title: GSM6659655: NoCFC_SD_S6_SUP_27; Mus musculus; RNA-Seq
library_construction_protocol: RiboTag TRAP was performed as previously described by indirect conjugation, separating MB and free-floating ribosomes. Following homogenization and centrifugation, the resulting supernatant (cytosolic fraction) was transferred to a new tube while the pellet (MB fraction) was resuspended in homogenization buffer. Both MB and cytosolic fractions were separated into Input, Camk2a+, and pS6+ fractions. For isolating ribosomes from Camk2a+ populations, fractions were incubated with 1:40 anti-HA antibody (Abcam, ab9110). To isolate ribosomes from highly active (pS6+) neurons, fractions were incubated with 1:25 anti-pS6 244-247 (ThermoFisher 44-923G). Homogenate-antibody solution was added directly to Protein G Dynabeads (ThermoFisher, 10009D) for incubation. After washing conjugated beads and removing the supernatant, RNA was eluted by vortexing the beads vigorously in 350 μL RLT (Qiagen, 79216). Eluted RNA was purified using RNeasy Micro kit (Qiagen). Amplified complementary DNA libraries were prepared using Takara's SMART-seq v4 Ultra Low Input RNA Kit (Takara 634888)