GSM7337056: Larvae-2,scRNAseq; Serratia marcescens; RNA-Seq
Up to half a million S. marcescens cells were collected by centrifuging at 4000 rpm for 5 min at 4℃.The supernatant was removed and cell pellets were resuspended in 2 mL ice-cold 4% formaldehyde (Sigma, 47608) and incubated with shaking overnight at 4℃. The fixative was removed by centrifuging at 4000 rpm for 5 min at 4℃ and cells were washed twice by 1 mL PBS-TRI (1x PBS supplemented with 0.1% Tween-20 and 0.1 U/mL Murine RNase inhibitor (Vazyme, R 301)). The supernatant was removed and cells were resuspended in 200 μL PBS-TRI (1x PBS supplemented with 0.1% Tween-20 and 0.2 U/mL Murine RNase inhibitor (Vazyme, R 301)). The cell suspensions were counted with a Moxi cell counter (ORFLO Technologies, Ketchum, Idaho) and diluted according to the manufacturer's instructions to obtain 2000-4000 single-cell per sample. Random primers were added to amplify the full length of RNA, and the resulting cDNA fragment was added with adaptor. The microbeads with cell barcode, UMI, and the cDNA fragments linked to the adaptor were combined with the mixture of enzymes and extended in the microfluidic system to obtain the labeled cDNA chain. The beads were lysed and the cDNA was used as a template for PCR amplification. All products were pooled to construct a standard sequencing library.