GSM5059678: E.timonensis-GABA B1; Emergencia timonensis; RNA-Seq
Cultures were centrifuged and cell pellets were resuspended in cold Trizol reagent, followed by flash freezing in liquid nitrogen. Total RNA was isolated by bead beating using 0.1 mm dia. Zirconia/Silica Beads at 10 m/s for 90 s, then using the Zymo Resarch Direct-Zol RNA MiniPrep Plus Kit. Illumina cDNA libraries were generated using a modified version of the RNAtag-Seq protocol (63). Briefly, 500 ng of total RNA was fragmented, depleted of genomic DNA, and dephosphorylated prior to its ligation to DNA adapters carrying 5ʹ-AN8-3ʹ barcodes with a 5ʹ phosphate and a 3ʹ blocking group. Barcoded RNAs were pooled and depleted of rRNA using the RiboZero rRNA depletion kit (Epicentre, Madison, WI). These pools of barcoded RNAs were converted to Illumina cDNA libraries in three main steps: (i) reverse transcription of the RNA using a primer designed to the constant region of the barcoded adaptor; (ii) addition of a second adapter on the 3' end of the cDNA during reverse transcription using SmartScribe RT (Clontech Biotechnologies, Mountain View, CA) as previously described (63); (iii) PCR amplification using primers that target the constant regions of the 3' and 5' ligated adaptors and contain the full sequence of the Illumina sequencing adaptors.