Total RNA of mouse brain of E15.5

A pregnant mouse was bred under a 12-hour light and 12-hour dark cycle with access to unlimited food and water. Embryos were removed after 15.5 days (E15.5) of pregnancy. The developing whole brain was dissected from one of the embryos for total RNA extraction. Total RNA was isolated from a whole brain dissected from an E15.5 embryo of C57BL/6 background using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Small RNAs with sizes ranging from 16-30nt were isolated from 10μg total RNA using polyacrylamide gel electrophoresis. The complementary small RNA library was constructed using the Small RNA Sample Prep Kit version 1.0 (Illumina) according to the manufacturer's protocol with 5’-GTTCAGAGTT CTACAGTCCG ACGATC-3’ and 5’- TCGTATGCCG TCTTCTGCTT GT-3’ adapters at the 5’ and 3’ ends, respectively.

将怀孕小鼠置于12小时光照与12小时黑暗交替的光周期环境中饲养，全程提供无限量的食物与饮水。待其妊娠至15.5天（E15.5）时，摘取胚胎。从其中一枚胚胎中分离完整的发育中脑组织，用于总RNA提取。使用TRIzol试剂（Invitrogen），严格按照厂商说明书的操作流程，从C57BL/6背景的E15.5胚胎的完整脑组织中分离总RNA。取10μg该总RNA，通过聚丙烯酰胺凝胶电泳分离出长度介于16-30nt的小RNA片段。随后使用Illumina公司的Small RNA Sample Prep Kit 1.0版本试剂盒，依照厂商操作流程，分别在5’端与3’端连接接头序列5’-GTTCAGAGTT CTACAGTCCG ACGATC-3’和5’-TCGTATGCCG TCTTCTGCTT GT-3’，完成互补小RNA文库的构建。