title: Illumina MiSeq paired end sequencing; Sphingobium Nickel resistance analysis by RNA-seq approach
design: Sphingobium Nickel resistance analysis by RNA-seq approach
library_construction_protocol: S. sp. ba1 was grown in either LB broth containing 30µg/ml kanamycin (hereafter simply LB) or in LB supplemented with 0.5 ml of 100 mM NiCl2 (hereafter LB-Ni) at 30 °C for two days, corresponding to a final OD600nm of 0.6-0.8. Both the cultures were grown in three biological replicates. Total RNA was isolated using the hot phenol method. Cells were centrifuged at 6,000xg for 4 min and the pellet was resuspended in 3 ml of 65 °C pre-warmed lysis solution (0.15 M sucrose, 1% SDS, 10 mM Na-acetate pH 4.7). Thereafter the samples were extracted twice with 3 ml of hot phenol pH 4.3 (65 °C) and once with phenol-chloroform-isoamyl alcohol (125:4:1) pH 4.3. RNA was precipitated with 1/10 volume of 3 M Na Acetate (pH 5.2) and 2.5 volumes of ethanol. After incubation at –80 °C for 15 min RNA was pelleted by centrifugation at 13000 x g for 15  min. Pellets were washed with 1 ml of 70% ethanol, re-centrifuged at 13000 x g for 5 min, washed again with 1 ml of cold absolute ethanol, air-dried and resuspended in 0.1 ml of DEPC-water. Solutions were then treated with RQ1 RNase-Free DNase (Promega) at 37 °C for 1 hour, and RNA precipitated in an equal volume of 4M LiCl. After incubation at – 20 °C for 30 min, RNA was pelleted and washed as described above. The pellet was then dissolved in 0.05 ml of DEPC-water.  Strand-oriented RNA libraries were prepared using the SureSelect Strand-Specific RNA Library Prep for Illumina Multiplexed Sequencing (Agilent Technologies, version A.3, September 2013) using c. 4 µg  of total RNA after depletion of ribosomal RNA (Ribo-Zero Magnetic kit for Gram negative bacteria (Epicentre)) and purification (RNeasy MinElute Cleanup kit).