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Sample GSM8240253

Query DataSets for GSM8240253

Status

Public on Jun 05, 2024

Title

fatbody of cricket, group B-1, 1st injection = heat-killed Bt, 2nd injection = live Bt, rep3

Sample type

SRA

Source name

fatbody

Organism

Gryllus bimaculatus

Characteristics

tissue: fatbody
treatment: 1st injection = heat-killed Bt, 2nd injection = live Bt

Treatment protocol

All crickets were selected 24 hours after molting from young to adult. Additionally, we removed inactive or unhealthy individuals (less than ~2%) after the first injection and used only healthy individuals that were very active and feeding for the injection of live Bt. In all groups, the crickets were almost identical in age (24 hours after adult molting), health (active movement and no infection), average size (~25±5 mm per cricket), and average weight (~1.1±0.5g per cricket). Immune priming was performed by diluting the OD 1.3 obtained from the LD50 value at a ratio of 1:999, and 2μl was inoculated (2ⅹ103 cfu/insect for Bt and 2.08ⅹ103 cfu/insect for E. coli). The reason for this is similar to preventive vaccination, where we aimed to ensure that there was no impact on survival due to the preventive inoculation. Therefore, we obtained values over the entire observation period of 96 hours where there was no difference between the control group and the survival group. Next, 48 hours after the immune cycle of crickets, all individuals were injected with 10μl of live pathogen at the concentration described in material and method section 2.3. Subsequently, survival analysis and microscopic examinations were conducted at different time intervals. For injection, the crickets were sterilized with 70% alcohol paper, and a finely pulled glass needle was shallowly inserted into the abdomen (Haematocrit capillaries, Sigma-Aldrich) (17, 18). The experiments were repeated three times independently.

Growth protocol

We collected 150 crickets from basement of a building or under rocks or log debris in mountain, mated them, and collected eggs. Female laid an average of 90±10 eggs, and hatched young were reared in a sterilized incubator (MIR-553, Sanyo Electric Biomedical, Japan; 25 ± 1 ℃, 40±10%, 16h light:8h dark) maintain a constant environment. We used second- and third-generation adults as experiment insects. We provided sterilized food (Chinese cabbage and wheat husk purchased from Milwormnara Ltd., Korea) and always maintained cleanliness. Healthy crickets consistently exhibit activity through antennae movement, while sick or weak crickets demonstrate diminished antennae mobility and a notable decline in overall activity levels. Therefore, when such crickets are found in a breeding cage, we immediately remove them.

Extracted molecule

total RNA

Extraction 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).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

2BTV2-3
s2BTV2_rep3

Data processing

Raw sequencing data was preprocessed using integrated software for adapter and quality trimming, Trim Galore v0.6.5.
Reads with a Phred33 score of less than 20 and a length of less than 20 base pairs were filtered out, and the standard Illumina adapter sequence was trimmed if found.
De novo full-length transcriptome reconstruction was performed by a software to reconstruct full-length transcriptome without a reference genome, Trinity v2.15.1, and ORF (open reading frame) prediction was performed by software to identified candidate coding region from de novo transcriptome assembly, TransDecoder v5.5.0 (25).
Predicted ORFs were assessed using homology search (BLAST), protein domain identification (PFAM), gene ontology assignment, and functional classification (e.g., eggnog) of sequence data using software to annotate de novo transcriptome assembly, Trinotata v4.0.2 (26).
Quantification of gene expression was performed using software to quantify expression of genes and transcripts from transcriptomic data, Salmon v1.10.2 (27).
Assembly: assembled_sequence.fasta
Supplementary files format and content: Normalized count matrix

Submission date

Apr 29, 2024

Last update date

Jun 05, 2024

Contact name

Saeyoull Cho

E-mail(s)

saeyoullcho@kangwon.ac.kr

Organization name

Kangwon Univ.

Street address

1, Kangwondaehak-gil