title: 16S rRNA gene full-length sequencing of human brain microbiome: entorhinal cortex|core|age-matched control
design: Full-length 16S rRNA gene was amplified using 1-2 g of DNA as template, the universal 16S rRNA bacterial primers 27F and 1492R (0.2 M each), and 1X Q5 Reaction Buffer Pack (New England Biolab). Cycling conditions were 94 C, 3 min; then 25 cycles of 94 C 30 s, 54 C 30 s, 72 C 2 min, and a final extension step at 72 C for 5 min. Barcodes were added to PCR amplicons through a second PCR reaction using Barcoded Universal F/R Primers (Pacific Biosciences, 100-629-100). PCR products were cleaned using High Prep PCR (Magbio) according to the manufacturers protocol. Cleaned barcoded 16S rRNA amplicons were quantified using AccuClear Ultra High Sensitivity dsDNA Quantitation Kit (Biotium) on BioTek FLx800 Microplate Fluorescence Reader. Based on quantification results, barcoded amplicons were then pooled in equimolar concentration into multiplexed sets.
identifiers.namespace: SUB15205659
identifiers.id: ua65