DOI: 10.5281/zenodo.18315521
title: BETTER-B: Transcriptomic data (fastq files & count files) of the hemocytes of Apis mellifera (from 1-day old nurse bees and L5-larvae)

description: In March 2024, honey bees (A. mellifera carnica) were collected by removing closed brood frames with ready-to-emerge honey bees from the UGENT apiary (51°1’29”N, 3°42’42”E). These frames were incubated in a temperature chamber at 34°C until emergence. Honey bees were collected at 24-hour intervals. Additionally, brood frames with 8-day-old worker larvae (fifth instar) were removed from the hive and the collected larvae were immediately used for hemolymph extraction.

RNA was extracted from hemocytes from 320 µL of hemolymph, equivalent to approximately 5 million hemocytes. The hemolymph was centrifuged at 0.5G for 5 minutes at 4°C and the plasma was discarded. RNA extraction from the resulting hemocyte pellets was carried out using the Qiagen RNeasy Micro Kit. The pellet was resuspended in 350 µl of RLT lysis buffer supplemented with 1% β-mercapto-ethanol and vortexed. The lysate was then passed through a 20G needle ten times to ensure thorough disruption of the hemocytes. The RNA was purified using the Qiagen RNeasy spin column following the manufacturer’s recommendations, including an on-column DNase step. Finally, the RNA was eluted in 14 µL of RNase-free water.RNA sequencing was performed by BGI Genomics (China). mRNA enrichment was conducted using oligo dT beads to target mRNA molecules with poly A tails. In the next step, mRNA was fragmented into smaller pieces and then converted into first-strand cDNA using random primers. The second-strand cDNA synthesis utilized dUTP. The resulting cDNA underwent end-repair and 3’ adenylation, followed by adapter ligation to the 3’ adenylated cDNA fragments. PCR amplification was employed for library fragment enrichment, followed by quality control and library circularization steps. Subsequently, the library underwent amplification to generate DNA nanoballs, enabling sequencing on the DNBSEQ platform with paired-end reads of 150 base pairs.




Library strategy
RNA-Seq


Library source
transcriptomic


Library selection
cDNA


Instrument model
DNBSEQ-G400




Raw data with adapter sequences or low-quality sequences was filtered. We first went through a series of data processing to remove contamination and obtain valid data. This step was completed by SOAPnuke developed by BGI. SOAPnuke software filter parameters: "-n 0.01 -l 20 -q 0.3 --adaMR 0.25 --ada_trim --polyX 50 -- minReadLen 150", steps of filtering: 1. Filter adapter: if the sequencing read matches 25.0% or more of the adapter sequence (maximum 2 base mismatches are allowed), cut the adapter; 2. Filter read length: if the length of the sequencing read is less than 150 bp, discard the entire read; 3. Remove N: if the N content in the sequencing read accounts for 1.0% or more of the entire read, discard the entire read; 4. Remove polyX: if the length of polyX (X can be A, T, G or C) in the sequencing read exceeds 50bp, discard the entire read; 5. Filter low-quality data: if the bases with a quality value of less than 20 in the sequencing read account for 30.0% or more of the entire read, discard the entire read; 6. Obtain Clean reads: the output read quality value system is set to Phred+33.

Raw data files: Larva_1.fq & Larva_2.fq // Adult_1.fq & Adult_2.fq

An index was constructed using the reference transcriptome of A. mellifera (GCF_003254395.2_Amel_HAv3.1_cds_from_genomic.fna) via the kallisto index command. Transcript abundance was subsequently estimated using Kallisto’s pseudo-alignment algorithm through the kallisto quant command.Assembly: GCF_003254395.2_Amel_HAv3.1_cds_from_genomic.fna

Count files are available through the GEO repository under the following link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE293998

Summary of analysis with Gene IDS & Gene names: 1-s2.0-S0965174825001420-mmc2.pdf




Mapping the Hemocyte Surfaceome of Apis mellifera by a Proteomic and Transcriptomic Approach
 


Organism
Apis mellifera


Experiment type
Expression profiling by high throughput sequencing


Summary
Transcriptomic analysis was performed on hemocytes from 1-day old nurse bees and L5-larvae.


Overall design
RNA-Seq of Apis mellifera hemocytes in different developmental stages: L5-larvae and (<1 day old) nurse bees.


Web link
https://pubmed.ncbi.nlm.nih.gov/40915621/


Contributor(s)
Braeckman M, de Graaf DC, De Smet L


Citation(s)






Braeckman M, De Smet L, Devreese B, de Graaf DC. Exploration of the hemocyte surfaceome of Apis mellifera by a proteomic and transcriptomic approach. Insect Biochem Mol Biol 2025 Nov;184:104398. PMID: 40915621







This work was supported by the Better-B project, which has received funding from the European Union, the Swiss State Secretariat for Education, Research and Innovation (SERI) and UK Research and Innovation (UKRI) under the UK government's Horizon Europe funding guarantee (grant number 10068544).