Bioanalyzer chip rna 7500 series 2

Total RNAs were isolated from the latex collected at each time-point using our previously described method [44 ]. RNA quality and quantity were determined with a Nanodrop 2000 instrument (Thermo Scientific, Wilmington, DE, USA) and a Bioanalyzer Chip RNA 7500 series II (Agilent, Santa Clara, USA). For enrichment of ET-responsive genes, total latex RNAs from two groups of rubber trees at three different time-points were equally pooled, respectively, and used for cDNA library construction.

For isolation of RNA, cells from 6 donors were cultured in 60 mm dishes until approximately 70% confluency was reached. MSCs were then exposed to experimental conditions. Both, control cells and 40 μM Vadadustat-treated cells were incubated at an atmospheric O₂ concentration. After 6 h treatment all cells were washed and disrupted in 350 μL of RLT buffer from the Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany). Samples were then stored in −80 °C until further use. RNeasy Mini Kit was used for total RNA extraction from MSCs according to the manufacturer’s isolation protocol. The concentration and integrity of collected RNA samples were determined spectrophotometrically using NanoDrop 1000 (NanoDrop Technologies, Thermo Fischer Scientific, Waltham, MA, USA) and Bioanalyzer Chip RNA 7500 series II (Agilent Technologies, Santa Clara, CA, USA).

The quantity of total RNA was determined using a Qubit fluorometer (Life Technologies). The quality of RNA was assessed by measuring RINs using Bioanalyzer Chip RNA 7500 series II (Agilent). One microgram of total RNA from each sample was used to prepare an mRNA-Seq library with TruSeq™ RNA Sample Prep Kit (Illumina), following the manufacturer’s instructions. Library quality control was performed with a Bioanalyzer Chip DNA High Sensitive (Agilent). Each library had an insert size of 300–400 bps, and 2 X 109 bps paired-end sequences were generated using Hiseq 1500 (Illumina).

2–4 × 10⁷ cells were collected by centrifugation for 5 min at 1400 x g, 4°C. RNA was extracted using the Trizol reagent as described previously (Strenkert et al., 2011 (link)). DNase treatment was performed using Turbo DNAse (Ambion), concentrating and cleaning with the Zymo Research RNA Clean and Concentrator−5 Kit according to the manufacturer’s instructions. For RNA-Sequencing, RNA quality and quantity were determined using a Nanodrop 2000 instrument (Thermo Scientific) and a Bioanalyzer ChiP RNA 7500 series II (Agilent). A stranded Illumina RNA-Seq library was prepared by the UCLA Clinical Microarray Core with standard Kapa RNAseq library preparation. Library quality control was performed with a Bioanalyzer Chip DNA 1000 series II (Agilent) and the libraries were quantified using Qubit. Libraries were sequenced on a HiSeq 1000 sequencer (Illumina) and single end 50 bp sequences were generated. Sequences were then aligned to the C. reinhardtii genome (v5.5) with RNA star. Relative expression estimates were generated using Cuffdiff 2.0.2.

Total RNA was extracted using TRIZOL reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). RNA degradation and contamination was monitored using 1% agarose gels. RNA quality was assessed by measuring the RNA integrity number (RIN) using a Bioanalyzer Chip RNA 7500 series II (Agilent, Santa Clara, CA, USA). Samples with an RIN value of greater with 8.0 were used for sequencing (Supplementary Figure S1). The total RNA concentration was determined with a Qubit fluorometer (Life Technologies).

Grapevine leaf disks (in biological triplicate) were ground in liquid nitrogen and total RNA was isolated from 200 mg of powder per sample, using the Spectrum Plant Total RNA Kit (MilliporeSigma). The quantity, integrity and purity of the RNA were determined using a Nanodrop 2000 (Thermo Fisher Scientific) and a Bioanalyzer Chip RNA 7500 series II (Agilent Technologies). The RNA was treated with DNase from the TURBO DNA-free kit (Thermo Fisher Scientific) and 1 µg was reverse transcribed using the SuperScript III kit (Invitrogen, Thermo Fisher Scientific). qPCR was carried out using the SYBR Green PCR Master Mix kit (Applied Biosystems, Waltham, MA, USA). The ubiquitin1-encoding gene (VIT_16s0098g01190) was used as the reference for expression normalization. The list of primers is provided in Supplementary Table S1. The “Comparative Quantitation” protocol was applied using the Mx3000P Real-time qPCR system (Stratagene, Agilent Technologies). The real amplification efficiency was calculated using LinRegPCR [58 (link)]. Mean normalized expression (MNE) values were calculated according to [59 (link)]. Standard errors (SE) were calculated from three biological replicates.

All the samples were collected in a thermo bottle containing liquid nitrogen and then immediately stored at −80 °C. Each sample included three independent biological replicates, and each biological replicate comprised the samples collected from six trees (cultivars) or two trees (Amazon wild germplasms). The dry rubber yield of the rubber trees was measured as described by Tungngoen et al. [25 (link)]. Total RNAs were isolated using the universal plant total RNA extraction kit (BioTeke, Beijing, China) and stored at −80 °C. The quality and integrity of the total RNAs were evaluated using Nanodrop 2000 (Thermo Scientific, Wilmington, DE, USA) and Bioanalyzer Chip RNA 7500 series II (Agilent, Santa Clara, CA, USA) instruments.