GSM5718383: ht_Saline_rep05_batchB; Rattus norvegicus; RNA-Seq
Using a microscope with an attached freezing plate, hypothalami were removed from the brains and frozen on dry ice in Eppendorf tubes immediately. Tissue was homogenized using QIAGEN Tissue Lyser II (85300; QIAGEN) and further purified using Proteinase K (PK) Solution (MC5005; Promega). Prior to extraction, RNA integrity and concentration were tested using NanoDrop™ 2000c spectrophotometer (Thermo Fisher Scientific). In the following step, mRNA was extracted using Promega's “Maxwell SimplyRNA Tissue Kit” (AS1340). RNA quality was checked using a 2100 Bioanalyzer with the RNA 6000 Nano kit (Agilent Technologies). The RIN for all samples was >7.2. DNA libraries suitable for sequencing were prepared from 100 ng of total RNA with oligo-dT capture beads for poly-A-mRNA enrichment using the TruSeq Stranded mRNA Library Preparation Kit (Illumina) according to manufacturer's instructions. After 15 cycles of PCR amplification, the size distribution of the barcoded DNA libraries was estimated ~285/335 bp (batch A/batch B) by electrophoresis on Agilent DNA 1000 Bioanalyzer microfluidic chips.