mRNA seq of Bacteria: Lactobacillus rhamnosus GG
For each RNA sample, double-stranded cDNA was synthesized from 10ng mRNA using the SMARTer Universal Low Input RNA Kit (Takara, catalog# 634940) for sequencing which included a 16-cycle PCR. Following quantitation with Qubit dsDNA HS Reagent (Thermo Scientific, Cat. # Q32854). 10ng dscDNA/sample was fragmented with the E220 Focused-ultrasonicators (Covaris). This fragmented cDNA was then prepared into libraries using KAPA Hyper Prep Kit (KAPA Biosystems, Cat. # KK8504). Libraries were then combined into equimolar pools, which were then measured for size and concentration. The pools were clustered onto a paired-end flowcell with a 20% v/v PhiX spike-in and sequenced on Illumina's HighSeq 2500.