title: GSM8714368: FXTAS, Rep4, 6m, Hippocampus; Mus musculus; RNA-Seq
library_construction_protocol: RNA of brain tissues were extracted by using TRIzol™ Reagent following the standard protocol. In TRAP procedure, cortex and cerebellum were dissected in pre-cold PBS buffer and homogenized in PBS containing 50 mM Tris (pH 7.5), 100 mM KCl, 12 mM MgCl2 and 1% Nonidet P-40, 1 mM DTT, 100 U/mL Ribolock RNase inhibitor, 100 μg/mL cycloheximide, and 1 tab/5 mL SigmaFast protease inhibitor cocktail (with EDTA). The homogenates were kept on ice for more than 15min and centrifuged at 13000 rpm for 10 min at 4 ℃ to remove nuclei and cell debris. Approximate 50 μL of S1 supernatant was kept for input RNA isolation and the remaining supernatant was pre-cleared with Protein G Dynabeads at 4°C on a rotator for 30 mins. The S2 supernatant was incubated with 10ul HA antibody (Roche, # Anti-HA (12CA5)) on a rotator at 4 ℃ for 2 hours followed by incubating with protein G beads for additional 2 hours at the same condition. Afterwards, the beads were collected and washed 3 times with High Salt Buffer (HSB: 50 mM Tris (pH 7.5), 300 mM KCl, 12 mM MgCl2 and 1% Nonidet P-40, 1 mM DTT, 50 U/mL Ribolock RNase inhibitor, and additional 3X wash by using Extra High Salt Buffer (EHSB: identical to HSB but containing additional 300 mM NaCl). Per each wash, beads were rotated gently at 4°C for 2 mins. During the last wash, 10% beads were saved for western blot, and remaining beads were used for RNA isolation by TRIzol™ Reagent. RNA libraries for bulk RNA-Seq and TRAP-Seq were prepared according to the manufacturer's protocol NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina.