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Sample GSM5176751

Query DataSets for GSM5176751

Status

Public on May 24, 2021

Title

C3_16S

Sample type

SRA

Source name

Midgut

Organism

insect gut metagenome

Characteristics

strain: w1118
tissue: Midgut of Drosophila
age: Adult
culture environment: Bacteria
treatment: Untreated
replicate: 3

Treatment protocol

For treatment group, 100mM sodium butyrate (Sigma) was added to Drosophila medium.

Growth protocol

The Drosophila melanogaster w1118 samples were reared on yeast-glucose medium (1L water, 100 g yeast, 100 g glucose, 1.2% agar, and 0.1% potassium sorbate) stocks at 25°C under 12 hours light/12 hours dark cycles in an artificial climate box.

Extracted molecule

total RNA

Extraction protocol

Total RNA of RNA-seq samples were homogenized in 1 ml TRIzol (Accurate Biotechnology) and precipitated by isopropyl alcohol. Total DNA of 16S-seq samples were extracted from Drosophila midgut using TIANamp genomic DNA kit (Tiangen, DP304) following the manufacturer's protocol.
After total RNAs of each sample were extracted, the mRNAs enriched by Oligo(dT) beads were fragmented using fragmentation buffer and reverse transcribed into cDNAs with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP, and buffer. The cDNA fragments were purified with QiaQuick PCR extraction kit (Qiagen), and were end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina HiSeq2500 by Genedenovo Biotechnology Co, Ltd.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

Adult flies, 3 to 4 days after eclosion.
16S rRNA
16S_OTU_counts.txt

Data processing

Library strategy: 16S rRNA sequencing
All raw sequencing data were quality controlled by fastq (version 0.18.0). Adapters, reads with more than 10% unknown nucleotide (N) content, and reads with more than half low quality (Q-value <20) bases were removed.
For RNA-seq data:
The sequencing data after quality control was firstly mapped to ribosome RNA (rRNA) database using Bowtie2 (version 2.2.8) to remove rRNA reads and get the clean sequencing data.
The clean sequencing data were mapped to the Drosophila melanogaster reference genome (dm6) using HISAT2 (version 2.2.4) with "-rna-strandness RF" and other default parameters after index building of the transcriptome.
The mapped reads of each sample were assembled and quantified by StringTie (version 1.3.1) in a reference-based approach. For each transcription region, a FPKM (fragment per kilobase of transcript per million mapped reads) value was calculated.
RNA differential expression analyses among groups were performed by DESeq2 (version 1.30.1), and RNA differential expression analyses between two samples were performed by edgeR (version 3.32.1). The genes/transcripts with a false discovery rate (FDR) < 0.05 and absolute fold change >2 were considered as differentially expressed genes/transcripts and used for downstream analyses.
For 16S-seq clean data:
Paired-end clean reads were merged as raw tags using FLASH (version 1.2.11) with a minimum overlap of 10 bp and a mismatch error rate of 2%.
Noisy sequences of the raw tags were filtered by QIIME (version 1.9.1) pipeline under specific filtering conditions to obtain the high-quality clean tags.
Clean tags were searched against the reference database (version r20110519, http://drive5.com/uchime/uchime_download.html) to perform reference-based chimera checking using UCHIME algorithm. All chimeric tags were removed to obtain the effective tags for downstream analyses.
Genome_build: dm6 (Release 6 plus ISO1 MT)
Supplementary_files_format_and_content: Sterile_RNA_counts.txt: Tab-delimited text file includes raw read counts for the RNA-seq data of sterile samples.
Supplementary_files_format_and_content: RNA_counts.txt: Tab-delimited text file includes raw read counts for RNA-seq samples.
Supplementary_files_format_and_content: 16S_OTU_counts.txt: Tab-delimited text file includes raw OTU counts for the 16S rRNA sequencing samples.

Submission date

Mar 17, 2021

Last update date

May 24, 2021

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