GSM3070853: 500nm-550nm_2; Xiphophorus maculatus; RNA-Seq
RNA isolation was performed following the Qiagen RNeasy RNA isolation protocol (Qiagen, Valencia, CA, USA). Skin samples harvested from fish were first homogenized using a hand-held homogenizer in a 1.5 mL microcentrifuge tube while the sample remained frozen in TRI Reagent (Sigma Inc., St Louis, MO, USA). After homogenization, 300 μL of fresh 4°C TRI Reagent was added to the samples followed by incubation (rt) for 5 min. Chloroform extraction was performed by adding 120 μL chloroform and shaken for 15 sec. Samples were centrifuged (16,100 rcf for 5 min at 4°C) for phase partition. The aqueous layer was transferred to a new 1.5 mL microcentrifuge tube and a second chloroform extraction performed (300 μL TRI Reagent, 60 μL chloroform). After extraction, nucleic acids in the aqueous phase were precipitated with 500 μL 70% EtOH in diethylpyrocarbonate (DEPC) treated water. The sample was then transferred to a Qiagen RNeasy mini spin column and on-column DNase treatment was performed for 15 min at 25°C. RNA samples were then washed and eluted in 100 μL RNase free water. RNA concentration was measured with a Qubit 2.0 fluorometer (Life Technologies, Grand Island, NY, USA). To further assess the RNA quality, a RNA integrity (RIN) score was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). All samples processed for RNA sequencing had a RIN score above 8. RNA libraries were prepared for sequencing using standard Illumina protocols
GEO Accession
GSM3070853