GSM5820976: lysR_B; Burkholderia sp. JP2-270; RNA-Seq
Briefly, a total of 3 µg RNA per sample was obtained and rRNA were depleted using Ribo-Zero rRNA removal kit (G- bacteria). The mRNA were fragmented using diavalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5 X). Then, the cDNA libraries were generated using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, USA) following manufacture's instructions. The cDNA fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA), and then 3µl USER Enzyme (NEB，USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then PCR was performed with Phusion HighFidelity DNA polymerase, Universal PCR primers and Index Primer. At last, products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The resultant samples were sequenced on an Illumina Hiseq 2000 platform. RNA libraries were prepared for sequencing using standard Illumina protocols