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Sample GSM2310291

Query DataSets for GSM2310291

Status

Public on Feb 22, 2017

Title

first instar larvae rep3

Sample type

SRA

Source name

first instar larvae

Organism

Helicoverpa armigera

Characteristics

developmental stage: larvae of first instar stage
tissue: whole body

Extracted molecule

total RNA

Extraction protocol

RNA was isolated from first instar larvae, fifth instar larvae and adults using the TRIzol (Invitrogen) reagent by following the company manual. Poly(A) mRNA was isolated from 20 µl total RNA using Oligo (dT) magnetic beads and then was broken into short fragments (about 200bp) in the presence of fragmentation buffer at 94 °C for 5 min.
The short fragments (about 200 bp) were used as templates for first-strand cDNA synthesis using random hexamer-primers. Subsequently, second-strand cDNAs were synthesized using buffer, dNTPs, RNaseH, and DNA polymerase I. After purification of short fragments with a QiaQuick PCR Purification Kit (Qiagen), samples werethen washed with EB buffer for end reparation and single nucleotide adenine addition. Finally, the short fragments were connected to sequencing adapters. Suitable fragments (about 200bp), as judged by agarose gel electrophoresis, were enriched with PCR amplification to prepare the sequencing library.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

first instar larvae rep3
1-lar-rep3

Data processing

5’ and 3’ low-quality ends of raw reads were trimmed using Fastx-tools. Adaptor sequences were also trimmed using Fastx-tools. Reads with over 5 Ns or the length of which is shorter than 80bp were removed. The remained pair-end reads were kept as clean reads, which were used for de novo assembly. Then, the Trinity (v2.0.6) software were used to assemble the clean reads with default parameters. All assemblies were performed on a server with 48 cores and 128 GB of memory. After assembly, the contigs longer than 200 bases were used for subsequent analysis.
The reads from 8 libraries were mapped to the assembled contigs using Bowtie 0.12.7 with no more than 2 mismatches within the ﬁrst 28 bp. The read counts accumulated on the contigs were normalized as fragments per kilobase of exon model per million mappedreads (FPKM) values. Quantitative analysis for each contig was estimated using FPKM values by RSEM (v1.1.17) software with default parameters.
BLASTx was performed to align the assembled contigs to the NR protein database for functional annotation. The e-value cut-off was set at 1E-5 for further analysis. Each assembled contig was assigned with the gene name and related function based on the best BLASTx hit (the smallest e-value). Assembled contigs assigned to the same gene were further compared, and the contig from the best e-value was adopted. If there was a tie between 2 assembled sequences, the one with the largest sequence identity was selected.
Supplementary_files_format_and_content: .txt file reports FPKM values

Submission date

Sep 14, 2016

Last update date

May 15, 2019

Contact name

Bao-Fa Sun

E-mail(s)

sunbf@big.ac.cn

Organization name

Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS)

Street address

Da-Tun Road