title: GSM1399324: WT animals 1, 2, 3 pooled (hippocampus); Mus musculus; RNA-Seq
library_construction_protocol: After cervical dislocation, cerebellum, midbrain and liver were dissected from homozygous wild-type (Atxn2+/+) and knock-out (Atxn2-/-) mice at 6, 12 and 24 weeks of age. Total RNA was extracted from these tissues by homogenization in 1 ml of Trizol® Reagent per 50 – 100 mg of tissue using a Pellet Pestle® Motor tissue homogenizer (Kontes, The Glass Company). One µg total RNA was digested with a DNase I Amplification Grade Kit (Invitrogen, Karlsruhe) in a reaction volume of 10 µl per tube in order to eliminate DNA during RNA purification prior to reverse transcription (RT-PCR) amplification. For preparation of MACE libraries, poly-adenylated mRNA was isolated from 1 μg of the large fraction of total RNA using Dynabeads® mRNA Purification Kit (Life Technologies GmbH) and cDNA was produced by first and second strand synthesis using the SuperScript® III First-Strand Synthesis System (Life Technologies GmbH). , but with modified, barcoded poly-T adapters that are suitable to bind on the Illumina Hiseq2000 flow cell and that are biotinylated at the very 5′ end. After cDNA production, the biotinylated cDNA was random-fragmented to reach an average size of 250 bps, the 3′ends were captured by streptavidin beads and GenXPro’s TrueQuant adapters were ligated. Ten barcoded samples were sequenced simultaneously in one lane of an Illumina Hiseq2000 with 1 × 100 bps.
identifiers.namespace: GEO
identifiers.id: GSM1399324