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Sample GSM4158836

Query DataSets for GSM4158836

Status

Public on Nov 20, 2019

Title

wBm Agilent SureSelect, vector, 8 dpi, b, SRX2508254

Sample type

SRA

Source name

B. malayi taken from A. aegypti 8 dpi

Organisms

Brugia malayi; Aedes aegypti; Wolbachia endosymbiont of Brugia malayi

Characteristics

brugia malayi strain: FR3
aedes aegypti strain: black-eyed Liverpool
enrichment method: wBm Agilent SureSelect capture

Extracted molecule

total RNA

Extraction protocol

For nematode samples, third stage larvae of Brugia malayi strain FR3 were isolated from infected mosquitoes 9-16 days post infection (dpi) using the NIAID/NIH Filariasis Research Reagent Resource Center (FR3) Research Protocol 8.4. Larvae were isolated in RPMI + P/S, enumerated, and either flash frozen in liquid nitrogen for storage at -80°C or used for the infection of male Mongolian gerbils (Meriones unguiculatus; Charles River, Wilmington, MA, USA). Gerbils were anesthetized with 5% isoflurane and infected by introducing 400 L3s into the peritoneal cavity using a butterfly catheter. Larvae, immature adults, and mature adults were recovered by peritoneal flush using a terminal procedure. Brugia malayi embryos were collected from live gravid females as previously described. For vector samples, the Aedes aegypti black-eyed Liverpool strain was obtained from FR3 and maintained in the Biosafety Level 2 Insectary at the University of Wisconsin Oshkosh. Desiccated mosquito eggs were hatched in deoxygenated water and the resulting larvae were maintained on a slurry of ground TetraMin fish food (Blacksburg, VA) at 27°C with 80% relative humidity. Female pupae were separated using a commercial larva pupa separator (The John Hock Company, Gainesville, FL), placed in mesh-covered paper soup cartons, and enclosed adults were fed with cotton pads soaked in sucrose solution. Females were deprived of sucrose approximately twelve hours prior to blood feeding. Mosquitoes were infected with the Brugia malayi FR3 strain by feeding microfilaremic cat blood (FR3) through Parafilm via a glass-jacketed artificial membrane feeder. When necessary microfilaremic cat blood was diluted with uninfected dog blood to achieve a suitable parasite density for infection (100-250 microfilariae/20 μL). Infected mosquitoes were collected at 18 hours post infection (hpi), 4 dpi, and 8 dpi. They were anesthetized with CO2 then transferred to chilled microscope slides where the legs, wings, heads and abdomens were removed. The thoraces with and without developing B. malayi larvae were flash frozen in liquid nitrogen and stored at -80 °C prior to RNA isolation. Vector-derived Infective B. malayi L3 were isolated from whole mosquitoes in bulk using the standard FR3 protocol. For RNA isolation, mosquito thoraces were combined with TRIzol (Zymo Research, Irvine, CA) at a ratio of 1 mL TRIzol per 50-100 mg mosquito tissue, while nematode samples were processed using a 3:1 volume ratio of TRIzol to sample. For both preparations, 1 µL β-mercaptoethanol was added for every 100 µL of sample. The tissues were homogenized using a bead beater and a TissueLyser (Qiagen, Germantown, MD) at 50 Hz for 5 min. The homogenate was then transferred to a new tube and centrifuged at 12,000g for 10 min at 4°C. After incubation at room temperature for 5 min, 0.2 mL chloroform was added for every 1 mL TRIzol. The samples were shaken by hand for 15 s, incubated at room temperature for 3 min, loaded into a pre-spun Phase Lock Gel heavy tube (5Prime, Gaithersburg, MD), and centrifuged at 12,000g for 5 min at 4°C. The upper phase was extracted, one volume of 100% ethanol was added, and then loaded onto a PureLink RNA Mini column (Ambion, Austin, TX). The samples were processed following manufacturer instructions, quantified using a Qubit fluorometer (Qiagen, Germantown, MD) and Nanodrop spectrometer (Nanodrop, Wilmington, DE), and sent to Institute for Genomic Sciences at the University of Maryland Baltimore for DNase treatment and library preparation.
Whole transcriptome libraries without AgSS capture were constructed for sequencing on the Illumina platform using the NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA). For targeting eukaryotic mRNA, polyadenylated RNA was isolated using the NEBNext Poly(A) mRNA magnetic isolation module. For the B. malayi and wBm AgSS capture designs, probes were designed using the SureSelect DNA Advanced Design Wizard for B. malayi and wBm. When targeting bacterial mRNA, samples underwent rRNA- and poly(A)-reductions. Probes were designed to capture every 120 bp for each coding sequence in both organisms with no overlap between baits. DustMasker was used to identify and mask low complexity regions of the genome from the probe design .For samples treated with the wBm AgSS capture, pre-capture libraries were constructed from 500-1000 ng of total RNA samples using the NEBNext Ultra Directional RNA Library Prep kit (NEB, Ipswich, MA, USA). First strand cDNA was synthesized without mRNA extraction to retain non-polyadenylated transcripts and fragmented at 94 oC for 8 min. After adaptor ligation, cDNA fragments were amplified with 10 cycles of PCR before capture. Wolbachia transcripts were captured from 200 ng of the amplified libraries using the Agilent SureSelectXT RNA (0.5-2 Mbp) bait library designed specifically for wBm. Library-bait hybridization reactions were incubated at 65 °C for 24 h then bound to MyOne Streptavidin T1 dynabeads (Invitrogen, Carlsbad, CA, USA). After multiple washes, bead-bound captured library fragments were amplified with 18 cycles of PCR. The libraries were loaded on an Illumina HiSeq4000, generating 151-bp paired end reads. For samples treated with the B. malayi AgSS capture, pre-capture libraries were constructed from 1000 ng of total RNA samples using the NEBNext Ultra Directional RNA Library Prep kit (NEB #E7420, Ipswich, MA, USA). After adaptor ligation, cDNA fragments were amplified for 10 cycles of PCR before capture. B. malayi transcripts were captured from 200 ng of the amplified libraries using an Agilent SureSelectXT Custom (12-24 Mbp) bait library designed specifically for B. malayi. Library-bait hybridization reactions were incubated at 65 °C for 24 h then bound to MyOne Streptavidin T1 Dynabeads (Invitrogen, Carlsbad, CA). After multiple rounds of washes, bead-bound captured library fragments were amplified with 16 cycles of PCR. The libraries were loaded on a Illumina HiSeq 4000 generating 151-bp paired end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

