Mirounga angustirostris muscle raw transcriptome assembly
Raw assembly of northern elephant seal (Mirounga angustirostris) skeletal muscle transcriptome. Experimental Design: Skeletal muscle tissue for transcriptome sequencing was collected from three anesthetized juvenile northern elephant seals at three time points during an acute stress challenge experi...
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figshare
2016
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Online Access: | https://dx.doi.org/10.6084/m9.figshare.2062518.v3 https://figshare.com/articles/dataset/Mirounga_angustirostris_raw_muscle_transcriptome/2062518/3 |
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ftdatacite:10.6084/m9.figshare.2062518.v3 |
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record_format |
openpolar |
institution |
Open Polar |
collection |
DataCite Metadata Store (German National Library of Science and Technology) |
op_collection_id |
ftdatacite |
language |
unknown |
topic |
60604 Comparative Physiology FOS Biological sciences |
spellingShingle |
60604 Comparative Physiology FOS Biological sciences Khudyakov, Jane Likit Preeyanon Champagne, Cory Ortiz, Rudy Crocker, Daniel Mirounga angustirostris muscle raw transcriptome assembly |
topic_facet |
60604 Comparative Physiology FOS Biological sciences |
description |
Raw assembly of northern elephant seal (Mirounga angustirostris) skeletal muscle transcriptome. Experimental Design: 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. RNA Isolation: 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. Library Preparation and Sequencing: 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. Assembly: Raw sequencing data were deposited at NCBI Sequence Read Archive under study accession [SRP045540]. 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 Reference: 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. |
format |
Dataset |
author |
Khudyakov, Jane Likit Preeyanon Champagne, Cory Ortiz, Rudy Crocker, Daniel |
author_facet |
Khudyakov, Jane Likit Preeyanon Champagne, Cory Ortiz, Rudy Crocker, Daniel |
author_sort |
Khudyakov, Jane |
title |
Mirounga angustirostris muscle raw transcriptome assembly |
title_short |
Mirounga angustirostris muscle raw transcriptome assembly |
title_full |
Mirounga angustirostris muscle raw transcriptome assembly |
title_fullStr |
Mirounga angustirostris muscle raw transcriptome assembly |
title_full_unstemmed |
Mirounga angustirostris muscle raw transcriptome assembly |
title_sort |
mirounga angustirostris muscle raw transcriptome assembly |
publisher |
figshare |
publishDate |
2016 |
url |
https://dx.doi.org/10.6084/m9.figshare.2062518.v3 https://figshare.com/articles/dataset/Mirounga_angustirostris_raw_muscle_transcriptome/2062518/3 |
long_lat |
ENVELOPE(-136.483,-136.483,60.788,60.788) ENVELOPE(-61.426,-61.426,-63.966,-63.966) ENVELOPE(-59.717,-59.717,-62.450,-62.450) |
geographic |
Champagne Grinder Ortiz |
geographic_facet |
Champagne Grinder Ortiz |
genre |
Elephant Seal Elephant Seals |
genre_facet |
Elephant Seal Elephant Seals |
op_relation |
https://dx.doi.org/10.6084/m9.figshare.2062518 |
op_rights |
Creative Commons Attribution 4.0 International https://creativecommons.org/licenses/by/4.0/legalcode cc-by-4.0 |
op_rightsnorm |
CC-BY |
op_doi |
https://doi.org/10.6084/m9.figshare.2062518.v3 https://doi.org/10.6084/m9.figshare.2062518 |
_version_ |
1766401250795978752 |
spelling |
ftdatacite:10.6084/m9.figshare.2062518.v3 2023-05-15T16:05:21+02:00 Mirounga angustirostris muscle raw transcriptome assembly Khudyakov, Jane Likit Preeyanon Champagne, Cory Ortiz, Rudy Crocker, Daniel 2016 https://dx.doi.org/10.6084/m9.figshare.2062518.v3 https://figshare.com/articles/dataset/Mirounga_angustirostris_raw_muscle_transcriptome/2062518/3 unknown figshare https://dx.doi.org/10.6084/m9.figshare.2062518 Creative Commons Attribution 4.0 International https://creativecommons.org/licenses/by/4.0/legalcode cc-by-4.0 CC-BY 60604 Comparative Physiology FOS Biological sciences dataset Dataset 2016 ftdatacite https://doi.org/10.6084/m9.figshare.2062518.v3 https://doi.org/10.6084/m9.figshare.2062518 2021-11-05T12:55:41Z Raw assembly of northern elephant seal (Mirounga angustirostris) skeletal muscle transcriptome. Experimental Design: 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. RNA Isolation: 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. Library Preparation and Sequencing: 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. Assembly: Raw sequencing data were deposited at NCBI Sequence Read Archive under study accession [SRP045540]. 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 Reference: 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. Dataset Elephant Seal Elephant Seals DataCite Metadata Store (German National Library of Science and Technology) Champagne ENVELOPE(-136.483,-136.483,60.788,60.788) Grinder ENVELOPE(-61.426,-61.426,-63.966,-63.966) Ortiz ENVELOPE(-59.717,-59.717,-62.450,-62.450) |