GSM8579656: PDS - 1 - age 32; Homo sapiens; RNA-Seq
RNA was isolated from 2 million cells in 1 ml RLT using the RNeasy Mini (QIAGEN) according to manufacturer's instructions with an elution volume of 30µl. The library preparation was done using the QIAseq Stranded mRNA Kit (QIAGEN). From 500 ng starting material, the mRNA was enriched. The RNA was heat fragmented. After first and second strand synthesis, the cDNA was end-repaired and 3' adenylated. Sequencing adapters were ligated to the overhangs. Adapted molecules were enriched using by 13 cycles or PCR and purified by a bead based cleanup. ). Library preparation was quality controlled using capillary electrophoresis (Tape D1000).High quality libraries are pooled based in equimolar concentrations. The library pool(s) were quantified using qPCR and optimal concentration of the library pool used to generate the clusters on the surface of a flowcell before sequencing on a MiSeq (Illumina Inc.) instrument (2x75, 2x10) according to the manufacturer instructions (Illumina Inc.).