title: GSM8825939: Pre-cachectic tumor mouse, Inguinal adipose tissue, Mouse #60; Mus musculus; RNA-Seq
library_construction_protocol: Pieces of mouse frozen tissues were homogenized in TRIzol (Life Technologies #15596018). RNA was then isolated by adding chloroform. After centrifugation at 12,000 x g for 20min, the upper phase containing RNA was collected and a volume of 0.6 absolute ethanol was added (e.g., 240 µL ethanol for 400µL of collected upper phase). RNA samples were then loaded onto an Econospin column (Econospin # 1920-250), washed three times with RPE buffer (Qiagen #1018013), dried and eluted by adding RNA/DNAse-free water. RNA samples were then sent to Novogene (Cambridge, UK) for RNA-seq. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount. Further detailed methods can be found in the original paper of the study.