Rn38 easyspin plus plant rna kit

Total RNA extraction from both inoculated and control tree samples was conducted utilizing the RN38 EASYSpin Plus Plant RNA Kit (Aidlab Biotech, Beijing, China). Quantification and assessment of purity for total RNA were performed using the Bioanalyzer 2100 and RNA 1000 Nano LabChip Kit (Agilent, CA, USA), ensuring a RIN number > 7.0. Poly(A) RNA was isolated from total RNA (5 μg) through two rounds of purification using Poly-T oligomer magnetic beads. The purified mRNA underwent decomposition into smaller fragments under high-temperature conditions in the presence of divalent cations. Then, according to the mRNASeq sample preparation kit (Illumina, San Diego, CA, USA), the cleaved RNA fragments were reverse-transcribed to obtain the final cDNA library. The average insertion length of the paired library was 300 bp ( ± 50 bp). Sequencing was carried out using Illumina Novaseq™ 6000 (LC Sciences, Houston, TX, USA) following the recommended procedures for double c-terminal sequencing.

Leaf, stem, and root samples taken from Populus clone NE-19 plants treated for 1 week with the three nitrate concentrations, and from different Populus GNC modified plants and then immediately frozen in liquid nitrogen and stored at −80 °C until RNA extraction. Three replications consisting of nine seedlings for each sampling time point were used. Total RNA was isolated using the RN38 EASYspin Plus Plant RNA Kit (Aidlab Biotech, Beijing, China). First-strand cDNA synthesis was performed using first-strand M-MLV Reverse Transcriptase and an oligo(dT) primer (Promega, Madison, WI, USA) following the manufacturer’s instructions. Samples were diluted 10 times (~100 ng μl⁻¹) prior to quantitative real-time PCR (qPCR) analysis. The specific primers used to quantify Populus NE-19 GATA genes, PdGATA genes, and differentially expressed genes by qPCR were designed according to the corresponding gene sequences in the Populus trichocarpa reference genome (see Supplementary Tables S1, S2 at JXB online). The PCR mixture consisted of 1 μl sample, 0.6 μl (10 μM) forward primer, 0.6 μl (10 μM) reverse primer, 5.8 μl RNase-free ddH₂O, 10 μl SuperReal PreMix Plus, and 2 μl ROX Reference Dye in a total volume of 20 μl. The reaction was amplified for 40 cycles at 95 °C for 10 s, 55 °C for 30 s, and 72 °C for 32 s.

Total RNA from each sample was isolated separately from appropriate amount of mixed leave powder using the RN38 EASY spin plus Plant RNA kit (Aidlab Biotech, Beijing, China). Sequencing libraries were prepared using a New England Biolabs (NEB) Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (Ipswich, MA, US) sequencing following the manufacturer's protocol. Sequencing was performed on an Illumina HiSeq™ 2000 sequencing platform in accordance with the manufacturer's instructions. An additional biological replicate of T2, T2 repeat (T2-r), was used to control for error.

Total RNA from each sample of inoculated trees and control trees was separately extracted using the RN38 EASYSpin Plus Plant RNA Kit (Aidlab Biotech, Beijing, China) following the manufacturer’s procedure [24 (link)]. The total RNA quantity and purity were analyzed with a Bioanalyzer 2100 and RNA 1000 Nano LabChip Kit (Agilent Technologies, Santa Clara, CA, USA) with RIN number > 7.0. Poly(A) RNA was purified from total RNA using poly-T oligo-attached magnetic beads using two rounds of purification. Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. Then, the cleaved RNA fragments were reverse transcribed to create the final cDNA library in accordance with the protocol for the mRNASeq sample preparation kit (Illumina, San Diego, CA, USA), and the average insert size for the paired-end libraries was 300 bp (±50 bp). Then, paired-end sequencing was performed on the Illumina HiSeq 4000 platform (LC Sciences, Houston, TX, USA) following the vendor’s recommended protocol.

Total RNA of each sample was extracted using the RN38-EASYspin Plus Plant RNA Kit (Aidlab Biotechnology Co., Ltd., Beijing, China). The quality and quantity of total RNA was analyzed using a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA), and integrity was further evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies Co. Ltd., Santa Clara, CA, USA). High-quality RNA separated from two independent samples (biological replicates) was pooled for library preparation. Construction of the libraries and RNA-Seq was performed by CapitalBio Technology Corporation (Beijing, China), and the cDNA library was sequenced using Illumina HiSeq™2500.

Total RNA was extracted separately from each sample using an RN38 EASYspin plus Plant RNA kit (Aidlab Biotech, Beijing, China). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, United States). The libraries were sequenced by Novogene (Novogene, Tianjin, China) with an Illumina HiSeq (Illumina, CA, United States) system. To gain more reliable data, reads from each data set with more than 10% N bases and low quality (Q ≤ 20) reads with more than 50% bases were removed (Chen et al., 2018 (link)). Eventually, the clean reads were mapped to the TM-1 reference genome (G. hirsutum, WHU_updated v1) (Huang et al., 2020 (link)) by Hisat2 v2.0.5. To calculate gene expression, the number of mapped clean reads for each gene was counted and normalized into Fragments Per Kilobase of transcript sequence per Millions (FPKM).

Total RNA was extracted separately from each sample using an RN38-EASY Spin Plus Plant RNA kit (Aidlab Biotech, Beijing, China) following the manufacturer's instructions. The concentration of purified RNA was determined by agarose gel electrophoresis and spectrophotometry (NanoDrop 5000, Thermo Scientific, Waltham, MA, USA). Only RNA samples with a 260/280 wavelength ratio between 2.0 and 2.2 and a 260/230 wavelength ratio greater than 1.8 were used for cDNA synthesis. The cDNA was synthesized using Superscript III RT (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. All cDNA synthesis reactions were performed at the same time so that the efficiency of reverse transcription was approximately equal among the samples. The cDNAs were diluted 1 : 10 with nuclease-free water for RT-PCR and amplification.