2100 bioanalyzer rna nanochip

Total RNA was isolated from different tissues of both sexes separately (20 each from males and females) using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. All the RNA samples were treated with RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) to eliminate the genomic DNA. Using a NanoDrop ND-8000 spectrophotometer (NanoDrop products, Wilmington, DE, USA) to measure the concentration of isolated RNA, their integrity was determined by agarose gel electrophoresis. RNA quality was verified using a 2100 Bioanalyzer RNA Nanochip (Agilent, Santa Clara, CA, USA). The high-quality RNA samples (OD260/280 = 1.8–2.2, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0, >10 μg) were placed at −80 °C and used to generate cDNA libraries. Synthesis of cDNA template was performed by PrimeScriptTM RT Reagent Kit with gDNA Eraser Kit (TaKaRa, Japan) for first-strand synthesis kit. PCR amplification was performed in a 20 µL volume containing 0.5 µL of each degenerate primer, 12.5 µL of Premix, and 10.5 µL of double distilled water (ddH₂O). The cycling conditions were an initial denaturation at 95 °C for 2 min, followed by 34 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, followed by a final extension at 72 °C for 10 min and storage at 4 °C. Samples were sent to Rui Bo Xing Ke Biotechnology Company (Beijing, China) to complete sequencing.

Tumors were excised, snap frozen and stored at − 80 °C. Tumors were subsequently pulverized with the automated CryoPrep instrument (Covaris) and 10 mg was transferred to a CK mix bead beating tube (Bertin, France) with 350 µL RLT buffer (Qiagen) supplemented with 1% β-mercaptoethanol (Sigma). Tissue was homogenized in the Precellys 24 instrument (Bertin) 6500 rpm for 20 s. Samples were centrifuged for 3 min at 16,000g and the supernatant was collected. RNA was extracted using the RNeasy Mini kit (Qiagen) following the manufacturer’s guidelines. RNA concentration was measured with the NanoDrop One (Thermo Scientific) and RNA integrity confirmed with the 2100 Bioanalyzer RNA Nano Chip (Agilent).

The total RNA of each sample (in three biological replicates) was isolated using Trizol reagent (Invitrogen, Carlsbad CA, USA). The quality and concentration of the total RNA were measured separately using a 2100 Bioanalyzer RNA Nanochip (Agilent, Santa Clara, CA, USA) and a NanoDrop ND-2000 Spectrophotometer (Nano-Drop, Wilmington, DE, USA). The synthesis of cDNA was carried out using a PrimeScript™ RT reagent kit with a gDNA Eraser (Takara, Dalian, China).

To construct RNA libraries, leaves collected from WW, and DS plants were subjected to RNA isolation with biological replication. Total RNA was prepared from each leaf using the RNeasy Plant Mini Kit (Qiagen). RNA quality and integrity was assessed using a 2100 Bioanalyzer RNA Nanochip (Agilent Technologies) prior to RNA-seq. RNA-seq was then performed using the Illumina HiSeq platform.

Total RNA was isolated from liquid nitrogen ground tissue of complete leaves of the selected individual plants according to TRIzol^® protocol (Thermo Fisher Scientific, Carlsbad, USA). The RNA yield and quality was spectrophotometrically verified assessing the A₂₈₀/A₂₆₀ ratio using a Nanodrop 2000 apparatus, and RNA integrity was evaluated by capillary electrophoresis using a 2100 Bioanalyzer RNA Nanochip (Agilent Technologies, Inc., Santa Clara, CA, USA). For each condition, total RNA from five selected plants was pooled in equimolar ratio to construct each cDNA library. The cDNA libraries containing ≈500 base pairs (bp) fragments were constructed using the TruSeq Stranded mRNA Sample Preparation kit (Illumina, San Diego, CA, USA) following the manufacturer´s instructions and sequenced separately (2 × 150 bp) on an Illumina NexSeq500 instrument MID-Output by Langebio-CINVESTAV, Irapuato facilities (Guanajuato, Mexico).

Samples of the 5th leaves of triploid-F, triploid-S, and diploid plants were taken at 9:00 a.m. and frozen in liquid nitrogen. An RNA 6000 Nano Assay Kit and 2100 Bioanalyzer RNA Nanochip (Agilent) were used to control the quality of purity and concentration of RNA, respectively (RIN ≥ 8.0), and a miRNeasy Mini Kit (Qiagen, shanghai, China) was used to purify the RNA. Similar to transcriptome sequencing, miRNA sequencing was completed on the Ion Proton platform (Life Technologies) by Shanghai Novelbio Biological Technology after the cDNA libraries were built using a mixture of the three candidate genotypes of triploid-F, triploid-S, and diploid plants, respectively. High-quality reads after filtering were mapped to the miRNA database of Populus trichocarpa (Release 21.0, http://www.mirbase.org/) using BWA (v0.7.5a). To obtain the new candidate miRNA precursor, unmapped reads were further mapped to the genome of P. trichocarpa. To obtain more homologous miRNAs, unmapped reads were mapped one by one onto the miRNA databases of Arabidopsis thaliana, Glycine max, Zea mays, and Oryza sativa. The target genes of miRNAs were predicted using psRNATarget (plantgrn.noble.org/psRNATarget).

Total RNAs were extracted using RNA prep Pure Plant plus Kit (Tiangen Biotech (Beijing) Co., Ltd., Beijing, China) and purified with the RNA easy Plant Mini Kit (Qiagen, Valencia, CA, USA). RNA quality was verified using a 2100 Bioanalyzer RNA Nanochip (Agilent, Santa Clara, CA, USA), and quantified using NanoDrop ND-1000 Spectrophotometer (Nano-Drop, Wilmington, DE, USA).
For full-length cDNA library, cDNA was synthesized using SMARTer PCR cDNA Synthesis Kit, and optimized for PCR amplification. The fragments for large-scale PCR were performed using magnetic beads to obtain sufficient total cDNA. The complete SMRT bell library was constructed with using a SMARTer PCR cDNA Synthesis Kit and assembly was performed on the PacBio Sequel platform, the second-generation sequencing and assembly was implemented on the Hiseq 2500 sequencing platform (Illumina) with PE150 by Novogene Co., Ltd. (Beijing, China).