title: GSM4435023: 9_12_29_17_2R; Homo sapiens; RNA-Seq
library_construction_protocol: BTCOE 4795 tumor punches from extracted mouse brains (pathologist verified) were homogenized using the Bead Ruptor 24 (Omni International, Tulsa, OK).  Approximately 10 - 50 mg of tissue was placed into a 1.4 mm ceramic bead tube with 1 ml QIAzol lysis reagent. The tissue sample tube was processed on the Bead Ruptor for 1 cycle at a speed of 4.7 m/s for 20 sec. The homogenized lysates were extracted for total RNA using the RNeasy Microarray Tissue Mini kit (Qiagen, Venlo, Netherlands). Extracted RNA was DNase-treated and purified using the RNA Clean and Concentrator-5 kit (Zymo Research, Irvine, CA) and assessed for RNA quality using an Agilent 2100 Bioanalyzer and the RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA). Total RNA from 10 samples was used to prepare cDNA libraries using the Illumina® TruSeq Stranded Total RNA with Ribo-Zero Gold Preparation kit (Illumina Inc., San Diego, CA).  RIN values for the RNA samples ranged from 9.6 to 10. Briefly, 750 ng of total RNA was rRNA depleted followed by enzymatic fragmentation, reverse-transcription and double-stranded cDNA purification using AMPure XP magnetic beads. The cDNA was end repaired, 3′ adenylated, with Illumina sequencing adaptors ligated onto the fragment ends, and the stranded libraries were pre-amplified with PCR.  The library size distribution was validated, and quality inspected using a Fragment Analyzer (Advanced Analytical, Santa Clara, CA). The quantity of each cDNA library was measured using the Qubit 3.0 (Thermo Fisher, Waltham, MA). The libraries were pooled and sequenced to a target read depth of 40M reads per library using single-end (SE) 1X75bp sequencing on the Illumina NextSeq 500.
attributes.tag: GEO Accession
attributes.value: GSM4435023