Persistence of homeostatic sleep drive is encoded by plasticity of a thalamic reuniens circuit

Purpose: snRNA-Seq was performed to characterize molecular differences that occur with sleep deprivation in the nucleus reuniens of the thalamus The nucleus reuniens of the thalamus (RE) were collected from 7-8 wk old male FosTRAP;tdTomato mice using a previously published protocol(Kim et al. 2021). In brief, the RE was dissected using a micropunch from a single mouse and placed into Hibernate-A media with a 2% B-27 and GlutaMAX supplement (0.5 mM final concentration). Collected tissues were then processed using a modified 10x Genomics protocol. Lysis buffer containing (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% Nonidet-P40, 0.01% Digitonin, 1 U/ml Rnase inhibitor, 1% BSA) was added into a tube containing the RE. The samples were incubated on ice for 15 min with gentle pestle grinding with 5 strokes every 3 minutes. Following this, a wash buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.2 U/ml RNase inhibitor, 1% BSA) was added, and nuclei were filtered through a 50 μm filter. Debris was removed using OptiPrep density gradient media, and nuclei morphology was accessed under a light microscope. Nuclei were then immediately processed for snRNA-Seq. The nuclei from the RE were loaded into the 10x Genomics Chromium Single Cell System (10x Genomics) and libraries were generated using v3.1 chemistry following the manufacturer’s instructions. Three biological replicates for each control and SD were conducted. Libraries were then sequenced on an Illumina NovaSeq6000. snRNA-Seq data were first processed through Cell Ranger (v.5.0.0, 10x Genomics) with default parameters with ‘include-introns’, aligned to the custom mm10 genome with tdtomato-WPRE (Ai14) sequence. Matrix files generated from these processes were used for subsequent analysis.