Signaling in hydrogel for biofilm mimics
TruSeq RNA sample preparation protocol (Illumina, San Diego, CA) with modifications was used to generate the next-generation sequencing library. The PCR amplification step, which selectively enriches for library fragments that have adapters ligated on both ends, was executed according to manufacturer’s recommendation with modifications. Each library was uniquely tagged with one of Illumina’s TruSeq LT RNA barcodes to allow library pooling for sequencing. Library quantitation was accomplished using Invitrogen’s Picogreen assay, and the average library size was determined by running the libraries on a Bioanalyzer DNA 1000 chip (Agilent). Library concentration was normalized to 2 nM, and was validated by qPCR on a ViiA-7 real-time thermocycler (Applied Biosystems), using primers recommended in Illumina’s qPCR protocol and Illumina’s PhiX control library as a standard. Libraries were then assembled at equal volumes and sequenced in two lanes of an Illumina HiSeq2500 rapid run at a final concentration of 7.2 pM and a read-length of 101 bp paired-end.