GSM5291572: NP_CreNeg_RNAseq_R4; Mus musculus; RNA-Seq
Microdissected CA1 from mouse brain hippocampal slices were sectioned into HBSS containing 0.1 mg/mL cycloheximide and resuspended in 0.5 mL ice-cold polysome buffer supplemented with 40 U/ml RNasin Plus (Promega) and cOmplete Mini EDTA-free Protease Inhibitor (Roche) and homogenized by mechanical homogenization with 10 strokes at 900 rpm. NP-40 was added to 1% final concentration, incubated on ice for 10 minutes and centrifuged at 2000 x g for 10 minutes. Supernatant was centrifuged at 20,000g for 10 minutes. 10% of the resulting lysate was used for bulk RNA-seq, and the remaining lysate was pre-cleared with 1.5 mg Protein G Dynabeads for 45 min. HA-tagged ribosomes were collected by indirect IP by adding 40 ug of anti-HA antibody (Abcam ab9110) to CB lysate pools and 5 ug to NP lysate pools. Immunoprecipitation proceeded overnight with rotation at 4°C, followed by addition of 7.2 mg (CB pools) or 4.44 mg (NP pools) Protein G Dynabeads and further incubated with rotation at 4°C for 1 hour.  Beads were washed with 1 mL polysome buffer containing 1% NP-40 once for 5 minutes and twice for 20 minutes, followed by 4 x 10 minute washes in 50 mM Tris pH 7.5, 500 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM DTT, 0.1 mg/mL cycloheximide. RNA was extracted from beads by incubating in 500 uL Trizol at room temperature for 5 minutes. RNA was quantified with RiboGreen Quant-IT assays (Invitrogen). Bulk RNA-seq samples were treated with RQ1 RNase-free DNase (Promega) prior to library preparation. Polyadenylated mRNA was collected by Dynabeads mRNA Purification Kit (Ambion). The libraries were prepared by TruSeq RNA Sample Preparation Kit v2 (Illumina) following manufacturer's instructions. High-throughput sequencing was performed on HiSeq (Illumina) to obtain 100 nucleotide paired-end reads. polyA selection, TruSeq library prep (unstranded)