4.8. RNA Affinity Purification Sequencing

The RNA affinity purification sequencing (RAP sequencing) was performed with reference to the DNA affinity purification sequencing (DAP sequencing) protocol as described previously [6], but with modifications. In brief, the full-length PeGRP2 was ligated into the pFN19K (HaloTag) T7 SP6 Flexi vector (Promega, Madison, WI, USA). The HaloTag-PeGRP2 protein was produced using the TnT SP6 High-Yield Wheat Germ Protein Expression System (Promega, USA) as a bait protein. The expression of the PeGRP2 protein was determined using Western blotting and purified using Magne HaloTag Beads (Promega, USA). Total RNA was extracted from the leaves of the WT P. × canescens and PeGRP2 transgenic lines using the EASYspin Plus Kit (Aidlab, Beijing, China). Subsequently, the mRNA was enriched using the Hieff NGS mRNA Isolation Master Kit (Yeasen, Shanghai, China). The covalently conjunct beads and HaloTag-PeGRP2 protein were incubated with mRNA from the WT or transgenic poplars in binding buffer (10 mM HEPES, 1 mM DTT, 1 mM MgCl₂, 20 mM KCl, pH 7.3). The bait protein was not added to the negative control but subjected to the same procedures as introduced for the HaloTag-PeGRP2 protein. The beads were washed with washing buffer (10 mM HEPES, 1 mM DTT, 1 mM MgCl₂, 0.1% Tween 20, 20 mM KCl, pH 7.3). Finally, the mRNA was eluted from the beads with nuclease-free water. The cDNA library was constructed using the RNA-Seq Library Prep kit (Illumina, San Diego, CA, USA), followed by sequencing using the Illumina NovaSeq platform. The P. × canescens sequenced genome (sPta717alba.fasta.gz, https://www.aspendb.org/downloads, accessed on 28 July 2023) was used as the reference genome. To identify the PeGRP2-binding mRNAs, we used fastp software version 0.21.0 to filter reads [63]. All clean reads were mapped using hisat2 version 2.1.0 to the reference genome [64]. Moreover, the enriched mRNAs were analyzed using the software featureCounts, version 2.0.4 [65].