Status
Public on Mar 18, 2024
Title
RoB-positive1
Sample type
SRA
Source name
RoB; body minus head and midgut tissues
Characteristics
tissue: RoB; body minus head and midgut tissues
genotype: WT
treatment: Ingestion of Gram-negative (Gr–) bacterium Escherichia coli
Treatment protocol
Microbes (Escherichia coli(K12/D31) andBacillus subtilis (ATCC 6633)) were grown in separate Lysogeny Broth (LB) (47) for 17 h at 30 °C in a shaking incubator (220 revolutions per minute). Subsequently, aliquots of these bacteria were reinoculated into fresh broth and incubated 4 h under the same conditions to reach the log phase of growth. The bacteria were then washed three times in sterile phosphate buffered saline (PBS; 0.01 M phosphate buffer, 2.7 mM potassium chloride, 0.137 M sodium chloride, pH 7.4), and were diluted in sterile defibrinated rabbit blood to a final concentration of ~1 ×ばつ 106cells/mL. Male bed bugs (food-deprived >20 days) fed 1 h on defibrinated, microbe-laced (E. coliorB. subtilis) rabbit blood in a water-jacketed membrane feeder (Thermo Fisher Scientific Isotemp 2150 B14, USA) set to 37 °C, with stretched out parafilm as the membrane. Control bed bugs ingested sterile blood. Fully engorged bed bugs were separated and housed in glass jars until analysis. Each experiment had five replicates, with each replicate containing five insects in treatment and control samples.
Growth protocol
Bed bug colonies were maintained as previously described (46). Briefly, colonies were kept in the insectary of Simon Fraser University (SFU) at a temperature of ∼24 °C, ambient relative humidity, and a photoperiod of 14 h light to 10 h dark. Groups of 150 bed bugs were maintained in 50-mL glass jars fitted with a square of cardboard (2 cm ×ばつ 2 cm) at the bottom and a strip of cardboard (2 cm ×ばつ 4 cm) diagonally across the jar. Bed bugs in separate jars were fed on the forearm of a volunteer (Regine Gries) once every month. For feeding, jars were covered with fine mesh, inverted, and pressed against the forearm so that the bed bugs could feed through the mesh.
Extracted molecule
total RNA
Extraction protocol
Midgut and RoB tissues (RoB = rest of the body: bodies without heads and midgut tissues) of treatment and control bed bugs were dissected 12 h after an immune challenge, and total RNA was extracted using TRizol reagent (Invitrogen) following the manufacturer’s recommendations. The samples were quantified on a Nanodrop 1000 spectrophotometer v. 3.7 (Thermo Fisher Scientific, USA). RNA samples were used to generate complementary DNA (cDNA) for analysis using quantitative real-time polymerase chain reaction (qPCR) and transcriptome assembly.
We created a de novo transcriptome assembly from RNA extracted from midgut and RoB tissues of male bed bugs 12 h after ingesting blood containing E. coli or B. subtilis. RNA purification, first and second strand synthesis, adaptor ligation, quantification, validation, and Illumina sequencing were all done at GENEWIZ LLC. (South Plainfield, NJ, USA). The RNA samples were sequenced using a paired-end (PE) configuration, consisting of two 150 base pair (bp) strands. HiSeq Control Software (HCS) was used for image analysis and base calling. Raw sequence data (.bcl files) generated from Illumina HiSeq were converted into fastq files and demultiplexed with bcl2fastq 2.17 software (Illumina Inc, San Diego, CA, USA). For index sequence identification, one mismatch was allowed. After examining the quality of raw data, adapter sequences and nucleotides of poor quality were removed from sequence reads using Trimmomatic v.0.36. The trimmed reads were mapped to the reference genome available on ENSEMBL, using STAR aligner v.2.5.2b which utilizes a splice aligner to detect splice junctions and incorporates them to help align the entire read sequences to generate Binary Alignment Map (BAM) files. In the Subread package version 1.5.2, we used the ‘Counts’ feature to calculate the unique gene count. Only reads predicted to fall within exons were counted.
Library strategy
RNA-Seq
Library source
transcriptomic
Library selection
cDNA
Instrument model
Illumina HiSeq 2500
Data processing
We used the gene hit counts table for downstream differential expression (DE) analysis, using DESeq2 (48) to compare gene expression among groups of samples. We used the Wald's test to generatep-values and Log2 Fold Changes (Log2FC). We considered genes to be differentially expressed (DE) when adjustedp-values were < 0.05 and the overall absolute Log2FC was > 1. The data for immune transcripts were mined to evaluate their expression levels, and are reported as transcripts per million (TPM).
Assembly: Cimex_lectularius_Clec_2.1 reference genome available on ENSEMBL
Supplementary files format and content: tab-delimited text files include raw counts for each sample
Submission date
Feb 16, 2024
Last update date
Mar 18, 2024
Contact name
Sanam meraj
Phone
6043454977
Organization name
simon fraser university
Department
Biological Sciences
Lab
Gries lab
Street address
8888 university drive
City
Burnaby
State/province
British Columbia
ZIP/Postal code
V5A 1S6
Country
Canada
Series (1)
GSE256026
Activation of immune pathways in common bed bugs, Cimex lectularius, in response to bacterial immune challenges - a transcriptomics analysis
Relations
Supplementary file
Size
Download
File type/resource
GSM8084338_RoB-positive1.counts.txt.gz
1.1 Mb
(ftp) (http)
TXT
Raw data are available in SRA