title: GSM8152333: UNCD3-22; Homo sapiens; RNA-Seq
library_construction_protocol: Extracted RNA was prepared using the Illumina TruSeq Stranded Total RNA Prep Kit 378 ng high quality total RNA (RIN > 7, OD260/280 > 1.75, OD260/230 > 1.75) was used in the Illumina TruSeq Stranded Total RNA Sample Prep Kit (Cat # RS-122-2201). ERCC Spike-in controls (Ambion, P/N 4456739) are added during the RNA normalization as a qualitative control. First rRNA is removed from the total RNA and the remaining RNA is purified, fragmented and primed. Next, the cleaved RNA fragments are reverse transcribed into first strand cDNA using reverse transcriptase and random primers. The RNA template is then removed and a replacement strand is synthesized generating double stranded cDNA. After purification, the result is blunt-ended cDNA. A single 'A' nucleotide is added to the 3' ends of the cDNA to prevent them from ligating to one another during the adapter ligation reaction. In this next reaction, a single T nucleotide on the 3' end of each adaptor allows for ligation of the adaptor to the cDNA. This is later used to hybridize the libraries to the flow cell and for de-multiplexing after sequencing is complete. A final PCR is then conducted to amplify and enrich those DNA fragments that have the adapter molecules on both ends. This process can be carried out manually, in the case of low sample number, or automated on the SciClone.  Libraries are QC'd by quantitation with picogreen, size analysis on an Agilent Bioanalyzer or Tapestation 2200 and qPCR quantitation against a standard curve. The assay used carried out in one batch on a Sciclone liquid handling system using the Illumina TruSeq Stranded Total RNA prep (Cat RS-122-2201) with ERCC spikein controls.  Three pools of 20 libraries were created and each was launched onto a HiSeq 2500 rapid 75PE flow cell.