HarborSealBrainTranscriptome2016fa.fa (39.39 MB)

Brain transcriptomes of harbor seals demonstrate gene expression patterns of animals undergoing a metabolic disease and a viral infection

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posted on 2016-08-15, 22:03 authored by Stephanie RosalesStephanie Rosales

Harbor seal (Phoca vitulina) transcriptome libraries were prepared by using 11 harbor seal brain tissue samples. A disposable pestle was used to homogenize ~0.5ng of frozen brain in Trizol (Life Technologies, CA). The homogenate was centrifuged for 10min at 12,000 x g at 4°C, and the supernatant transferred to a clean tube. For every 1 mL of Trizol, 0.2mL of chloroform was added to the supernatant, vortexed briefly, and centrifuged at 10,000 x g for 18 minutes at 4°C. The aqueous layer was then transferred to a clean tube and equal volumes of 100% ethanol were added to samples, and loaded onto an RNeasy column for extraction as recommended by the manufacturer (Qiagen, CA). To remove DNA, samples were exposed to 2U of Turbo DNase (Life Technologies, CA) for 9 hours at 37°C. Harbor seal rRNA was removed using the Ribo-Zero Kit Gold (Human-Mouse-Rat) from Epicentre (WI, USA) following the manufacturer’s directions. High quality RNA was converted to cDNA using superscript II Reverse Transcriptase (Life Technologies, CA). Libraries were prepared for each of the 11 samples using the TruSeq paired end cluster kit v.3 from Illumina (San Diego, CA). Libraries were sequenced on two lanes of the Illumina Hi-Seq 2000 platform.

Harbor seal  brain transcriptome assembly was conducted using a combination of transcriptome guided and de novo methods. Eleven quality assured sequence libraries were combined and aligned to the hypothetical Weddell seal, Leptonychotes weddellii (NCBI accession: PRJNA232772) transcriptome, using the program Bowtie2-2.2.3 (21). Aligned sequences were used to build a de novo harbor seal transcriptome using Trinity 2.0.6 with parameters --single, and –full-cleanup. The longest representative transcript for each component or subcomponent in the transcriptome assembly was selected using ( ). 

The National Marine Fisheries (NMFS) authorized the collection of tissue samples from stranded marine mammals and satisfies The Marine Mammal Protection Act (MMPA) regulation 50 CR 216.22 and 216.37.


SeaGrant Starter Grant # NA010AR4170059, and NSF Graduate Research Fellowship #201213629