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Sample GSM4851409

Query DataSets for GSM4851409

Status

Public on Jul 30, 2021

Title

PA germ-free hindgut 3 [F_H3]

Sample type

SRA

Source name

P.americana.Germfree

Organism

Periplaneta americana

Characteristics

tissue: germ-free hindgut

Extracted molecule

total RNA

Extraction protocol

Total RNA from two different regions (midgut and hindgut) of P. americana guts (pools of 10 gut tissues) were obtained by the RiboZol™ RNA Extraction Reagent (VWR) and cleaned with the PureLink RNA Mini Kit (ThermoScientific) following the on-column DNase digestion kit.
Poly(A) mRNA were filtered using the NEBNext Poly(A) mRNA Magnetic Isolation Module according to the manufacture’s instruction (NEB).
cDNA libraries were generated with the Ultra II directional RNA library kit (NEB). All samples were sequenced using an Illumina HiSeq 4000 sequencer (Illumina) with an average of 20 million pair-end reads (2x150 bp) at The James Cancer Center sequencing facilities (The Ohio State University, Columbus OH USA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Quality check of all raw reads was completed by FastQC 0.11.7. Reads within a ‘phred’ quality score below 30 and Illumina adaptors were removed from sequences by TrimGalore 0.4.5 (--illumina -q 30 --retain_unpaired --paired parameters). To discard possible bacterial contamination in samples, only reads that mapped to the publicly-available P. americana genome. For this, high quality reads from each treatment were mapped against the P. americana genome ((19) PGRX00000000.1) using Hisat2 2.1.0 (60) with the following parameters -p 8 -x P.americana.genome.index -1 <Tissue>.<replicate>.1.fq -2 <Tissue>.<replicate>.2.fq -S <file>.sam. All ‘sam’ files were converted to ‘bam’ files with SAMtools 1.6
For this, high quality reads from each treatment were mapped against the P. americana genome (PGRX00000000.1) using Hisat2 2.1.0 with the following parameters -p 8 -x P.americana.genome.index -1 <Tissue>.<replicate>.1.fq -2 <Tissue>.<replicate>.2.fq -S <file>.sam. All ‘sam’ files were converted to ‘bam’ files with SAMtools 1.6
Properly paired mapped reads were retrieved by fastq tool from SAMtools with the following parameters SAMtools fastq -@ 8 -f 2 -1 <Tissue>.<replicate>.1.mapped.fq -2 <Tissue>.<replicate>.2.mapped.fq <file>.bam. Finally, these mapped paired reads were used for de-novo transcriptomic assembly of the P. Americana gut using Trinty 2.5.0 with the following parameters: Trinity --SS_lib_type RF --seqType fq --max_memory 900G--SS_lib_type RF --seqType fq --max_memory 900G--SS_lib_type RF --seqType fq --max_memory 900G –CPU 48 –full_cleanup. The completeness of the P. americana gut transcriptome assembly was estimated by the BUSCO v 3.2.0 (63) pipeline (-m trans -l insecta_odb9 -c 16 parameters)using the insecta_odb9 orthologue database.
For transcriptome annotation, similarities to known proteins were retrieved by BLASTX (90) (Trintiy transcripts) and BLASTP (Transdecoder protein coding translated sequences) searches to UniRef90 database and for conserved domains (pfam) by HMMER. All BLAST and HMMER tables were merged and sorted using the Trinotate pipeline. All transcripts with at least one annotation by Trinotate were manually retrieved and used to create a P. americana gut master annotation table. As a last quality filter, all transcripts with at least one bacterial hits annotation were manually removed from the assembled transcripts. These no-bacterial quality filter transcript assemblies were considered for further analysis. Additionally, all transcripts were annotaded using blastx searches to all protein coding genes of pathways (20,AMPK,Chitin,DPP,GRH,Growthfactor,Hedgehog,Hippo,IMD,Insulin,JACKSTAT,JH,JNK,Notch,Pathway,PPO,Toll,TOR and Wingless) described in "The genomic and functional landscapes of developmental plasticity in the American cockroach paper" PMID: 29559629.
Differential expression analysis was performed with DESeq2 pipeline, briefly, treatments were classified depending tissue (midgut M or hindgut H) and germ-free, gnotobiotic or conventionalized conditions. Counts of RNA-seq reads from each treatment mapping to Trinity assembled genes and isoforms were quantified by Salmon v 0.9. Total “raw” transcript counts matrix was generated by tximport Bioconductor library in R. The software DESeq2 was used to detect differentially expressed isoforms and genes using as a contrast each bacterial treatment (germ-free, gnotobiotic and conventionalized) in the midgut and the hindgut. Isoforms/genes were considered differentially expressed if the adjusted p-value (Benjamini-Hochber [BH] multiple test correction) was less than or equal to 0.05 and an absolute fold-change above 1.5.
Genome_build: De-novo transcriptome assembly (trinity.fasta file)
Supplementary_files_format_and_content: transcriptome assembly in fasta format
Supplementary_files_format_and_content: Transcritpome annotation tab separate format
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
Supplementary_files_format_and_content: Matrix table with the FPKM normalized values by DEseq2 for every gene and every sample
Supplementary_files_format_and_content: Matrix table with DESeq2 values (log2FoldChange, p-value and p-adjust value) for every gene and every contrast
Supplementary_files_format_and_content: Annotations of P. americana transcripts involved in metabolic pathways described in PMID: 29559629

Submission date

Oct 23, 2020

Last update date

Jul 30, 2021

Contact name

Aturo Vera Ponce de Leon

E-mail(s)

[email protected]

Organization name

The Ohio State University

Department

EEOB