GSM4083463: HFD4_IP-GFP; Mus musculus; RIP-Seq
The hypothalamus was rapidly dissected using a stainless steel brain matrixand immediately frozen in liquid nitrogen. RNA was purified after ribosomal immunoprecipitation using the RNeasy Micro Kit (QIAGEN). RNA integrity was assessed using an Agilent 2100 bioanalyzer. Due to low amount of input material, pre-amplification using the Ovation RNASeq System V2 was performed. Total RNA was used for first strand cDNA synthesis, using both poly(T) and random primers, followed by second strand synthesis and isothermal strand-displacement amplification. For library preparation, the Illumina Nextera XT DNA sample preparation protocol was used, with 1 ng cDNA input.