GSM7719808: Ketamine 1; Mus musculus; RNA-Seq
To pull down the EGFP-tagged polyribosomes, we used an affinity matrix containing Dynabeads (Thermo Fisher Scientific, Waltham, MA, USA), Biotinylated protein L (Genscript, Piscataway, NJ, USA), and GFP antibodies (HtzGFP-19F7 and HtzGFP-19C8, Heintz Lab, Rockefeller University, New York, NY, USA). The supernatant with EGFP-tagged polyribosomes was incubated with this affinity matrix, and after incubation, the beads (now with EGFP-tagged polyribosomes) were washed in high salt buffer (20 mM HEPES, 10 mM MgCl2, 350 mM KCl, 1% NP-40, 0.5 mM DTT and 100 μg/ml cyclohexamide). RNA from the complex was isolated using the Agilent Absolutely RNA Nanoprep kit (Agilent, Santa Clara, CA, USA) following the manufacturer's instructions. Further, before sequencing the RNA was quantified in Drop Sense (Perkin Elmer, MA, USA) and Qubit HS RNA (Thermo Fisher Scientific) and checked for quality in 2100 Bioanalyzer (Agilent). The Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus sample preparation kit (Illumina, San Diego, CA, USA) was used for library preparation. Library concentrations were measured, and the average size was determined using the Qubit dsDNA HS kit (Thermo Fisher Scientific) and the 2100 Bioanalyzer (Agilent). The four libraries were then combined equally based on molar concentrations, and the pool's concentration was measured using the KAPA Illumina Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA).