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| Status |
Public on Jan 25, 2019 |
| Title |
2-4 hpo embryos |
| Sample type |
SRA |
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| Source name |
2-4 hpo embryos
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| Organism |
Bactrocera dorsalis |
| Characteristics |
age: 2-4 hpo
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| Extracted molecule |
total RNA |
| Extraction protocol |
0-1, 2-4 and 5-8 post-oviposition (hpo) embryos were collected and immediately stored in RNAlater® Solution (Ambion). Total RNAs were extracted using Trizol reagent (Invitrogen, CA, USA). Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with total RNA for the construction of sequencing libraries.
RNA libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250-300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.4 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.4. We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
HTSeq v0.9.1 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels.
Genome_build: ASM78921v1
Supplementary_files_format_and_content: Excel files reporting readcounts and fpkms
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| Submission date |
Aug 13, 2018 |
| Last update date |
Jan 25, 2019 |
| Contact name |
Wei Peng |
| E-mail(s) |
pengweijack@163.com
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| Organization name |
Huazhong Agricultural University
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| Street address |
Shizishan Street No. 1, Hongshan District, Wuhan,Hubei 430070, P.R. China
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| City |
Wuhan |
| ZIP/Postal code |
430070 |
| Country |
China |
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| Platform ID |
GPL21756 |
| Series (1) |
| GSE118472 |
Transcriptome analysis of the oriental fruit fly Bactrocera dorsalis early embryos |
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| Relations |
| BioSample |
SAMN09813456 |
| SRA |
SRX4548871 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3330636_2-4_hpo_embryos_gene_expression.xlsx |
591.6 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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