title: GSM4306572: KO1 IP: P24 TRAP-Seq - cKO 1 IP; Mus musculus; OTHER
library_construction_protocol: For Bulk RNA-Seq, RNA was extracted from flash-frozen cerebral cortex of sex-matched littermate mice using Trizol (Thermo Fisher) followed by RNeasy Mini Kit (Qiagen) according to the manufacturers' instructions. For sequencing, RNA was reverse-transcribed with oligo-dT priming, and the cDNA was sequenced on an Illumina HiSeq 2000. For TRAP-Seq, the brain was homogenized in 2 ml homogenization buffer (20mM Hepes-KOH, 150mM Kcl, 10mM Mgcl2) with RNase inhibitor, and 10% NP40 and 300mM DHPC (Avanti Polar Lipids) was added to the supernatant. After spinning down, 10% of the supernatant was used as for the input fraction with the rest being subjected to immunoprecipitation. GFP-ribosome positive ribosomes were pulled down by incubation with affinity antibody matrix (19C8 and 19F7, Memorial Sloan-Kettering Monoclonal antibody facility) at 4C overnight. RNA was collected by Qiagen miRNA kit. RNA quality and concentration were assessed using high sensitivity RNA ChiP on the Agilent BioAnalyzer. Intact RNA (RIN>8) was used for the library using NEBNext UltraRNA library preparation kit (E7530) as described 45,46. Briefly RNA was purified by oligdT beads and cDNA was synthesized by reverse transcriptase. After DNA fragmentation, end repair, adaptor ligation and PCR amplification, libraries were used for Illumina deep sequencing.
attributes.tag: GEO Accession
attributes.value: GSM4306572