title: GSM4769560: JpB.skin.14; Xiphophorus maculatus; RNA-Seq
library_construction_protocol: For RNA isolation, fish were anesthetized in an ice bath and upon loss of gill movement were sacrificed by cranial resection. Skin and liver tissues were dissected directly into TRI reagent (Sigma Inc., St Louis, MO, USA) and flash frozen in an ethanol dry ice bath. For JpA, skin samples and liver samples were collected; For JpB, skin samples, and liver samples  were collected for RNA isolation and sequencing library preparation. RNA isolation was performed following the Qiagen RNeasy RNA isolation protocol (Qiagen, Valencia, CA, USA). Tissue 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 room temperature incubation for 5 min. Chloroform extraction was performed by adding 120 μL chloroform and shaken for 15 s. Samples were centrifuged (16,100 rcf for 5 min at 4°C) for phase partitioning. 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, an 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
attributes.tag: GEO Accession
attributes.value: GSM4769560