Description page of RNA-Seq

Description:

The RNA-seq tracks show mapping of organ-specific RNA sequencing reads to X. maculatus genome.

Display Convention and Configuration:

Methods:

1. Fish Utilized and RNA Isolation: X. maculatus Jp 163 A in the 116th inbred generation (i.e., sibling mating) were provided by the Xiphophorus Genetic Stock Center (http://www.xiphophorus.txstate.edu/). All fish were maintained in aquaria filled with filtered aquifer water from San Marcos, TX on a 13 hr light/11 hr dark cycle under 10,000 K fluorescent light (Coralife T8 Lamp 10,000 K, 32 W). All fish were maintained and samples taken in accordance with approved protocols (IACUC# 2015107711).

2. RNA isolation: Total RNA was isolated from brain, skin, liver and heart. Briefly, RNAlater was removed from respective microcentrifuge tubes followed by addition of 750 μL of QIAzol (Qiagen) in 2.0 mL collection tubes designed for automated tissue homogenization and RNA isolation stations. Organs were homogenized using the TisseLyser II (Qiagen) facilitated by stainless beads (Qiagen) for 10 min at 25 Hz. RNA isolation was subsequently performed using a QIAcube HT (Qiagen) automated bio-sample isolation system. The isolation system is equipped with a robotic arm with 8 pipettes. Each pipette is able to pick and eject pipette tips, self-clean, and transfers liquids between well/columns, or between reagent reservoirs and well/columns in standard 96-well plate formats. Each sample was independently maintained throughout the isolation process. Briefly, 150 µL of chloroform was added to each isolation tube and the samples were vigorously shaken for 15 sec and then phases partitioned by centrifugation (12,000×g for 15 min at 4°C). The aqueous phase containing nucleic acids was transferred to a new sample tube by a rack of automated pipettors. After extraction, nucleic acids were precipitated with 500 µL 70% ethanol. RNA was then purified using a Qiagen RNeasy mini RNA kit (96-well plate) and eluted following the manufacturer’s protocol. RNA was quantified with a Qubit 2.0 fluorometer (Life Technologies, Grand Island, NY, USA) and RNA quality was measured on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) to confirm RIN scores were above 8.0 prior to sequencing. Concentrations of RNA samples were adjusted to 100 ng/μL with RNase-free water (Qiagen) prior to RNA sequencing.

3. RNA-Sequencing: Each RNA sample was used to construct an independent sequencing library using Illumina TruSeq library preparation kit (Illumina, Inc., San Diego, CA, USA). Libraries were sequenced as 150 bp paired-end fragments using Illumina Hi-Seq 2000 system (Illumina, Inc., San Diego, CA, USA). Short sequencing reads were filtered using an in-house data processing pipeline. Briefly, sequencing adaptors, if detected, were first removed from sequencing reads. Processed sequencing reads were subsequently trimmed and filtered based on quality scores by using a filtration algorithm that removed low-scoring sections of each read and preserved the longest remaining fragment. Processed sequencing reads were subsequently mapped to X. maculatus genome version 5.0 using Tophat2.

Credits:

RNA isolation, quantification, quality control, RNA-Seq reads quality control, data filtration, genetic mapping and UCSC browser configuration were performed by the Xiphophorus Genetic Stock Center staff/faculty and trainees.

References:

*For question about data accessibility, contact XGSC staff/faculty: xiphophorustsu@gmail.com