title: GSM6433338: iWAT, female, infected, rep4; Mus musculus; RNA-Seq
library_construction_protocol: iWAT was harvested from mice and stored in TRIzol (Cat. #15596026, Invitrogen, Massachusetts, USA). Total RNA was then purified from iWAT using an RNeasy Kit (Cat. #74004, Qiagen, Dusseldorf, Germany) as per the manufacturer's recommendations. The RNA was purified in 30 µL of nuclease-free water (Cat. #129112, Qiagen, Dusseldorf, Germany), and RNA concentration measured on a NanoDrop 2000 (Cat. #ND2000, Thermo Fisher Scientific, Massachusetts, USA). Samples were shipped to Novogene (Cambridge, UK) to undergo quality control, library preparation and sequencing. Library preparation was performed by Novogene (Cambridge, UK). Messenger RNA (mRNA) was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in a First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was then performed using DNA Polymerase I and RNase H. Remaining overhangs were converted to blunt ends via exonuclease/polymerase activity. Following adenylation of 3' ends of DNA fragments, adaptors with hairpin loop structures were ligated. To select cDNA fragments of 370~420 bp in length, library fragments were purified using AMPure XP beads (Beckman Coulter, Beverly, USA), as per the manufacturer's instructions. PCR was then performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) primers. Finally, PCR products were purified (AMPure XP system) using AMPure XP beads (Beckman Coulter, Beverly, USA), as per the manufacturer's instructions, and library quality was assessed using a Bioanalyzer 2100 (Agilent Technologies, CA, USA).