GSM5742556: CAD_S15.C; Homo sapiens; RNA-Seq
Frozen tissues of approximately 50 mg were homogenized in Qiazol Lysis Reagent (Qiagen, cat# 79306) with a cooled 5 mm stainless steel bead (Qiagen, cat# 69989) using a TissueLyser II (Qiagen, cat# 85300). Downstream RNA isolation was then performed using the Qiagen RNeasy Mini protocol on the Qiacube according to manufacturer's instruction. After elution, 1 µL of each sample was evaluated for total RNA concentration using standard 260 nm absorbance on a NanoDrop 1000 (Thermo Scientific) UV-Vis spectrophotometer. The quality of the RNA in each sample was assessed using 2 µL of each sample via electrophoresis on the Agilent Tapestation. The remaining sample was stored at -80°C until analysis.  Total RNA from each kidney sample was prepared for sequencing using the Illumina TruSeq Stranded mRNA Sample Preparation Kit (Illumina, cat# 20020595) according to manufacturer's instructions on the Perkin Elmer Sciclone NGS prep system. 1,000 ng of total RNA was used to prepare the RNA libraries and fifteen cycles of library amplification PCR was performed. After library generation, 2uL of the cleaned cDNA library was used to quantify the library concentration on the ThermoFisher Scientific Qubit 4 Fluorometer (ThermoFisher Scientific, cat# Q33238). Additionally, the quality of each cDNA sample was assessed using 2 µL of each sample via electrophoresis on the Agilent Tapestation. Samples were binned according to Qubit readings to make a library pool for sequencing, which was diluted to 4 nM before denaturing and further dilution prior to loading onto the Nextseq 500. The remaining individual cDNA libraries were stored at -20°C.  Sequencing was performed at 2 x 150 bp, paired end read setting using a 300-cycle sequencing reagent kit & high output flow cell on a NextSeq 500 Sequencing System (Illumina, San Diego, CA). Condensed and quality controlled paired end fastq files were generated for data analysis.