alias: GSM2809912
title: GSM2809912: HFD_liver_Control1; Mus musculus; RNA-Seq
library_construction_protocol: RNA was isolated with TRIzol reagent, as per the manufacturers instructions. 5 μg of total RNA was depleted of rRNA using a Ribo-Zero Gold kit (Epicentre) and first strand cDNA was made using 200 ng RNA, 3 ug random hexamers, 1 μg Superscript III, and standard dNTPs in the presence of Actinomycin D. First strand cDNA was purified by PCI extraction, ethanol precipitated, and resuspended in water. Second strand cDNA was synthesized with E. coli DNA polymerase, in the presence of E. coli DNA ligase and RNase H (all from NEB), using dNTPs in which dUTP was substituted for dTTP, at 16°C for 2 hrs. After purification on a Clean and Concentrate column (Zymo Research), cDNA was fragmented using Fragmentase (NEB), PCI extracted and precipitated as above. End repair, A-tailing, and barcoded adapter ligation were performed as described (Illumina), and the second strand cDNA was digested with USER enzyme (NEB). Libraries were amplified from the resulting adapter-ligated first-strand cDNA using Phusion (NEB) and 16 cycles of amplification. Libraries were size-selected on 1.5% agarose gels to a size range of ~180-250 bp.
identifiers.namespace: GEO
identifiers.id: GSM2809912