title: NextSeq 550 paired end sequencing; Cellular heterogeneity of the developing worker honey bee (Apis mellifera) pupa: a single cell transcriptomics analysis
design: Cellular heterogeneity of the developing worker honey bee (Apis mellifera) pupa: a single cell transcriptomics analysis
library_construction_protocol: Honey bees are holometabolous and worker prepupae at day 11 (S1) and pupae at day 15 (S2) were chosen for this study in order to capture the key developmental stages between capping of the larval cell (day 9) and the emergence of the imago on day 21 (Oertal, 1930) (Figure 1A). To gather samples, a piece of brood comb containing appropriately staged pupae was collected from a single honey bee colony at the Easter Bush Campus apiary in August 2018. Pupae were removed from the comb and placed in microcentrifuge tubes on ice. Each pupa was placed in 0.5 ml HyQTase (GE Healthcare, Chicago, Illinois, USA), finely chopped with small spring scissors for one minute and incubated for 5 min at 25°C. Samples of each stage were centrifuged at 400 RCF for 5 minutes at 4°C. Cell pellets were resuspended in 1 ml WH2 medium by drawing liquid into and out of pipette tip fifteen times (Goblirsch et al., 2013). Samples (n = 4 per stage) were pooled (total volume 4ml), and the cells passed into a 5ml tube through a 70 μm strainer cap (Becton, Dickinson and Company, New Jersey, USA) to remove debris and aggregated cells. Following centrifugation of the filtered cells at 400 RCF for 5 minutes at 4°C, the supernatant was discarded and the cells resuspended in 2ml WH2 medium. After further centrifugation at 400 RCF for 5 min at 4°C, cells were resuspended in 1 ml WH2 medium and stained with 1:2,000 Sytox Red (Thermo Fisher, Waltham, Massachusetts) for downstream cell viability analysis during cell sorting. Gating strategies sorted cells on the basis of their size (forwards vs side scatter area to exclude debris), single cells (forward scatter area vs height to exclude doublet cells) and viability using a 633nm laser and 660/20 band pass emission filter on an Aria IIIu FACS (Becton, Dickinson and Company, New Jersey, USA (Figure 1B and C). Before sequencing the cells were counted and tested again for viability using a TC20 automated cell counter (Bio-Rad, Hercules, California, USA). Approximately 7,000 cells at each stage were used for cDNA library preparation using the Chromium platform v2.0 (10X Genomics, Pleasanton, California, USA), as per the manufacturer's instructions. Library quality was confirmed with a LabChip Gx24 bioanalyzer (PerkinElmer, Waltham, Massachusetts, USA). Approximately 7,000 cells at each stage were used for cDNA library preparation using the Chromium platform v2.0 (10X Genomics, Pleasanton, California, USA), as per the manufacturer's instructions. Library quality was confirmed with a LabChip Gx24 bioanalyzer (PerkinElmer, Waltham, Massachusetts, USA).