title: GSM7868049: Vglut, Sublibrary 2, Replicate 3, polysome RNA, CreON; Mus musculus; OTHER
library_construction_protocol: For HEK experiments, cells were lifted into plating medium, spun down, and washed in ice-cold PBS containing 100 µg/mL cycloheximide. Cells were resuspended in lysis buffer (20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 1% NP-40, 100 µg/mL cycloheximide, 0.02 U/µL rRNasin, 0.02 U/µL Superasin, EDTA-free protease inhibitor) containing 1% NP-40 and triturated by passing ten times through a 27 gauge needle. Lysate was clarified by centrifugation at 20,000 xg. For in vivo experiments, cortex tissue was dissected and washed in ice-cold lysis buffer and homogenized using a hand-operated tissue grinder. Crude lysates were clarified and then incubated on ice following the addition of  1% NP-40 and 30 mM DHPC. Lysates were clarified again by spinning at 20,000 xg. In both experiments, a fraction of the clarified lysate was fractionated using a 10-50% sucrose gradient. Fractions containing 40S subunits, monosomes, and polysomes were harvested for HEK experiments; only monosome and polysome fractions were taken for in vivo experiments. For the HEK experiment, a fraction of lysate was used for Translating Ribosome Affinity Purification (TRAP). Lysates for TRAP were further incubated with 30 mM DHPC before spinning at 20,000xg. Streptavidin-coated magnetic beads (Invitrogen) conjugated to anti-eGFP IgG antibodies (clones 19C8 and 19F7) via biotinylated Protein L were added to lysate supernatants and incubated for 6 hours at 4°C. Beads were washed four times in wash buffer (20 mM HEPES, 350 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 1% NP-40, 100 µg/mL cycloheximide, 0.02 U/µL rRNasin, 0.02 U/µL Superasin, EDTA-free protease inhibitor) before collecting in lysis buffer. RNA from lysates, TRAP immunoprecipitation, 40S fractions, and monosome fractions was extracted using Trizol LS and RNA clean up kit (Zymo). RNA from polysome fractions was extracted by ammonium acetate precipitation and RNA clean up kit (Zymo). DNA from lysate pellets for both HEK and in vivo experiments was extracted using Trizol LS. For HEK and "CreOFF" (from Cre-ngative cells) RNA-seq libraries, cDNA was prepared by reverse transcription with a "CreOFF RT" primer. For "Cre-ON" (from Cre-positive glutatmetergic neurons) RNA-seq libraries, a "Cre ON RT" reverse transcription primer was used instead. For all RNA libraries, cDNA was amplified using "FivePrime_Sense" and "FivePrime_Antisense" primers followed by an indexing PCR using Illumina TruSeq adapters. DNA-seq for HEK cells and "CreOFF" samples were prepared by first amplifying with "CreOFF RT" and "FivePrime_Antisense" primers followed by an indexing PCR. DNA-seq libraries from "CreON" samples were prepared by first amplifying with "CreON RT"  and "FivePrime_Antisense" primers followed by an indexing PCR.