Identifying synaptically enriched mRNA by using next generation sequencing

Preparation of Synaptosomes by density gradient and compare synaptically enriched mRNA to total homogenate transcriptome In brief, mouse brains were homogenized in 5 ml homogenization buffer (0.32 M sucrose, 1 mM EDTA pH 7.4, 1 mM dithiothreitol, phenylmethanesulfonyl fluoride solution (Sigma, 93482-50ML-F), complete mini-protease inhibitor (Roche Diagnostics) for 10 sec using a polytron. The homogenate was centrifuged at 1,000g for 10 min at 4°C yielding the nuclear fraction (Nuc) and the supernatant (Sup). The supernatant was centrifuged at 31,000g for 5 min at 4°C using a discontinuous Percoll gradient. The layer between 3% and 10% of Percoll were collected, washed in 30 ml of homogenization buffer and further centrifuged at 22,000 x g for 15 min at 4°CT. The pellet was resuspended in in EBC buffer (50 mM Tris-HCl pH 8.0, 120 mM NaCl and 0.5% NP-40) containing complete mini-protease inhibitor (Roche Diagnostics) and phosphatase inhibitor cocktail 1 and 2 (Sigma-Aldrich)) for Western blot analysis or lysis buffer for RNA extraction (GenElute Mammalian Total RNA Miniprep Kit, Sigma).通过密度梯度离心法制备突触小体（Synaptosomes），并比较突触富集mRNA与全匀浆转录组的表达特征。简言之，将小鼠脑组织置于5 mL匀浆缓冲液（含0.32 M蔗糖、1 mM pH 7.4的EDTA、1 mM二硫苏糖醇、苯甲基磺酰氟溶液（Sigma，货号93482-50ML-F）、完整型迷你蛋白酶抑制剂（Roche Diagnostics））中，使用polytron匀浆器匀浆10秒。将匀浆物于4℃下以1000g离心10分钟，得到细胞核组分（Nuc）与上清液（Sup）。将上清液置于不连续Percoll分层液体系中，于4℃下以31000g离心5分钟。收集3%至10% Percoll分层区间的组分，用30 mL匀浆缓冲液洗涤后，于4℃下以22000g再次离心15分钟。将所得沉淀重悬于含完整型迷你蛋白酶抑制剂（Roche Diagnostics）及磷酸酶抑制剂混合液1和2（Sigma-Aldrich）的EBC缓冲液（含50 mM Tris-HCl pH 8.0、120 mM NaCl及0.5% NP-40）中，用于蛋白质免疫印迹（Western blot）分析；或重悬于裂解缓冲液中，用于RNA提取（采用GenElute哺乳动物总RNA微量制备试剂盒，Sigma）。