Dnase 1

RNA pull-down was performed as previously described
[19] (link). First,
lncRP11-675F6.3 and its antisense RNA were transcribed
in vitro using the pGEM-3Z vector and T7 RNA polymerase RNA production system (Promega, Madison, USA). RNA was purified with an RNeasy® Mini kit (Qiagen, Valencia, USA) and treated with DNase I (Quanta Biosciences, Beverly, USA). RNAs were labelled with biotin using T4 RNA ligase sense, and the labelled RNA was captured with streptavidin magnetic beads. HepG2 cells were lysed and the extracts were mixed with the magnetic beads. The binding proteins on the beads were eluted and subsequently separated through SDS-PAGE followed by silver staining (Thermo Scientific, Waltham, USA). The differential band was cut and then subjected to be analyzed by Q Exactive LC-MS/MS (Thermo Scientific). The candidate binding protein was further verified by western blot following RNA pull-down.
RNA immunoprecipitation (RIP) was performed according to the manufacturer’s protocol (Millipore). Briefly, cells were lysed and immunoprecipitated using an antibody against HK1 (1:50; Millipore) with protein A/G magnetic beads. The complexes bound to the magnetic beads were immobilized with a magnet, and the beads were washed to remove unbound materials. Finally, binding RNA was extracted and analyzed by real-time fluorescence quantitative PCR (RT-qPCR).

Cellular mRNA was analyzed by isolating for total RNA with TRIzol^TM (Invitrogen) as per manufacturers’ instructions and eluted into 20 μL of RNase-free water. Potential DNA contaminants were removed with DNase I (Quanta Biosciences) before cDNA synthesis using the qScript cDNA supermix (Quanta Biosciences). APOBEC3 mRNA fold change was calculated by amplification and normalization alongside the internal reference, GAPDH, using the iTaq Universal SYBR Green (Bio-Rad Laboratories, primer sequences can be found in Supplementary Table S3). Negative controls for quantitative real-time (qRT)-PCR include no template controls (water), reverse transcriptase negative, and mock RNA extraction samples. All samples were performed in triplicate.

For quantitative RT-PCR, barley plants were grown on sterile perlite supported with ¼ MS (Murashige and Skoog) medium in glass jars. The plant roots were drenched with bacterial suspension (10⁷ CFU/g perlite). Samples were harvested from roots before and 24 h after bacterial inoculation. Each treatment was performed in four independent biological replicates.
Total RNA was extracted from plant samples using TriFast (peqGOLD, USA) and DNase I (Quanta Biosciences, USA) kits. cDNA synthesis was performed using the qScript cDNA Synthesis Kit (Quanta Biosciences, USA). The qRT-PCR reaction was run with the following program: initial denaturation at 95 °C for 60 s, denaturation at 95 °C for 15 s, and extension at 60 °C for 30 s with 40 cycles, and an additional melting curve from 60 to 95 °C. The expression of candidate genes was assessed with the primers listed in Supplementary Table S1.