Supplementary materials for papper “Transcriptomic analysis reveals low-quality queen bees (Apis mellifera) by commercial queen rearing.”.
Description
These are supplementary materials for the paper “Transcriptomic analysis reveals low-quality queen bees (Apis mellifera) by commercial queen rearing.” Fig. S1: Measurement of queen cell length. Table S1: Statistics quality of RNA sequencing of all libraries in each type of group (E, L1, L2 and L3). Table S2: Mapping reads of all RNA-Seq libraries in each type of group (E, L1, L2 and L3). Table S3: Pearson correlation coefficient among queen samples. Table S4: Differentially expressed genes in different functional classes, when comparing queens reared from larvae (L1, L2 and L3) with E group. Table S5: KEGG analysis result of DEGs of larvae groups (L1, L2 and L3) compared with E group. Of them, Fig. S1, Table S1,Table S2, Table S3,and Table S5 are all in the Supplementary materials, while Table S4 was shown in a extra excel.
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Steps to reproduce
The single-drone mated queen was imprisoned on a plastic worker bee frame to lay eggs for 6h . On the 2nd, 4th, 5th and 6th day after laying, we transplanted eggs (refer to worker eggs, E), one-day old larvae (refer to worker larvae, L1), two-day old larvae (refer to worker larvae, L2) and three-day old larvae (refer to worker larvae, L3) from worker cells to queen cells respectively to rear queen samples. For newly emerged queens in each type of queen group (refer to E group, L1 group, L2 group and L3 group respectively), the first six queens were killed immediately in liquid nitrogen and stored in a -80 ℃ refrigerator for next step of RNA-Seq. Four of them were selected randomly for RNA-Seq. Brain, thorax and ovary (dissected over ice) of each queen were pooled as one sample for total RNA extraction. Total RNA was extracted by using TRIzol reagent (Life technologies, California, USA). The integrity and concentration of all samples were checked by using Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA) in CapitalBio. The cDNA library was constructed following the manufacturer’s instructions for the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, E7530S), NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490S), NEBNext Multiplex Oligos for Illumina（Index Primers Set1）(NEB, E7335S) and the NEBNext Multiplex Oligos for Illumina (NEB, E7500S). Raw data filtering and quality control Evaluate the quality of data by using FastQC software and filter control of the data according to the quality control result. HISAT2 software were used for sequence alignment. Reference genome files is GCF_000002195.4_Amel_4.5_genomic.fa. Reference gene annotation file is GCF_000002195.4_Amel_4.5_genomic.gtf. Then, we did uniformity analysis and saturation analysis for estimating whether the measured data volume can meet the analysis needs. After transcript assembly, calculate the expression readcount by using StringTie software. Then, count the FPKM (Fragments Per Kilo bases per Million fragments) value for each gene. All DEGs and background genes were mapped to the GO database (Http://www.geneontology.org/), and the number of genes in each term were counted. The q-value of both significance enrichment analysis of GO function and KEGG Pathway were calculated through hypergeometric test, the correct q-value (q-value) were calculated using the BH method. Then, find the pathway of significant enrichment based on the q-value or q-value. Predict the main biochemical metabolism pathways and signal transduction pathways involved in DEGs through significant enriched pathway term. Annotate the genes expressed in the project by integrating multiple databases (ENSEMBL、NCBI、Uniprot、GO、KEGG) by using Apis mellifera (Amel _ 4.5) reference genes.