GSM4476066: VMH_refeed_WT_1; Mus musculus; RNA-Seq
2-4 independents hypothalamic nuclei punches were pooled together per biological replicate. Hypothalamus punches were homogenized manually by Pellet-Pestle (Kimble, 749515) for 1 minutes with 1mL QIAzol Lysis Reagent (Qiagen). Samples were then centrifuged at 4°C for 10min at 15,000 x g. In a new tube, 200uL chloroform was added to homogenate and mixed thoroughly before repeating the previous centrifugation. The aqueous chloroform phases were pipetted into new tubes, followed by addition of one volume of 70% EtOH to each sample. Total RNA was purified and collected from RNeasy Mini spin columns according to RNeasy Lipid Mini Kit (Qiagen, 74804). RNA libraries were prepared for sequencing using standard Illumina protocols