title: GSM4827529: high fat diet 2; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated and purified from cell or tissue samples using TRIzol reagent (Invitrogen, USA) following the manufacturer's protocol. RNase R (Lucigen, USA) was used to isolate circRNA from total RNA. We reverse-transcribed the cleaved RNA fragments into cDNA and synthesized U-labeled second-strand DNA with reverse-transcribed cDNA, E. coli DNA polymerase I, RNase H and dUTP. An A-base was then added to the blunt ends of each strand, preparing for ligation to the indexed adapters. Single- or dual-index adapters were ligated to the fragments, and AMPureXP beads were used for size selection. After heat-labile UDG enzyme treatment of the U-labeled second-strand DNA, the ligated products were amplified by PCR. The average insert size for the final cDNA library was 300 bp (±50 bp). Finally, we performed paired-end sequencing on an Illumina HiSeq 4000 (LC Bio, China) following the manufacturer's recommended protocol.