GSM1246371: MQ 60min rep2; Listeria monocytogenes EGD; RNA-Seq
RNA samples were stabilized with RNAprotectTM (Qiagen 76506) and stored at – 80°C prior to RNA purification with RNeasy Mini Kit (Qiagen 74104) including DNase treatment with RNase free DNase (Qiagen 79254) according to the manufacturer’s instructions. Quantity and quality of the purified total RNA was evaluated by Nanodrop and Agilent 2100 Bioanalyzer using the RNA 6000 Nano kit. Five µg total RNA from each sample was ethanol precipitated, dissolved in 15 ul RNase free water and used as input in MICROBExpressTM Kit (Ambion AM1905) to remove of 16S and 23S rRNA. mRNA samples were evaluated on Qubit RNA assay kit (Invitrogen Q32852) and on Agilent 2100 Bioanalyzer with the RNA 6000 Nano kit. Libraries for RNA Seq were prepared using TruSeq RNA Sample preparation Kit v2 (set A: RS-122-2001 and set B: RS-122-2002) according to the manufactures protocol. In brief, cDNA was generated from mRNA, which had been fragmented to approximately 180 bp. Specific adaptors were ligated to each of the 20 samples. The library was validated by measuring quantity with Qubit dsDNA BR assay kit and quality with Agilent 2100 Bioanalyzer on DNA-1000 chip. Due to a peak at 1500 bp, an E-gel size select (Invitrogen G6610-02) was performed to isolate the cDNA sequences of 200-400 bp. Lastly, each library was normalized to 10 nM and pooled yielding a final concentration of 7.92 nM with an average base pair size of 290.
GEO Accession
GSM1246371