Adult mouse brain tissue
Both laser microdissection and laser pressure catapulting were employed to selectively collect sleep-ensemble (mCherry) neurons and neighboring unidentified (mCherry) neurons from the ZI within the same tissue sections. Dissected cellular material was collected into the caps of 0.5-mL RNase-free PCR tubes preloaded with 5 L of DNase/RNase-free distilled water (VICMED VR909, Xuzhou, China). Approximately 100 cells from 3 mice were pooled and homogenized. RNA sequencing of recovery sleep (RS)activated ZI sleep-ensemble (mCherry) neurons (n = 3 mice) and unidentified (mCherry) neurons (n = 3 mice) was performed using the SMART-Seq v4 Ultra Low Input RNA Kit (Takara/Clontech) by Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China), in strict accordance with the manufacturers protocols. RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kitfollowing manufacturer's protocols.