GSM1243003: SB2_methane_BR2; Methylocystis sp. SB2; RNA-Seq
RNA was extracted from cultures grown with either methane or ethanol as the sole growth substrate.  Cultures were collected in the late-exponential phase by centrifugation at 4500 x g for 10 min at 4ºC. Cell pellets were re-suspended in 0.75 ml of RNA extraction buffer (0.2 M NaH2PO4/Na2HPO4 buffer, pH 7.5; 5% CTAB in 2.4 M NaCl). The resuspended cell pellets were subjected to bead-beating (1 min at 4800 rpm) in 2 ml plastic tubes containing 0.5 g of 0.1 mm zirconia-silica beads (Biospec Products), 35 µl of SDS 20%, 35 µl of laurylsarcosine 20% and 750 µl of phenol:chloroform:isoamylic alcohol (25:24:1). The samples were then centrifuged at 14000 rpm for 5 min at 4ºC. The aqueous phase was mixed with an equal volume of chloroform:isoamylic alcohol (24:1) and centrifuged at 14000 rpm for 5 min at 4ºC. RNA was precipitated by adding MgCl2 (final concentration, 2.5 mM), 0.1 volume of 3 M sodium acetate and 0.7 volume of isopropanol and incubating overnight at -80 ºC. RNA was then recovered by centrifugation at 14000 rpm for 30 min at 4ºC. The DNase treatment was carried out using the RNase free DNase set (Qiagen) in accordance with the manufacturer’s instructions. The RNA samples were further purified using the RNeasy Plus Kit (Qiagen) using gDNA eliminator columns and RNeasy Mini Spin columns according to manufacturer’s instructions. The rRNA content was reduced using a MICROBExpress Bacterial mRNA purification kit (Ambion). To check for any DNA contamination, PCR was performed with extracted RNA as template. RNA was reverse transcribed to obtain cDNA by using Superscript III Reverse Transcriptase (Invitrogen) following the manufacturer’s instructions. RNA libraries were prepared for sequencing using TruSeq mRNA preparation kit
GEO Accession
GSM1243003