wbm_counts.txt
wbm_tpm.txt

Data processing

Reads aligned to a combined A. aegypti, B. malayi, and wBm reference using TopHat v1.4 (for poly(A) selection and B. malayi Agilent SureSelect samples) or Bowtie v0.12.9 (for poly(A) depletion and wBm Agilent SureSelect samples)
Protein-coding genes for B. malayi and A. aegypti quantified using HTSeq v0.5.3p9 with mode set to union. Protein-coding genes for wBm quantified using FADU v1.2.
Differentially expressed genes for all three organisms were individually identified using edgeR v3.20.1
TPM values calculated for protein-coding genes
Expression modules for differentially expressed genes for all three organisms identified using WGCNA v1.61
Significantly over-represented functional terms identified using Fisher's exact test for each WGCNA module
Genome_build: Brugia malayi WS259, Wolbachia endosymbiont strain TRS of Brugia malayi (NC_006833.1), and Aedes aegypti AaegL3.3
Supplementary_files_format_and_content: tab-delimited files containing counts, TPM, WGCNA module assignments for differentially expressed genes, functional term enrichment analyses for WGCNA modules for both B. malayi and A. aegypti

Submission date

Nov 12, 2019

Last update date

Nov 20, 2019

Contact name

Suvarna Nadendla

Organization name

University of Maryland School of Medicine

Department

Institute for Genome Sciences

Street address

670 W.Baltimore Street