RNA extraction and library preparation

All RNA-seq experiments were performed under RNA clean conditions. For RNA-seq, live cells were sorted into RNase-free PBS, pelleted at 600g, for 5 min at 4 °C, then resuspended in 500 μl TRIzol, flash-frozen in liquid nitrogen and stored overnight at −80 °C. RNA extraction was performed the next day. TRIzol suspensions were thawed on ice, 1/5 V of 1-bromo-3-chloropropane was added, and tubes were shaken vigorously to combine phases. Phases were allowed to separate for 2 min at room temperature, then tubes were centrifuged at 10,500 rpm, for 15 min at 4 °C, in an Eppendorf centrifuge C5424R. We collected the upper aqueous phase and transferred to a new tube. Then, 1/2 V of isopropanol and 1 μl of linear polyacrylamide (Sigma Aldrich 56575) were added, the tube was inverted to mix the contents, and RNA was allowed to precipitate for 10 min at room temperature. Tubes were centrifuged for 10 min at 10,500 rpm and 4 °C. The supernatant was removed, and 1 V of 75% ethanol was added to the pellet, which was dislodged by flicking the tube. Tubes were centrifuged for another 5 min, at 10,500 rpm and 4 °C. Ethanol was removed, and tubes were allowed to air dry for 5 min until the pellet turned clear. Next, we added 26 μl of RNase-free water, 3 μl of Ambion DNase I 10× buffer and 1 μl of DNase I (AM2222) to remove all DNA and incubated tubes at 37 °C for 30 min. RNA was purified by a 1.5× AMPure bead clean-up, measured on a nanodrop and used as the input for library preparation with a SMARTER Stranded Total RNA-Seq Kit - Pico Input Mammalian v2 (TaKaRa Bio USA). OMP-tTA>tetO-GFP, gg8-tTA>tetO-GFP and two gg8-tTA>tetO-P2 libraries were prepared with the TruSeq kit. However, mOSN samples were compared with both OMP-tTA>tetO-GFP (TruSeq prep) and OMP-IRES-GFP (TaKaRa Bio USA), which label the same neurons, and produced the same results (Extended Data Fig. 10c,g–i). Libraries were sequenced on either a NextSeq550 or a NextSeq2000 and were sequenced to a targeted coverage of approximately 25 million reads. All RNA-seq experiments were performed with two to three biological replicates.