Experion

Total RNA was extracted from frozen sections with the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Total RNA yield was measured with an A260/280 ratio of 1.7-1.9, demonstrating purity. Quality was evaluated on nanochips with the Bio-Rad Experion automated electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA). Samples had a 28S/18S ratio of 1.5 and did not show evidence of ribosomal peak degradation.

7- to 8-week-old male CD1 nude athymic mice were anesthetized and injected with 3 × 10⁵ HCT-116 cells via tail vein. Treatments by day zero and for 24 days, were performed with vehicle, CP and iVR1 (50 mg/kg), or bevacizumab, following the schedule reported before. Mice were sacrificed on day 25 and genomic DNA was extracted from all the lobes of mice lungs using QIAamp DNA Mini Kit (Qiagen). The concentration of double-stranded DNA was determined using BIORAD Experion and 1K Experion DNA Kit (Biorad). To quantify human Alu sequences in DNA extracted from mice lungs, specific primers reported by Schneider et al. [40 (link)] were used to perform qRT-PCR as previously described [41 (link)].

The young leaves on each HLB tolerant or susceptible citrus plant were collected and frozen in liquid nitrogen using properly labeled tubes. Total RNA was extracted from whole leaves of each sample according to the RNeasy Plant Mini Kit standard protocol (Qiagen Inc., Valencia, CA). The quantity and quality of RNA was evaluated using Nanodrop ND-1000 spectrophotometer. A total of 20–30 μg RNA were sent to BGI-Hong Kong (China) for RNA sequencing.
We constructed the RNA-Seq libraries following the Illumina protocol of mRNA-sequencing sample preparation (Illumina Inc., San Diego, CA). The quality of each library was examined using a BioRad Experion (BioRad, Hercules, CA). The high-throughput sequencing was carried out by BGI using HiSeq2000 (Illumina, San Diego, CA).

The FastDNA ®SPIN Kit for Soil (MP Biomedicals, Solon, OH) was used for extraction of total DNA. Total RNA was extracted from slurries using a previously described method [66 (link)]. Briefly, fresh soil (0.5 g) was mixed with the same volume of glass beads and suspended in 700 μl of TPM buffer (0.5 M Tris pH 7.0, 1.7% polyvinylpyrrolidone, 0.2 M MgCl₂). The mixture was shaken at 6.0 m s⁻¹ for 45 s followed by centrifugation at 20,000×g for 4 min. The pellet was resuspended in 700 μl of PBL buffer (0.05 M Tris pH 7.0, 0.05 M Na₂EDTA, 0.1% SDS w/v, 6% v/v phenol) and the lysis procedure was repeated as described above. Supernatants were purified by two-step phase extraction with phenol-chloroform-isoamylalcohol and chloroform-isoamylalcohol. RNA was precipitated with precooled isopropanol and resuspended in 50 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]). RNA extracts were treated with DNase I (Ambion) and purified using the RNA Clean and Concentrator kit (ZymoResearch) according to the manufacturer’s instructions. The integrity of the purified RNA was checked by Bio-Rad Experion™ and RNA HighSens Chips (Bio-Rad).

RNA from each abdominal biopsy sample was isolated using the Qiagen RNeasy Mini Kit (Cat No. 74104). The quantification of samples was done using a Microfluidic-based capillary electrophoresis system (Bio-Rad Experion, Bio-Rad Laboratories, Inc., Philadelphia, PA, USA). Total RNA was made to undergo reverse transcription to synthesize the first-strand cDNA. The cDNA, thus formed was then converted to a double-stranded cDNA template during second-strand cDNA synthesis. Biotinylated ribonucleotide was subsequently incorporated by in vitro transcription reaction and then purified by the bead-based purification method. The purified biotin-labeled-cRNA was then fragmented using a fragmentation buffer, and then the sample was hybridized onto Affymetrix GeneChipPrimeView (Affymetrix Inc., Santa Clara, CA, USA). The obtained CEL files per sample were subjected to normalization and non-specific filtering using Bioconductor packages affy and genefilter. As primeview annotation package primeview.db was not available at Bioconductor, annotations file “PrimeView.na36.annot” was downloaded from the Affymetrix website, and the package was created using AnnotationForge and Human.db0 packages. Raw CEL files and series matrix files have been submitted to NCBI Genomic Expression Omnibus database (GEO Accession # GSE78721).

Total RNA was purified using RNeasy Lipid Tissue Mini Kit (Qiagen) and treated with RNase-free DNase I (Qiagen) on-column according to the manufacturer’s recommendations. RNA concentrations and 260/280 and 260/230 ratios were measured with a Nanodrop. RNA Quality Indicator (RQI) was measured using Bio-Rad Experion (Bio-Rad Laboratories) with Eukaryote Total RNA StdSens assay according to the manufacturer’s protocol. Five hundred nanograms of RNA was reverse-transcribed to complementary DNA (cDNA) in duplicates with the High Capacity RNA-to-cDNA kit (Applied Biosystems) according to the manufacturer’s recommendations.