title: GSM5472118: Bursa RCT-009 Traumatic; Homo sapiens; RNA-Seq
library_construction_protocol: Samples received in the laboratory were stored at 4oC for 12-24 h for complete immersion of RNAlater solution to protect RNA integrity and to avoid RNA degradation. Samples were the held at -80oC until extraction of total RNA. Total RNA was isolated using RNAeasy Mini Kit (Thermo Fisher Scientific, Carlsbad, CA) according to manufacturer's protocol. Briefly, tissues were allowed to thaw, then dissected into 23 mm3 cubes using two scalpels under the tissue culture hood. RNAlater was replaced with 1 mL TRIzol reagent (Life Technologies, Carlsbad, CA) and tissues were individually homogenized at 30,000 rpm for 2 min in 1.5 mL Eppendorf tubes with the use of an electric homogenizer. After 10 min of incubation of lysate at room temperature, 200 µL of chloroform was added followed by 30 sec of vigorous vortexing. Samples were then incubated at room temperature for 10 min, followed by centrifugation at 13,000 rpm for 10 min at 4oC. Next, aqueous upper phase containing total RNA was transferred to a fresh 1.5 mL Eppendorf tube and  an equal volume of RNase-free isopropanol (2-propanol) was dispensed prior to brief but extensive shaking. The contents were incubated at room temperature for 10 min, after which centrifuged at 13,000 rpm for 15 min at 4oC to obtain a pellet. Next, supernatant was removed and the pellet was washed once with RNase-free 70% ethyl alcohol (1 mL volume) by centrifugation (13,000 rpm for 5 min). Then, the supernatant was removed, and residual ethyl alcohol was allowed to air dry for a few minutes. Lastly,  the pellet was eluted in 30 L RNase-free water. The qulity of total RNA was assessed using RNA integrity number (RIN) system with the use of an Agilent 2100 BioAnalyzer (Agilent technologies, Santa Clara, CA) and the concentration of each samples was measured with a NanoDrop ND-1000 instrument (NanoDrop Technologies). All samples were stored at 80°C until use for RNA-seq or real-time qPCR. Qiagen FastSelect