title: Illumina HiSeq 4000 paired end sequencing; RNAseq of scWAT obtained from normoxic (21% oxygen) and hypoxic (10% oxygen) mice
design: RNAseq of scWAT obtained from normoxic (21% oxygen) and hypoxic (10% oxygen) mice
library_construction_protocol: The scWAT was removed from the inguinal region, snap frozen and stored at -80°C until further analysis RNA extraction from mice scWAT was performed by homogenisation in RNA STAT-60. Samples were broken up using a pestle and mortar under liquid nitrogen. The broken pieces were then put in 2 mL screw-top tubes with Lysing Matrix D (MP Biomedicals). 1 mL STAT-60 was added to the tissue samples, followed by homogenisation using Precellys 24 Touch. The homogenate was then transferred to 1.5 mL eppendorfs. Tubes were spun at 13,000 rpm for 5 min at 4°C to remove debris. The supernatant was removed and transferred to fresh tubes containing 200 µL of Chloroform. Samples were vortexed at maximum speed for 15 s, and spun at 12,000 g for 15 min at 4°C. A clear aqueous supernatant of around 600 µL for every 1 mL of STAT-60 added will have formed. The aqueous phase was transferred to fresh tubes containing 500 µL of isopropanol. Tubes were mixed by inversion several times and left at -20°C overnight. After overnight incubation, the precipitates were spun at 12,000 g for 10 min at 4°C to pellet RNA. After removing the supernatant, 1 mL of cold 70% ethanol (stored at -20°C) was added to the pellet and washed by vortexing briefly. Samples were then spun at 8000 g for 5 min at 4°C before discarding the supernatant. Pellets were air-dried for 10-15min on ice until transparent. Nuclease-free water was added to the pellet and incubated at 60°C for 10 min. Samples were placed on ice and re-suspended by pipetting up and down. 1 μg of total RNA was quality checked (RIN >7) using an Agilent Bioanalyser 2100 system and then used to construct barcoded sequencing libraries with Illumina's TruSeq Stranded mRNA Library Prep Kit following manufacturer's instructions