title: GSM3728381: FS_1162.1_Saline; Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted from a sagittal section of a half-kidney. Kidney tissue was homogenized using tissue homogenizer  in 1 mL of TRIzol™ Reagent. Following 5 min incubation, 0.2 mL chloroform was added, and samples were vigorously mixed for 30 sec, then centrifuged at 12,000 x g at 4°C for 15 min. The upper aqueous phase containing RNA was then vigorously mixed with 0.5 mL of isopropanol for 30 sec. After 10 min incubation at RT, the samples were centrifuged at 12,000 x g at 4°C for 10 min. The pellet was resuspended in 1 mL of 75% ethanol and centrifuged at 12,000 x g 4°C for 5 min. The RNA pellet was then air dried for 15 min,  dissolved in 50 μL of Nuclease-Free Water, treated with  DNase I, Amplification Grade following the manufacturer's  protocol and assessed for yield and purity by NanoDrop™ 2000. Concentrations of purified RNA were measured with the QuantiTTM RiboGreen® RNA Assay Kit  and normalized to 5 ng/μL. An automated variant of the Illumina TruSeqTM Stranded mRNA Sample Preparation Kit was used for library preparation from a 200 ng aliquot of RNA. This method preserves strand orientation of the RNA transcript and uses oligo dT beads to select mRNA from the total RNA sample. Following cDNA synthesis and enrichment, cDNA libraries were quantified with qPCR using the KAPA Library Quantification Kit for Illumina Sequencing Platforms and then pooled equimolarly.