DOI: 10.6084/m9.figshare.2062518
title: Mirounga angustirostris muscle raw transcriptome assembly (single ACTH challenge)

description: Raw assembly of northern elephant seal (Mirounga angustirostris) skeletal muscle transcriptome.<br><br>Experimental Design:<br>Skeletal muscle tissue for transcriptome sequencing was collected from three anesthetized juvenile northern elephant seals at three time points during an acute stress challenge experiment: before 0.25 U/kg ACTH injection (“0 hr”), 2 hours after injection (“2 hr”), and 24 hours (“24 hr”) after injection. <br><br>RNA Isolation:<br>Tissue samples were stored at −80 °C until extraction. In the laboratory, 75–165 mg of muscle tissue were minced with a scalpel on ice, transferred to a glass tissue grinder (Kimble-Chase Kontes Duall, USA), and homogenized with 1 ml of TRIzol Reagent (Life Technologies, USA). RNA was extracted according to the manufacturer’s protocol and purified with the RNeasy mini kit including a 30-minute on-column DNase I digest (Qiagen, USA). RNA was treated with TURBO DNase I (Ambion, Life Technologies, USA) for 30 minutes according to manufacturer’s protocol. Phenol:chloroform:isoamyl alcohol (Affymetrix, USA) extraction was performed to remove DNase I. RNA concentration was quantified on a Qubit fluorometer (Life Technologies, USA). Total RNA integrity was evaluated using 2100 Bioanalyzer RNA 6000 kit (Agilent, USA). All RNA samples had integrity values (RIN) of 7.6 - 9.0.<br><br>Library Preparation and Sequencing:<br>Libraries for sequencing were prepared according to TruSeq protocol (Illumina, USA). Specifically, mRNA was isolated from total RNA samples using oligo-d(T)25 magnetic beads (Dynabeads: Invitrogen, USA) and used as template for first-strand cDNA synthesis. After double-stranded (ds) cDNA synthesis, overhang fragments were end-repaired by incubation in the presence of T4 DNA polymerase and Klenow polymerase. The polished fragments were phosphorylated by T4 PNK, followed by the addition of a single ‘A’ base to the 3′ end of the blunt-ended phosphorylated fragments. This ‘A’ base prepared the cDNA fragments for ligation to proprietary adapter oligonucleotides (Illumina, USA) that have a ‘T’ base at their 3′ end. Ligation products were subjected to a final PCR amplification step (8–10 cycles) before library quantification and validation. Individual libraries were prepared with barcode and all nine samples (biological triplicates of 0 hr, 2 hr, and 24 hr samples) were pooled for sequencing on one lane. Sequencing was carried out for 100 cycles on the Illumina HiSeq 2500 platform with paired-end 100 bp reads and library insert size of approximately 500 bp. The average number of reads generated per sample was 28.5 ± 7.5 million with total reads numbering 256 million, with 25.6 billion total bases and 66.3 GB of data. Fastq files were generated using the Illumina Casava pipeline v1.8.2.<br><br>Assembly:<br>Raw sequencing data were deposited at NCBI Sequence Read Archive under study accession [SRP045540]. <br>Transcriptome assembly was performed using the Eel Pond mRNAseq Protocol (https://​khmer-protocols.​readthedocs.​org/​). Analysis was conducted in the cloud using an Amazon EC2 x-large machine (m1.xlarge), except for assembly, for which a 2x-large machine (m2.2xlarge) was required. Downloaded reads were trimmed of sequencing adapters and poor quality sequences using Trimmomatic v0.30 with TruSeq3-PE adapter sequences. Reads with quality scores <br>Reference:<br>Khudyakov JI, Preeyanon L, Champagne CD, Ortiz RM, Crocker DE. (2015) Transcriptome analysis of northern elephant seal (Mirounga angustirostris) muscle tissue provides a novel molecular resource and physiological insights. BMC Genomics 16: 64. <br><br>