GSM8502730: ATN_neg_4, No-rejection, DSA-negative; Homo sapiens; RNA-Seq
Total RNA was isolated from kidney tissue using Trizol-chloroform extraction, concentration was measured by Qubit fluorimetr and RNA integrity number was checked using Agilent Bioanalyzer 2100. From 400 ng of total RNA, mRNA was isolated using poly (A) magnetic selection NEBNext® Poly (A) mRNA magnetic isolation module (New England, BioLabs, Inc). Transcriptome libraries were prepared using the NEBNext® Ultra™ II Directional RNA Library Prep with Sample Purification Beads according to the manufacturer's protocol (New England, BioLabs, Inc).In brief, mRNA was randomly fragmented by heat digestion in the presence of a divalent metal cation (Mg2+). Sheared RNA was reversibly transcribed making 1st strand of cDNA using random hexamers as primers and reverse transcriptase. The second strand was created using dUTPs, purified with NEBNext Sample Purification Beads, and ligated with diluted NEBNext adapters. After removal of the 2nd strand by uracil-DNA-dependent glycosylase, the final amplification of adaptor-ligated DNA was done using NEBNext® Multiplex Oligos for Illumina®. Library was purified with NEBNext Sample Purification Beads and its quality was assessed on a Bioanalyzer 2100 using the Agilent DNA 1000 assay.