GSM7730276: Male pupal forewing, 24h APF, scRNAseq; Bicyclus anynana; RNA-Seq
The preparation of a single-cell suspension from 22.5-25-hour male pupal forewings of Bicyclus anynana butterflies has been documented in-depth in a protocol paper (Prakash and Monteiro, Methods Protoc, 2020). Briefly, 14 male pupal forewings were dissected into multiple tubes with sterile PBS, washed, then dissociated in warm 5X TrypLE with continuous trituration for a total of 15-20 minutes. The dissociated cell mixture was filtered with a 41 µm filter and collected by centrifuging at 300 g, 4 ˚C for 5 minutes. Samples were pooled and gently resuspended into 1mL of cold 1X PBS + 0.01% BSA. Cells were counted in a Countess Automated cell counter by mixing 5 µl of sample with 5 µl of 0.4% trypan blue and loading it into a Countess cell counting chamber. Before going for fluorescence-activated cell sorting (FACS), 5 µl of a 300 µM intermediate concentration of DAPI (Invitrogen by Thermo Fisher Scientific, Cat. no.: D1306) was added per mL of sample. Cells larger than 6 µm in diameter were sorted using FACS and collected in a 1.5 mL LoBind Eppendorf tube pre-filled with 50 µl of cold 1X PBS + 0.01% BSA. Around 104,000 cells were collected and precipitated by centrifuging at 900g, 4 ˚C for 3 minutes. Cells were resuspended in 30 µl of cold 1X PBS + 0.01% BSA, with a final cell count of 1650 cells/µl and 88% viability. Cells were processed via the 10X Genomics microfluidics system at the Genome Institute of Singapore. Single-cell libraries were generated using the Chromium Single cell 3' Library Kit V3 with a targeted cell recovery of 7000 cells. The quality of the generated cDNA library was quantified using an Agilent Bioanlyzer and sequenced on one lane of HiSeq4K 2x151bp (multiplex) at the Genome Institute of Singapore.