organism: Sousa chinensis
description: Protocols: The blood sample of the Indo-Pacific humpback dolphin was obtained from a wild male adult individual The fresh blood were collected into EDTA-containing tubes, and then centrifuged for 5 min at 400-500g. The red supernatant was discarded and then 18 ml of Red Blood Cell Lysis Buffer (Beyotime Institute of Biotechnology) were added, mixed gently by flicking the tubes and lasted for 10 min. The tubes were then centrifuged at 1000g for 5 minutes at 4 degrees C to collect the leukocyte pellet. Total RNA was extracted from the leucocytes of Indo-Pacific humpback dolphin using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Illumina sequencing was performed at Beijing Genomics Institute (BGI)-Shenzhen, China. mRNA with poly(A) tail was isolated from 20ug total RNA treated with DNase I using Magnetic Oligo (dT) Beads (Illumina). The mRNA was fragmented into small pieces (200-700 bp) by treatment with divalent cations at 94 degrees C for 5 minutes. With random hex-amer primers (Illumina), the double-stranded cDNA was synthesized using the SuperScript double-stranded cDNA synthesis kit (Invitrogen) and was further subjected to end-repair using T4 DNA polymerase, the Klenow fragment, and T4 polynucleotide kinase followed by a single A base addition using Klenow 3' to 5' exo-polymerase, then ligated with an adapter or index adapter using T4 DNA ligase. To select the proper templates for downstream enrichment, the products of ligation reaction were purified on 2% agarose gel. The cDNA fragments (about 200 bp) were excised from the gel. Fifteen rounds of PCR amplification were carried out to enrich the purified cDNA template using PCR primer PE 1.0 and 2.0 (Illumina) with phusion DNA polymerase. Finally, the cDNA library was constructed with 200 bp insertion fragment.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2013-08-30T17:00:54Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T16:36:41Z
attributes.tag: External Id
attributes.value: SAMEA2149044
attributes.tag: INSDC center name
attributes.value: College of Life Science, Sun Yat-Sen University
attributes.tag: INSDC first public
attributes.value: 2013-08-30T17:00:54Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T16:36:41Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-1748:leucocyte
attributes.tag: age
attributes.value: 40 year
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: Indo-pacific humpbacked dolphin
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: organism part
attributes.value: Leucocyte
attributes.tag: sample name
attributes.value: E-MTAB-1748:leucocyte
attributes.tag: scientific_name
attributes.value: Sousa chinensis
attributes.tag: sex
attributes.value: male
attributes.tag: specimen with known storage state
attributes.value: fresh specimen