RNA-Seq of Halobacterium salinarum: RNase R treatment, replicate 2
Halobacterium salinarum NRC-1 cells were grown in complex media (CM) (NaCl 250g/L, MgSO4.7H2O 20g/L, KCl 2g/L, sodium citrate 3g/L, peptone 10g/L) until OD600 ~ 0.5. We extracted small RNAs (<200nt) using the mirVana miRNA Isolation kit (Ambion). RNA was treated with RNase R as described in Danan et al., 2012. 120U of RNase R (Biosearch Technologies) was added to 20 g of RNA for 45 minutes at 37oC. The samples were cleaned with the RNeasy MinElute Cleanup kit (QIAGEN) and eluted in 30uL of DEPC water. The RNase R treatment was repeated twice, using 3U of RNase R for a g of purified RNA for subsequent treatments. Control samples underwent the same treatment, but substituting the enzyme for water. We prepared the sequencing library using the TruSeq mRNA Stranded RNA Sample Preparation kit (Illumina), and they were sequenced using the MiSeq Reagent v2 50 cycles kit (Illumina) on the Illumina MiSeq (Illumina).