title: GSM5907732: cortex, CPN_hemi5_RP; Mus musculus; OTHER
library_construction_protocol: nanoRibo-seq: RPFs were gel purified, then converted into libraries using the QiA-seq miRNA kit. We performed neuronal FACS using established approaches (Catapano et al., 2001), but with the addition of translational inhibitors to prevent ex vivo elongation. For the CPN sorts, all buffers contained 100 ug/mL cycloheximide to inhibit translational elongation. We dissected brain into HBSS buffer+CHX, visualized the labeled region of cortex using an epifluorescent dissection microscope, and dissected the labeled cortical region into Disassociation Solution (DS)+CHX. We washed cortex tissue pieces twice in DS+CHX, then enzymatically digested twice by incubation in Enzyme Solution (DS with cysteine and papain)+CHX for 15 minutes each time (with inversion every 5 minutes to keep tissue pieces mixed). We washed twice in Wash Solution (WS)+CHX, triturated 15-20 times in ~1 mL of WS+CHX using fire-polished glass pipettes. We diluted the cell suspension with 4 mL WS+CHX, spun down cells for 5 minutes at 1000g, triturated again in 1 mL WS+CHX, and passed the cell suspension through a strainer cap. We added 1:1000 SYTOX Blue to be able to screen out dead cells. We then FACS-sorted the red cells, and collected them in a DNA LoBind Eppendorf Tube containing 2X polysome buffer+CHX (25 mM HEPES pH=8.0, 200 mM KCl, 10 mM MgCl2, 4 mM CaCl2, 1% nonidet P-40, 100 ug/mL CHX). For the SCPN sort, we performed all steps as described above, except that we omitted CHX until the SCPN were sorted into 2X polysome buffer+CHX.