Circadian liver_wt and Clock mutants

One hundred ninety wildtype male C57BL/6J mice age 7-10 weeks were purchased from Jackson Laboratory and entrained to a 12:12 light:dark cycle for 2 weeks. Mice were placed in light-tight boxes on a 12:12 LD cycle for 4 weeks, then released into constant darkness. Starting 30 hours after entry into DD (CT18), tissues from 5 (skeletal muscle) or 10 (liver or SCN) wildtype mice were collected every 4 hours for 48 hours, for a total of 12 timepoints. At timepoints 34 through 58 hours in DD, tissues from age-matched male C57BL/6J Clock/Clock homozygous mutant mice that had been treated with the same light entrainment protocol as the wildtype were collected. Tissues were collected from 5 Clock/Clock mutants at each timepoint except for 34 and 46 hours after the onset of DD, when tissues from 10 Clock/Clock mice were collected and run as independent replicates. Mice were sacrificed by cervical dislocation, and the optic nerves were severed in complete darkness; brain dissection was performed using illumination from an infrared viewer (FJW Industries, Palatine, IL). SCNs were dissected out, pooled at a density of 5 per tube in 100 ul RNAlater (Ambion, Austin, TX), frozen on dry ice, and stored at 80 degrees C until use. Keywords: time course duplicates consisted of pools from independent mice本实验共使用190只7~10周龄的野生型雄性C57BL/6J小鼠，购自杰克逊实验室（Jackson Laboratory），先经12小时光照:12小时黑暗光暗循环（light:dark cycle，简称LD）驯化2周。 随后将小鼠置于不透光箱中，维持12:12 LD光暗循环4周，再将其释放至持续黑暗（constant darkness, DD）环境中。 自小鼠进入持续黑暗后的30小时（即昼夜节律时间（circadian time, CT）18）起，每4小时采集一次小鼠组织，持续48小时，共计12个时间点；其中骨骼肌组织每组采集5只小鼠的样本，肝脏与视交叉上核（suprachiasmatic nucleus, SCN）则每组采集10只小鼠的样本。 在持续黑暗环境下的34至58小时时间点，同步采集与野生型小鼠周龄匹配的雄性C57BL/6J Clock/Clock纯合突变型小鼠的组织样本，该突变小鼠采用与野生型一致的光驯化方案。 除持续黑暗启动后的34小时与46小时两个时间点外，其余每个时间点均采集5只Clock/Clock突变小鼠的组织样本；在上述两个特殊时间点则采集10只突变小鼠的组织，作为独立重复样本开展实验。 小鼠通过颈椎脱臼法处死，且需在完全黑暗环境下切断视神经；脑组织解剖操作借助红外成像观察仪（FJW Industries，美国伊利诺伊州帕拉廷市）完成。 解剖获取视交叉上核后，以每管5个的密度将其置于100 μL RNAlater保存液（Ambion，美国得克萨斯州奥斯汀市）中混匀，随后置于干冰上速冻，并储存于80摄氏度环境直至使用。 关键词：本时间序列实验的重复样本均由独立小鼠的组织混合池构成。