title: GSM6165021: KO26; Mus musculus; RNA-Seq
library_construction_protocol: RNA was isolated from aliquots of liver tissue (10 mg) procured from euthanized mice (male myeloid cell conditional FoxO1-knockout and male wild-type littermates, n=4 per group) after 25 weeks of NASH diet feeding, using the RNeasy Micro Kit (QIAGEN). RNA purity was first checked on 1% agarose gel and was then validated, using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA). Aliquots of liver RNA (1 µg) were used for the preparation of sequencing libraries from each sample, using NEBNext UltraTM RNA Library Prep Kit for Illumina (New England Biolabs, USA). Briefly, mRNA was purified from total RNA, using poly-T oligo-attached magnetic beads. Fragmentation was carried out, using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X) (New England Biolabs, USA). Random hexamer primer and M-MuLV Reverse Transcriptase (RNase H) were used for the first strand cDNA synthesis. Second strand cDNA synthesis was performed, using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments of 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then USER Enzyme (New England Biolabs, USA) was used with size selected and adaptor-ligated cDNA at 37 °C for 15 min, followed by 5-min incubation at 95 °C. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. RNA-Seq was conducted at the Novogene (Novogene, CA, USA).