title: RNA-seq of Homo spaiens: 49,XXXXY skin fibroblast
design: Cells were collected and frozen in RNA Protect Cell Reagent (Qiagen) until RNA extraction with the RNeasy Plus Mini Kit (Qiagen), which included a genomic DNA removal step with a gDNA Eliminator Column (Qiagen). 10ul of a 1:100 dilution of ERCC Spike-Ins were added per 1 million cells.  Libraries were prepared using the TruSeq mRNA library kit (Illumina) according to manufacturer's instructions with the following modifications: Libraries were size-selected using 2% agarose gels on the PippinHT system (Sage Science) with a capture window of 300-600 bases. The size-selected material was then subjected to one additional cycle of PCR with fresh reagents and P5/P7 primers (Illumina) to ensure all library fragments were fully double-stranded. This step significantly reduced the percentage of smaller fragments co-purifying in the final gel size-selection. After PCR, libraries were size-selected a second time on the PippinHT with the same settings.