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Sample GSM5335138

Query DataSets for GSM5335138

Status

Public on Aug 06, 2021

Title

Antibiotics-3_RNA

Sample type

SRA

Source name

Midgut

Organism

Spodoptera frugiperda

Characteristics

strain: 3-day-old sixth instar larvae
culture environment: Feed on artificial diet
treatment: with antibiotics
replicate: 3

Treatment protocol

The newly hatched larvae were fed on the artificial diet with four antibiotics mixtures consisted of 500 µg/mL each of penicillin, gentamicin, rifampicin, and streptomycin, and the control larvae were fed on the artificial diet without antibiotics.

Growth protocol

The larvae were fed on the artificial diet including soybean powder, wheat germ, and yeast as described previously (Jia et al., 2009) for three generations at 27±1°C, 70±5% relative humidity, and the photoperiod light: dark =16 h: 8 h.

Extracted molecule

polyA RNA

Extraction protocol

The genomic bacterial DNA was extracted by using the classical Phenol/Chloroform/Proteinase K method (Bargues et al., 2007). Total RNA from the midguts of S. frugiperda in the control and antibiotics-treated group was extracted by using the TRIzol reagent according to the manufacturer’s protocol (Invitrogen, California, USA).
RNA concentration and purity were measured using NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). 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 a random hexamer primer and M-MuLV Reverse Transcriptase. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. The library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). The library preparations were sequenced on an Illumina platform nova-seq6000 and paired-end reads were generated.
RNA-seq and 16S rDNA-seq

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

PolyA RNA
The midguts were dissected from 3-day-old sixth instar larvae

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, GC-content, and sequence duplication levels of the clean data were calculated. All the downstream analyses were based on clean data with high quality.
For RNA-seq data, the adaptor sequences and low-quality sequence reads were removed from the data sets. Raw sequences were transformed into clean reads after data processing. These clean reads were then mapped to the S. frugiperda genome sequence (NCBI: ZJU_Sfru_1.0). Only reads with a perfect match or one mismatch were further analyzed and annotated based on the reference genome. Hisat2 (version 2.0.4) tools soft were used to map with reference genome by the parameters “--dta -p 6 --max intronlen 5000000”.
For RNA-seq clean data, the mapped reads were assembled with the StringTie software (version 1.3.4d) with the parameters “--merge -F 0.1 -T 0.1”.
For RNA-seq clean data, after detecting transcriptome library was eligible, The library preparations were sequenced on an Illumina platform nova-seq6000. The FPKM（Fragments Per Kilobase of transcript per Million fragments mapped）value representing the expression level of transcripts was calculated.
For RNA-seq clean data, differential expression analysis of the control and antibiotics-treated groups was performed using the DESeq2 (version 1.6.3) with the parameter "default:test="Wald",fitType="parametric". DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P values were adjusted using Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P-value < 0.01 found by DESeq2 were assigned as differentially expressed.
For 16S-seq data, the gut microbiota was sequenced on the Illumina nova-seq6000 platform with the paired-end method. The raw reads were filtered by using the software Trimmomatic (version 0.33).The primer sequences were identified and removed by using the CutAdapt (version 1.9.1) software.
For 16S-seq data, high-quality reads were spliced by using the software FLASH (version 1.2.7), and clean reads were obtained.
For 16S-seq clean data, chimerism was removed by using the software Uchime (version 4.2), and the effective reads were obtained.
Genome_build: Spodoptera frugiperda genome sequence (NCBI: ZJU_Sfru_1.0).
Supplementary_files_format_and_content: Tab-delimited text file (.txt) includes raw read counts for the RNA-seq data and OTU counts for the 16S-seq data of all samples. In addition, the sequences for the counted genes of the RNA-seq data are provided by a FASTA file (.fa).

Submission date

May 24, 2021

Last update date

Apr 20, 2022

Contact name

Xiaoyun Wang

E-mail(s)

wang_xiaoyun@gibh.ac.cn

Organization name

Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences

Department

Infection and Immunity