title: Illumina HiSeq 2500 sequencing; RNA sequencing of iPSC and Human dermal fibroblasts
design: RNA sequencing of iPSC and Human dermal fibroblasts
library_construction_protocol: Cells grown to ~90% confluency in 10cm dishes were collected by trypsinization and washed once in PBS. For fractionation the cells were lysed in lysis buffer, followed by separation of the nucleus from the cytoplasmic material by centrifugation in a sucrose cushion. The isolated nucleus was rinsed once in PBS-EDTA and lysed by adding glycerol buffer and urea buffer in equal volumes. The precipitate, which contained the chromatin-RNA complex, was isolated by centrifugation and washed once in PBS-EDTA. RNA from each of the three subcellular compartments was isolated by Trizol™ (Thermo Fisher®). Prior to preparation of sequencing libraries, rRNA was removed by poly(A)+ selection, construction as in https://www.nature.com/articles/nature12787