Nextflex directional rna seq kit

S. pneumoniae precultures were centrifuged at 8,000 × g at 4°C for 8 min and resuspended 1:10 in C+Y; 10-ml culture volumes were incubated at 37°C at 5% CO₂ for 20 min; and the cultures were divided in two, with one half being treated with sSHP (1 µM, N-DIIIIVGG) and the other half being treated with the same volume of 1× PBS. Cultures were incubated for 2 h at 37°C in 5% CO₂ and harvested at 8,000 × g at 4°C for 8 min. RNA was extracted with the mirVana microRNA isolation kit (Life Technologies, Inc.). After a first extraction, lysed cells were treated with TURBO DNase (Life Technologies, Inc.) and a second RNA extraction was performed. RNA quality was tested on a bioanalyzer with the RNA 6000 Nano kit. The 16S and 23S rRNAs were then removed with the MICROBExpress Bacterial mRNA Enrichment kit (Thermo Fisher Scientific). RNA was treated with the NEXTFlex Directional RNA-Seq kit (dUTP based) for preparation of the DNA library for paired-end sequencing with Illumina HiSeq by the Norwegian Sequencing Centre (http://www.sequencing.uio.no). A sequence in FASTQ format was derived from each sample. Analyses were performed in accordance with previously reported protocols (63 (link), 64 (link)).

Poly-A RNA was enriched from 0.6 to 1.0 µg total RNA using NEXTflex Poly(A) Beads from Bioo Scientific. Strand-specific cDNA libraries were prepared using the Bioo Scientific NEXTflex Directional RNA-Seq Kit (dUTP Based) for Illumina following manufacturer instructions with a 12-min fragmentation time and 15 PCR cycles. Libraries were barcoded with Bioo Scientific adaptors so they could be pooled for sequencing. For the 20 adult samples, two libraries were created per individual: one for whole brain and one for abdominal tissue. In total, 60 libraries were created for the 40 individuals in the study. Library concentrations were quantified using a Qubit dsDNA High Sensitivity Assay Kit (Life Technologies), and library size was assessed using a Bioanalyzer High Sensitivity DNA chip (Agilent). Libraries were pooled into four groups at equal concentration and diluted to final pooled concentrations of 10 nM. Library pools were quantified using the Illumina-compatible kit and KAPA standards for real-time PCR by the W. M. Keck Center for Comparative and Functional Genomics at the Roy J. Carver Biotechnology Center (University of Illinois).

Poly-A RNA was enriched from 1–2 μg of total RNA by using Dynabeads Oligo(dT)25 (Life Technologies), following the manufacturer’s protocol. Two rounds of poly(A) enrichment were performed with a final elution in 14μL of water. The poly-A–enriched RNA was used to prepare RNA-Seq libraries, using the NEXTflex Directional RNA-seq Kit (dUTP based) with Illumina compatible adaptors (Bioo Scientific). Manufacturer’s instructions were followed and 13–15 cycles of PCR amplification were performed depending on the starting input of total RNA. Libraries were quantified on a Qubit fluorometer, using the dsDNA High Sensitivity Assay Kit (Life Technologies), and library size was assessed on a Bioanalyzer High Sensitivity DNA chip (Agilent). Libraries were pooled and diluted to a final concentration of 10 nM. Final library pools were quantified using real-time PCR, using the Illumina compatible kit and standards (KAPA) by the W. M. Keck Center for Comparative and Functional Genomics at the Roy J. Carver Biotechnology Center (University of Illinois). Single-end sequencing was performed on an Illumina HiSeq 2500 instrument by the W. M. Keck Center for Comparative and Functional Genomics at the Roy J. Carver Biotechnology Center (University of Illinois). The samples were sequenced on 20 lanes.