Illumina MiSeq paired end sequencing; Response of Leptospirillum ferriphilum DSM14647 to elevated NaCl concentration
Response of Leptospirillum ferriphilum DSM14647 to elevated NaCl concentration
Centrifugation of culture in late exponential phase at 8000 x g and 4C for 15 minutes. Washing of cells with cold 10 mM H2SO4, and twice with 10 mM sodium citrate pH 7.0. Growth of L.ferriphulum in DSMZ 882 medium (pH1.8) supplemented with 20 g L-1 ferrous sulfate in a shaker at 37C and 180 rpm. Isolation of total RNA by RNeasy Mini Kit from Qiagen. DNA removed by treatment with DNase I (New England Biolabs). Evaluation of purity and integrity of total RNA with Agilent Bioanalyzer 2100 and an RNA 600 Nano Kit (Agilent Technologies.  For each sample 3 RNA preparations with RMNA integrity number above 7 from independent biological replicates pooled into one. Depletion of ribosomal RNA by MICROBExpress kit (Thermo Fisher). Preparation of cDNA library by TruSeq stranded mRNA library prep kit (Illumina).