Young [2.5m] Tg NaCl mouse 20
Total RNA was isolated from homogenized hippocampus samples by QIAzol Lysis Reagent (Qiagen, Netherlands) and further isopropanol precipitation. The RNA concentration was performed with Qubit2.0 Fluorometer (Invitrogen, USA) and using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., USA). The A260/A280 ratio of the RNA samples was 1.82.0. The integrity of the isolated RNA (RNA integrity number, RIN) was determined using the Bioanalyzer Agilent 2100 (Agilent Technologies, USA). An RIN value for all samples was not less than 8.0. All samples were treated with DNase I (Fermentas, Lithuania). To prepare samples for the mRNA sequencing libraries, we used the Illumina TruSeq RNA Sample Preparation Kit. Total RNA from each sample (2.5 g) was used to purify the poly-A containing mRNA molecules by poly-T oligo-attached magnetic beads, with two rounds of purification. During the second elution of poly-A RNA, the RNA was also fragmented and primed for cDNA synthesis according to the manufacturers protocol. Then the fragmented mRNA samples were subjected to cDNA synthesis using a SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen, USA). The cDNA was converted into double-stranded (ds) cDNA and then Ampure XP beads were used to separate ds cDNA from the second-strand reaction mix. The ds cDNA was blunt-ended through an end-repair reaction. Next, a single A nucleotide was added to the 3 ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. The cDNA fragments were then ligated to specific RNA Adapter Indexes supplied in the kit. The In-Line Control DNA was added to each enzymatic reaction. To selectively enrich DNA fragments with adapter molecules on both ends and to amplify the amount of DNA in the library, the PCR process was used according to the manufacturers protocol (15 cycles). The quantity of libraries was determined using a qPCR method on a Rotor-Gene 6000 PCR System (Qiagen, USA) according to the manufacturers protocol (Sequencing Library qPCR Quantification Guide). The primers matched sequences within adapters flanking an Illumina sequencing library. Before starting qPCR, a control template was selected to measure the libraries for quantification. The quality of libraries was determined on an Agilent 2100 Bioanalyzer (Agilent, USA) per the manufacturers instructions. The size and purity of the samples were checked. The final product was a band at approximately 260 bp. Indexed cDNA libraries were normalized to 2nM and pooled together in equal volumes. Sequencing was performed using HiSeq2000/2500 platform (Illumina, USA).