GSM2898211: 28_ANS_6HR_J; Escherichia coli; RNA-Seq
RNA was isolated for all the various time-point samples using Trizol method followed by Dnase I treatment. The total RNA concentration and quality  was determined by Agilent bioanalyzer 2100 expert. The good quality and quantity of total RNA was given to AgriGenome for further library preparation & RNA Sequencing. 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.
GEO Accession
GSM2898211