Design: Sample collection, RNA extraction and sequencing: Five 10-month old individuals of E. sinensis were collected from the Chongming aquafarm (31º19`22" N; 121º45`52"E). An approximately 500 μL hemolymph sample was collected from the third pereiopod of each crab, diluted in an equal volume of anti-coagulant solution (NaCl 510 mmol l−1; glucose 100 mmol l−1; citric acid 200 mmol l−1; Na-citrate 30mmol l−1; EDTA-Na2 10 mmol l−1; pH 7.3) and centrifuged for 10 min at 800 rpm at 4 °C. The cell suspension medium was removed and resuspend the cell pellet in trizol. Total RNA was extracted using Trizol reagent (Invitrogen, USA) following manufacturer’s instructions. The total RNA quantity and quality were determined by Agilent Technologies 2100 Bioanalyzer (Agilent Technologies). Genomic DNA was digested with DNase I (Takara, Japan). Then poly (A) mRNAs were purified for cDNA library construction using UltraTM RNA Library Prep Kit (NEB, USA). Illumina HiSeq library construction was performed according to the manufacturer’s instruction (Illumina, USA). RNA fragments were used to synthesize first-strand cDNA using random hexamer primers, which were transformed into double-strand cDNA with RHase H and DNA polymerase I. A paired-end library was constructed from the cDNA synthesized using a Genomic Sample Prep kit (Illumina, USA). The fragments in desirable lengths were purified using a QIAquick PCR purification kit (Qiagen, Germany), end repaired, and linked with sequencing adapters. AMPureXP beads (NEB, UK) were used to remove the unsuitable fragments, and the sequencing library was constructed with PCR amplification. After being checked with PicoGreen staining and fluorospectrophotometry and quantified using an Agilent 2100 Bioanalyzer, the multiplexed DNA libraries were mixed in equal volume with normalized concentration of 10 nM. The sequencing library was then sequenced with the Illumina HiSeq 2000 platform using paired-end strategy in a single run. Data filtering, de novo assembly, and bioinformatics analysis: The raw sequencing reads of the three samples were mixed together to perform a stringent filtration process and subsequent de novo assembly. The adaptor contamination was removed, and the reads were screened from the 3′-end to the 5′-end to trim the bases, with a quality score of Q < 20 using 5 bp windows. The reads with final length less than 25 bp were removed. All clean reads from the two libraries were jointly assembled into contigs using Trinity software. The assembled contigs were conducted to predict protein-coding regions through the GetORF module of EMBOSS package . All the protein-coding sequences were submitted for the basic local alignment search tool (BLAST) similarity searches against the NCBI non-redundant protein database and EuKaryotic Orthology Groups database with the e-value of the top hit lower than 1 e−5. Furthermore, GoPipe software was used for blastp (cutoff value of <1 e−5) against the Swiss-Prot and TrEMBL databases. The blastp results showed that GO annotation associated with “biological process,” “molecular function,” and “cellular component” was obtained using gene2go. Similarly, the predicted protein sequence was submitted for bidirectional blastp (cutoff e-value of <1 e−3) similarity searches against the KEGG database to assign KO numbers. Metabolic pathways were generated according to the KO assignment, with tools supplied by KEGG. The mapped read count of the given gene was affected by its length and sequencing depth; the reads per kb per million reads (RPKM) were calculated to standardize gene expression level. Comparative expression analysis: RPKM was directly used to compare the expression levels of all genes. This process was completed using the DEGseq package based on the MA plot-based with random sampling model. The Benjamini–Hochberg method was used to determine the threshold of the E value in multiple tests. In the present study, “q < 10−3” and “|log2 (RPKM a / RPKM b)|≥ 2” were chosen to identify organ-biased genes.
Submitted by: Shanghai Ocean University
Study:
Eriocheir sinensis breed:Chen Raw sequence readsshow Abstracthide AbstractThe hemocytes of Eriocheir sinensis are divided into three types: hyalinocytes, migranulocytes, and granulocytes. Additional information is necessary to elaborate the mechanisms involved in gene expression of those cells.
Sample:
The Chinese mitten crab (Eriocheir sinensis) possesses an open circulatory system, in which hemolymph moves through interconnected sinuses or spaces surrounding organs called hemocoels. Nutrients, oxygen, hormones, and cells are distributed throughout the circulatory system.Library:
Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: unspecified
Layout: PAIRED
Spot descriptor:
1 forward151 reverse
Runs:
1 run, 11.9M spots, 3.6G bases, 1.6Gb