title: GSM4876576: P9808_OB_L; Mus musculus; RNA-Seq
library_construction_protocol: Six samples from two Glioma_CKO mice (one normal brain tissue, two tumor tissues per mouse) were performed Whole Exome Sequencing by Novegene. The insert size of library was tested by Agilent 2100. After the validation of library quality, sequencing was performed by Illumina HiSeq PE150. Raw data was filtered by removing reads with adaptor, reads with more than 10% undetermined bases and read with more than 50% low quality bases. Effective sequencing data was aligned by BWA and Samblaster and processed by Samblaster further. Somatic SNV was processed by muTect. Somatic INDEL was performed by Strelka. Somatic CNV was performed by freec (http://bioinfo-out.curie.fr/projects/freec/) RNA libraries were prepared for sequencing using standard Illumina protocols.Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. First strand cDNA was synthesize dusing random oligonucleotides and SuperScript II. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 200 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter,Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The sequencing library was then sequenced on a Hiseq platform (Illumina)