GSM6259869: LF_MUT1_IP; Mus musculus; RIP-Seq
Whole P7.5 cortices were rapidly dissected, rinsed, and Dounce homogenized in 500 uL of ice-cold RNase-free buffer polysome buffer (50 mM Tris pH 7.5, 100 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM DTT, 200U/ml Promega RNasin, 1 mg/ml heparin, 100 ug/ml cycloheximide, and protease inhibitors). Lysate was cleared via centrifugation at 10,000g for 20min at 4ºC. 60 ul of supernatant (“Input”) was collected and lysed with 350 ul of buffer RLT from Qiagen RNeasy Kit supplemented with 10μl/ml bME. The remaining supernatant was gently mixed with 5ul of rabbit anti-HA antibody (#71–5500 Thermo Fisher Sci) and incubated with gentle rocking at 4°C for 2hrs before isolation with 50 ul of protein A/G magnetic beads previously equilibrated in polysome buffer and a magnetic stand. Immunoprecipitates were rinsed 3x with 800 ul of a high salt buffer (50 mM Tris pH 7.5, 300 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM DTT, 100 ug/ml cycloheximide) and then lysed in 350 ul of buffer RLT.  RNA isolation from Input and Immunoprecipitated fractions was performed following manufacturers instructions using Qiagen's RNeasy extraction kit with DNAse digestion. Following RNA quantitation using a Ribogreen RNA reagent and exclusion of samples with an RNA integrity number < 7 on an Agilent Bioanalyzer, 2 ng of RNA were used for single primer isothermal amplification (SPIA) amplification and generation of double-stranded cDNA with the Ovation RNAseq System V2 (Nugen Technologies). Library construction and sequencing was performed on the Illumina NextSeq 500 platform at the Arizona State University's Genomics Core facility. Trimmed 50 bp single-end reads were quality checked and aligned to the mouse genome GRCm38.p6 primary assembly from Ensembl using RNA-STAR.