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 ÎŒm 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.研究目的：通过单细胞核RNA测序（single nuclear RNA sequencing, snRNA-Seq）解析丘脑reuniens核（Nucleus Reuniens, RE）在睡眠剥夺后出现的分子差异。实验样本采集自7-8周龄雄性FosTRAP;tdTomato小鼠的丘脑reuniens核（RE），采用已发表的实验方案（Kim等，2021）完成组织收集。简言之，通过显微穿刺术从单只小鼠体内分离RE组织，将其置于添加有2% B-27添加剂与终浓度0.5 mM GlutaMAX补充剂的Hibernate-A培养基中。收集得到的组织采用改良的10x Genomics实验流程进行处理：向装有RE组织的离心管中加入裂解缓冲液，其组分包含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抑制剂以及1%牛血清白蛋白（BSA）。将样本置于冰上孵育15分钟，期间每隔3分钟使用研磨棒轻柔研磨5次。孵育完成后，加入洗涤缓冲液（组分：10 mM Tris-HCl pH 7.4、10 mM NaCl、3 mM MgCl₂、0.1% Tween-20、0.2 U/ml RNase抑制剂、1% BSA），随后将细胞核通过50 μm滤膜过滤。使用OptiPrep密度梯度介质去除细胞碎片，并通过光学显微镜观察细胞核形态。随后立即对分离得到的细胞核开展单细胞核RNA测序：将RE来源的细胞核上样至10x Genomics Chromium单细胞系统（10x Genomics），采用v3.1化学试剂盒并严格遵循制造商说明书构建测序文库。对照组与睡眠剥夺（sleep deprivation, SD）组均设置3次生物学重复。构建完成的文库在Illumina NovaSeq6000测序平台上完成测序。snRNA-Seq测序数据首先通过Cell Ranger（v5.0.0，10x Genomics）以默认参数处理，并开启"include-introns"参数；随后将测序数据比对至带有tdtomato-WPRE（Ai14）序列的定制化mm10基因组。上述流程生成的矩阵文件将用于后续的生物信息学分析。