title: GSM2886450: WTD/WT rep2; Lactococcus lactis subsp. cremoris; RNA-Seq
library_construction_protocol: Total RNA was isolated by slight modification of the method described by(Dijkstra et al., 2016)  for L. lactis. Cells from the late-log phase were snap frozen and harvested at (5000 x g, 10 mins, 40C) for the wild type and the adapted strains. Cell pellets were resuspended in 500ul of ice-cold TE buffer pH 7.5 and transferred to ice-cold 2ml microfuge tube (Eppendorf), after which 500 μl phenol/chloroform mixture (1:1), 30 μl 10% SDS, 30 μl 3 M sodium acetate (pH 5.2), and 500 mg glass-beads (150-212 μm, Sigma-Aldrich, St. Louis, USA) were added. The mixture was vortexed for 4 minutes at maximum speed and then 400 μl of chloroform isoamyl alcohol (24 :1) was added. The mixture was spun at 10000 x g at 20 degrees for 2 minutes. The supernatant was transferred to fresh microfuge tube and equal volume of RNAzol (Sigma) was added and steps were followed according to manufacturer's protocol. The total RNA concentration and quality was determined by Agilent bioanalyzer 2100 expert. Illumina HiSeq 2000/2500 Paired end run; 2 X 100 bp 30 million reads (3GB) per sample. ~3 ug of total RNA was used to prepare the RNA seq library. In short, mRNA molecules were purified using magnetic beads. Folllowing purification, mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were used to synthesize first strand cDNA using reverse transcriptase and random primers followed by second strand cDNA synthesis using DNA polymeraseI and RNase H. These cDNA fragments were used to generate RNA libraries. Bioanalyzer plots were used at every step to assess mRNA quality, enrichment success, fragmentation sizes,and final library sizes. The size distribution of the sequencing library was determined by AgilentTapestation or Bioanalyzer. After the libraries were constructed, paired end run were performed on Illumina's HiSeq 2500 platform to obtain 2 x100bp reads.
attributes.tag: GEO Accession
attributes.value: GSM2886450