title: GSM1533391: Δhns (OD 2.0) rep2; Vibrio cholerae; RNA-Seq
library_construction_protocol: Total RNA was extracted using RNeasy Plus Mini Kit (Qiagen). To this end, the cell pellets were resuspended in 200 uL of bacterial lysis buffer [30 mM Tris-HCl pH 8.0, 1 mM EDTA, 15 mg/mL lysozyme (Sigma-Aldrich)], supplemented with  15 uL of proteinase K (20 mg/mL, Qiagen), and the samples were incubated at room temperature for 10 min with vortexing every 2 min. Qiagen RLT Plus buffer (750 uL) containing 1 % v/v 2-mercaptoethanol was added to each sample and the tubes were briefly vortexed. The bacterial lysates were homogenized using QIAshredder spin columns (Qiagen) and genomic DNA removed using gDNA Eliminator spin columns (Qiagen). Finally, total RNA was recovered using the RNeasy spin column method (Qiagen) following the manufacturer's protocol. RNA integrity was determined by formaldehyde agarose gel electrophoresis and RNA was stored at -80°C. Contamination with DNA was further eliminated using the TURBO DNA-free kit (Invitrogen), which involves a second treatment with DNase for 30 min at 37°C. Reactions were terminated by addition of 0.2 volumes of the DNase inactivation reagent and RNA was purified using the Agencourt RNAClean XP kit (Beckman) following the manufacturer's instructions. Total RNA was eluted in 60 uL of RNase-free water. The absence of DNA contamination was confirmed by PCR with 16S RNA sequence-specific primers. Next, rRNA was removed using Ribo-Zero Magnetic Kit (Epicentre) following the manufacturer's instructions. Briefly, 6 ug of total RNA sample and 20 uL of Ribo-Zero rRNA Removal Solution were combined in a final reaction volume of 80 uL. Samples in Ribo-Zero rRNA removal solution were incubated at 68°C for 10 min followed by a 5 min incubation at room temperature. To remove the rRNA molecules from the mRNA, reactions mixtures were incubated with the magnetic beads provided in the kit, mixed and placed at room temperature for 5 min and then at 50°C for 5 min. The rRNA bound to the beads was then removed by magnetic separation. Finally, mRNA was purified using Agencourt RNAClean XP kit and eluted in 15 uL of RNase-free water. RNA-seq was performed on the Illumina HiSeq2000 using the latest versions of sequencing reagents and flow cells providing up to 300 Gb of sequence information per cell. Briefly, the quality of the total RNA was assessed using the Agilent 2100 Bioanalyzer and samples were subsequently converted to cDNA. Libraries were constructed using the TruSeq library generation kits as per the manufacturer's instructions (Illumina, San Diego, CA). The cDNA libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Biosystems kit for library quantitation (Kapa Biosystems, Woburn, MA) prior to cluster generation. Clusters were generated to yield approximately 725K - 825K clusters/mm2
attributes.tag: GEO Accession
attributes.value: GSM1533391