GSM6796912: RNA-seq of NFAT5 siRNA-treated primary RPTEC, rep 2; Homo sapiens; RNA-Seq
RNA was extracted from the proximal tubule cell cultures with the RNeasy Mini Kit (Qiagen, 74104) following manufacturer instructions. Total RNA integrity was determined by Agilent 4200 Tapestation. Library preparation was performed with 0.5-1 ug of total RNA. Ribosomal RNA was removed by RNase-H method using RiboErase kits (Kapa Biosystems). mRNA was fragmented in reverse transcriptase buffer and heated to 94 degrees for 8 minutes. mRNA was then reverse transcribed using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers to produce cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using a 2x150bp configuration.