title: GSM9161741: Endostatin-1; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted from 100 mg of eWAT which was homogenized with a mortar and pestle in liquid nitrogen and separated with 1 mL trizol (Qiagen, 73906) with repeated vortex steps to dissociate lipid phase. The lipid layer was removed from the surface of the trizol after 5 minute incubation at room temperature and a 12,000g spin for 10 minutes at 4°C. The clear homogenate was then transferred to a new tube, and 200 µL of chloroform was added. Samples were vortexed vigorously, incubated for 10 minutes at room temperature and centrifuged for 15 minutes for 12,00g at 4°C. RNA was then collected from the aqueous phase and 500 µL isopropanol was added. RNA was vortexed, incubated for 10 minutes, and then centrifuged as per the first centrifugation step. Following centrifugation, the supernatant was removed and the pellet was washed with 75% ethanol with a final centrifugation at 7500g for 5 minutes at 4°C. Once RNA was collected, cleanup was performed using the RNeasy Plus Mini Kit columns (74136) according to manufacturer's recommendations. Approximately 500ng Total RNA was used for Total RNA-seq library preparation by following the NEB Directional RNA library preparation guide with NEBNext rRNA depletion kit v2 (human/mouse/rat) (New England Biolabs, Ipswich, MA). The first step in the workflow involved the depletion of rRNA by hybridization of complementary DNA oligonucleotides, followed by treatment with RNase H and DNase to remove rRNA duplexed to DNA and original DNA oligonucleotides, respectively. Following rRNA removal, the left RNA was fragmented into small pieces using divalent cautions under elevated temperature and magnesium. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Strand specificity was achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM). The incorporation of dUTP in second strand synthesis effectively quenches the second strand during amplification, since the polymerase used in the assay will not incorporate past this nucleotide. These cDNA fragments then went through an end repair process, the addition of a single 'A' base, and then ligation of the adapters. The products were then purified and enriched with PCR to create the final RNA-Seq library.