title: GSM2413621: 0Cu25Ce_a; Methylosinus trichosporium; RNA-Seq
library_construction_protocol: RNA was extracted from cultures grown with methane as the sole growth substrate. Cultures were collected in the mid-exponential phase. 20ml from each flask was first mixed with 2.5 ml stop solution [5 % buffer equilibrated phenol (pH 7.3) in ethanol] to stop synthesis of new mRNA and then centrifuged at 4,000 g for 10 minutes at 4 °C 20mL. The cell pellets were then resuspended in 0.75 ml of extraction buffer [100 mM Tris-HCl (pH 8.0), 1.5 M NaCl, 1% (w/v) hexadecyltrimethylammonium bromide (CTAB)] and subjected to lysis by bead beating using 0.5 g 0.1-mm zirconia-silica beads at 4,800 rpm for 1 minute (Biospec Products) in the presence of 35 ml 20% SDS, 35 ml 20% laurylsarcosine, and 750 ml phenol-chloroform-isoamylic alcohol (25:24:1). The samples were then centrifuged at 14,000 rpm for 5 min at 4°C and the upper aqueous phase was transferred to a new tube and mixed with an equal volume of chloroform-isoamylic alcohol (24:1). The mixture was centrifuged again and the upper aqueous phase containing RNA was precipitated in MgCl2, sodium acetate, and isopropanol overnight at -80°C. RNA were recovered the next day by centrifugation at 14,000 rpm for 30 min at 4°C and washed by 75% ethanol. DNA was removed by Qiagen RNase-free DNase through at least two treatments, followed by purification using a Zymo RNA Clean & Concentrator kit (Zymo Research, Irvine, CA) following the manufacturer’s instructions. Removal of all traces of DNA was confirmed by the absence of a 16S rRNA PCR product in reactions using 2 μl of RNA template and 32 PCR cycles. RNA concentrations were estimated by using an ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE). Following the purification of total RNA, ribosomal RNA were removed using the RiboZero kit for Gram-negative bacteria (Illumina, San Diego, CA). The qualities of the samples were checked by Agilent bioanalyzer (Santa Clara, CA). mRNA-seq libraries were prepared by University of Michigan DNA Sequencing Core (http://seqcore.brcf.med.umich.edu) and sequenced in one lane using Illumina HiSeq4000 SE 50.
attributes.tag: GEO Accession
attributes.value: GSM2413621