GSM8240250: fatbody of cricket, group A, 1st injection = PBS, rep3; G... - SRA

SRX24389845: GSM8240250: fatbody of cricket, group A, 1st injection = PBS, rep3; Gryllus bimaculatus; RNA-Seq
1 ILLUMINA (Illumina NovaSeq 6000) run: 62.7M spots, 12.7G bases, 3.7Gb downloads

External Id: GSM8240250_r1

Submitted by: Kangwon Univ.

Study: Granulocyte dynamics: A key player in the immune priming effects of crickets

PRJNA1105776 • SRP504554 • All experiments • All runs

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This study investigates immune priming effects associated with granulocytes in crickets through a comprehensive analysis. Kaplan-Meier survival analysis reveals a significant contrast in survival rates, with the heat-killed Bacillus thuringiensis (Bt)-primed group (B-1) exhibiting an impressive ~80% survival rate compared to the PBS buffer-primed group (A-1) with only ~10% survival 60 hours post live Bt infection. Hemocyte analysis underscores elevated hemocyte counts, particularly in granulocytes of the B-1 group, suggesting a correlation between heat-killed Bt priming and heightened immune activation. Microscopy techniques further explore granulocyte morphology, unveiling distinctive immune responses in the B-1 group characterized by prolonged immune activation, heightened granulocyte activity, phagocytosis, and extracellular trap formation, contributing to enhanced survival rates. In particular, after 24 hours of injecting live Bt, most granulocytes in the A-1 group exhibited ETosis (death with release of extracellular traps), while in the B-1 group, the majority of granulocytes were observed to maintain highly activated extracellular traps, sustaining the immune response. Gene expression analysis supports these findings, revealing differential regulation of immune-related genes such as antibacterial humoral response, detection of bacterial lipopeptides, and cellular response to bacteria lipopeptides. Additionally, groups A (preinjected with heat-killed Bt), B (preinjected with heat-killed E. coli), and C (preinjected with PBS) were re- injected with live Bt and evaluated for survival rates over two- and nine-day intervals. Two days later, only group C displayed low survival rates. After injecting live Bt 9 days later, group B surprisingly showed a similarly low survival rate, while group A exhibited a high survival rate of ~60% after 60 hours, with actively moving and healthy crickets. In conclusion, this research provides valuable insights into both short-term and long-term immune priming effects in crickets, contributing to our understanding of invertebrate immunity with potential applications in public health. Overall design: We divided 1,800 crickets into 8 groups and conducted the experiments with 75 crickets per group, 3 repetitions (8?75?3=1,800 crickets). In this study, immune priming was induced using two groups, A and B. PBS buffer and heat-killed Bacillus thuringiensis (Bt) were utilized for this purpose, resulting in the induction of immune priming. Two days later, live Bt was injected again to create groups A-1 and B-1. Subsequently, the immune priming effect was assessed by examining survival rates, the immune activation of granulocytes, and conducting RNA-Seq analysis for both the A-1 and B-1 groups. Next, group C, D, and E was the first injection of PBS (control), heat-killed E. coli, and heat-killed Bt. After 2 days, live Bt was injected into all groups and they were named group C-1, D-1, and E-1. 9 days after the first injection, we injected live Bt into group C, D, and E (called C-2, D-2, and E-2).

Sample: fatbody of cricket, group A, 1st injection = PBS, rep3

SAMN41109860 • SRS21145484 • All experiments • All runs

Organism: Gryllus bimaculatus

Library:

Name: GSM8240250

Instrument: Illumina NovaSeq 6000

Strategy: RNA-Seq

Source: TRANSCRIPTOMIC

Selection: cDNA

Layout: PAIRED

Construction protocol: To perform RNA sequencing, we extracted fatbodies from group A, B, A-1, and B-1. We selected 10 crickets from each group and prepared them in one tube for each group. Since each group was repeated 3 times, fatbodies were extracted from a total of 30 crickets and prepared in 3 tubes per group (12 tubes in total). Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, USA) (all operations were performed using sterile spaces and tolls to minimize contamination). To briefly summarize the procedures, 100mg of fat bodies were mixed with 1ml of TRIzol reagent (Thermo Fisher Scientific, Waltham, USA). Plastic beads of 0.6 mm, 2 mm, and 7 mm were added to the mixture, and it was homogenized for 10 minutes with a vortex (DAIHAN Ltd., Korea). The sample were centrifuged at 14,000 rpm for 3 min at 4℃ and 1mL of supernatant was extracted. The extracted supernatant was mixed with 200 µL chloroform and reacted at 4℃ for 20 min (Thermo Fisher Scientific, Waltham, USA). The sample were centrifuged at 14,000 rpm for 3 min at 4℃ for 15min and then the supernatant (~600 µL) was loaded into a gDNA elimination column to remove gDNA (Thermo Fisher Scientific, Waltham, USA). The sample was centrifuged at 14,000 rpm for 1 min at 4℃ and then total RNA was precipitated by adding 600 µL of cold isopropanol/70% ethanol (maintained at -20℃, Sigma-Aldrich, USA). The sample was centrifuged at 14,000 rpm for 1min at 4℃ using RNeasy Plus Mini column, mixed with ~700 µL of RW1 buffer (10mM Tris-Hcl, 1mM EDTA, 50mM NaCl, 0.5% Tween-20, pH 8.5, Sigma-Aldrich, USA), and centrifuged at 14,000rpm for 1min. Finally, the sample was mixed with 500 RPE buffer (10mM Tris-Hcl, 80% ethanol, 0.5mM EDTA pH 7.5), centrifuged, and total RNA was extracted by placing ~50 µL RNeasy-free water in a new sterilized tube (Sigma-Aldrich, USA). The cDNA library was generated from the total RNA, and paired-end sequencing was performed using the Illumina RNA-seq technology (Macrogen Ltd, Korea).

Runs: 1 run, 62.7M spots, 12.7G bases, 3.7Gb

Run	# of Spots	# of Bases	Size	Published
SRR28826984	62,694,017	12.7G	3.7Gb	2024-06-05

ID:: 32704826

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