GSM7099119: NMP peptides APV rep3; Mus musculus; RNA-Seq
RNA was harvested using RNeasy mini plus kit (Qiagen). 1 µg of total RNA was used for the construction of sequencing libraries. Illumina's TruSeq Total RNA V2 protocol was used to generate libraries.  Specifically, ribosomal depleted RNA is converted to cDNA and size selected to 150 to 200 bp in length with 3' or 5' overhangs. End repair is performed where 3' to 5' exonuclease activity of enzymes removes 3' overhangs and the polymerase activity fills in the 5' overhangs. An 'A' base is then added to the 3' end of the blunt phosphorylated DNA fragments which prepares the DNA fragments for ligation to the sequencing adapters, which have a single 'T' base overhang at their 3' end. Ligated fragments are subsequently size selected through purification using SPRI beads and undergo PCR amplification techniques to prepare the 'libraries'. The BioAnalyzer is used for quality control of the libraries to ensure adequate concentration and appropriate fragment size.  The resulting library insert size is 120-200bp with a median size of 150bp. Libraries were uniquely barcoded and pooled for sequencing.DNA Sequencing: DNA sequencing was performed on an Illumina® HiSeq 2500 instrument using standard protocols for paired end 100bp sequencing.