Qubit dsdna kit

Fresh frozen human glioma samples (n = 21) were obtained from the University Health Network Brain Tumor Bank (Toronto, Canada). Samples were prospectively collected between 2002 and 2015 with written informed consent prior to surgery for use of tissues for research purposes. Local institutional review board approval for the work outlined in this manuscript was obtained prior to study initiation. DNA was extracted from specimens using the PureLink Genomic DNA kit (Invitrogen), and quantified using the Qubit dsDNA kit (Invitrogen). RNA was extracted using the Ambion WT kit (Ambion). RNA quality was assessed using Agilent 2100 Bioanalyzer.

Cell lines used in this study are listed in Table 1. Cells were maintained in fibroblast medium [DMEM high glucose (Gibco) with 10% foetal bovine serum (Gibco) and no antibiotics] at 37°C in a humidified 5% CO₂ atmosphere. Cells were grown until ∼80% confluence. When ready, cells were washed with PBS (Gibco), then incubated with 0.05% trypsin (Gibco) for 5 min at 37°C. Cells were collected by centrifugation (1500 rcf for 5 min) and pellets were washed once with PBS, before being snap frozen in liquid nitrogen and kept at −20°C until further use. All DNA from cell lines was extracted from snap-frozen pellets using the QIAmp DNA mini kit (QIAGEN) following the manufacturer's instructions. DNA was quantified using the Qubit dsDNA kit (Invitrogen) following the manufacturer's instructions.

To analyze the effect of R. intestinalis on the structure of the intestinal flora, mice were gavaged with R. intestinalis at a concentration of 1 × 10⁹ CFU/ml for 14 days. On days 7 and 14, mouse feces were harvested for 16S rRNA sequencing.
A MagPure Stool DNA kit (Magen, China) was used to extract DNA from the microbial communities. DNA was quantified using a Qubit Fluorometer with the Qubit dsDNA Kit (Invitrogen, United States), and quality was checked using a 1% agarose gel. Polymerase chain reaction (PCR) primers 341F (5’-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5’-GGACTACHVGGGTWTCTAAT-3′) were used to amplify the variable regions V3–V4 of the bacterial 16S rRNA genes. The primers were tagged with Illumina adapter, pad, and linker sequences. PCR enrichment was performed using a 50 μl reaction containing 30 ng template, fusion PCR primer, and master mix. The PCR cycling conditions were 94°C for 3 min, 30 cycles of 94°C for 30 s, 56°C for 45 s, 72°C for 45 s, and 10 min at 72°C for extension. PCR products were purified using AmpureXP beads and eluted with elution buffer. The libraries were qualified using an Agilent 2,100 bioanalyzer (Agilent, United States). The validated libraries were sequenced on an Illumina MiSeq platform (BGI, Shenzhen, China) following the standard Illumina pipeline and 2 × 300 bp paired-end reads were generated.

Isolation and library generation was done as previously described (52 (link)). Briefly, RNA was extracted using the standard protocol within the AllPrep DNA/RNA Micro kit (Qiagen, Germantown, MD, USA). RNA quality from sorted nuclear RNA was determined based on Bioanalyzer Picochip analysis (Agilent, Santa Clara, CA, USA). Isolated RNA was amplified, made into cDNA, sheared, and libraries were made. The library was quantified using the Qubit dsDNA kit (Invitrogen) and Kapa library quantification kit (KapaBiosystems, Boston, MA). cDNA libraries were pooled, clustered on the cBot and subject to 100 or 125 base pairs paired end reads on the HiSeq 2000 or 2500 (Illumina, San Diego, CA, USA).

Tissues used in this study are listed in Table 1. Tissues were obtained from 7 different healthy individuals. All DNA from human tissues was extracted using QIAmp Fast DNA Tissue Kit (QIAGEN), following the manufacturer's instructions. DNA was quantified using the Qubit dsDNA kit (Invitrogen) following the manufacturer's instructions.