protein interaction probabilities generated by http://pridb.gdcb.iastate.edu/RPISeq/about.php (RNA–Protein Interaction Prediction, RPISeq) ranged from 0 to 1. Reaction probabilities were represented as values of random forest (RF) and support vector machine (SVM), and only those values where both RF >.5 and SVM >.5 were identified as ‘positive’, displaying that the possible interaction of protein with RNA.

The double-strand RNA (dsRNA) was synthesized using a Transcript Aid T7 High Yield Transcription Kit (Thermo Scientific, Wilmington, DE, United States) according to the manufacturer’s instructions (Supplementary Table S1). The dsRNA was diluted with Nuclease-free water to a final concentration of 5,000 ng/μL.
Newly emerged green and red form adults (≤12 h old) from the NY strain were used in the RNAi experiments. The dsRNA injection was accomplished as in a previous study (Ye et al., 2019 (link)). Briefly, 600 ng of dsGGPPS and dsGFP (negative control) were injected into the aphids using an M3301 micromanipulator (World Precision Instruments, Sarasota, FL, United States). In each treatment, 25 aphids were injected as one biological replicate and 4 biological replicates were completed. After injection, the aphids were moved to broad bean leaves. After 36 h, in each biological replicate, 5 aphids were collected to detect gene expression levels and 20 aphids were collected for analyzing carotenoids content.