Agilent bioanalyzer hs dna kit

The Fluidigm C1 system was used to prepare single cells for RNA-Seq. RNA-Seq-IFCs were selected to capture all major cell populations from all cell size ranges observed using IFCs which capture cells of different sizes: 5–10 μM (embryonic), 10–17 μM (embryonic, neonatal), 17–25 μM (>3 weeks of age). No batch effects were observed between chips of the same size. Onboard cell lysis, reverse transcription and cDNA synthesis were performed using the SMART-Seq v4 Ultra Low RNA Kit for the Fluidigm C1 System (Takara) reagents, following the manufacturer’s protocol. The resulting cDNAs from individual cells were used for the construction of NGS libraries with the Nextera XT DNA sample preparation kit (Illumina). Libraries were pooled, quantified with qubit HS DNA spectrophotometer and quality control was performed with the Agilent Bioanalyzer HS DNA kit. Approximately 1 Million 2 × 150bp Paired End Reads were generated for each single-cell RNA-Seq library in Illumina NovaSeq system following the manufacturer’s standard protocol. Count data were normalized to counts per million and transformed to Log2(CPM + 1). Single-cell libraries with >500,000 reads and <5% in mitochondrial genes were used for further analysis.

RNA from both the ex vivo and in vitro samples were processed identically from all subjects and MRSA isolates. Total RNA was extracted using the Mirvana kit (Life Technologies, Grand Island, NY), with the inclusion of a bead-beating step for 20 min with Lysing-Matrix B (MP Bio, Santa Ana, CA). Total RNA then was enriched for microbial RNA using MicrobEnrich (Life Technologies), and further enriched for mRNA using MicrobExpress (Life Technologies) and MegaClear (Life Technologies), which are designed to remove ribosomal RNAs. Enriched RNA then was prepared for sequencing through the construction of cDNA libraries using the Ion Total RNA-Seq kit (Life Technologies), and subjected to successive rounds of Ampure bead purification (Beckman-Coulter, Brea, CA) to remove small cDNAs. Libraries were quantified using an Agilent Bioanalyzer HS DNA Kit (Agilent, Santa Clara, CA) and then were sequenced on a 314 chips using an Ion Torrent Personal Genome Machine (Rothberg et al., 2011 (link)), producing an average of 559,129 reads per subject of mean length 106 nucleotides. All sequence data produced in this study are available in the MG-Rast database (metagenomics.anl.gov/) under the project name “MRSA_RNAseq_Study” or project #2278.