NextSeq 500 sequencing; scRNA-Seq of Phallusia mammillata from 2-cell to 16-cell stage
scRNA-Seq of Phallusia mammillata from 2-cell to 16-cell stage
Adult P. mammillata animals were procured from the Roscoff Marine Station (France). P. mammillata embryos were dissociated with pronase for 20 minutes and individual dissociated embryos were transferred to calcium-free artificial sea water in imaging plates (Mat-tek) coated with a solution of 0.1%PFA and 0.1% gelatin. Dissociated embryos were kept on ice before collection. Individual cells were manually collected in 0.3 μl with pipet tips coated with a solution of 0.1%PFA and 0.1% gelatin and cells were directly lysed in 4μl of lysis+dNTP+oligodT mix (2 μl of lysis buffer – 0.2% Triton X-100+RNase inhibitor–, 1 μl of 10 μM oligo-dT primer – sequence 5'AAGCAGTGGTATCAACGCAGAGTACT30VN –, 1 μl of 10mM dNTP mix) according to the Smart-Seq2 protocol from Picelli et al. (2014). Cells were directly lysed in 4μl of lysis+dNTP+oligodT mix (2 μl of lysis buffer – 0.2% Triton X-100+RNase inhibitor–, 1 μl of 10 μM oligo-dT primer – sequence 5'AAGCAGTGGTATCAACGCAGAGTACT30VN –, 1 μl of 10mM dNTP mix) according to the Smart-Seq2 protocol from Picelli et al. (2014). cDNA from single cells was prepared following the Smart-Seq2 from Picelli et al. (2014). 500 pg of cDNA was then used for the tagmentation reaction using a home-made Tn5 transposase (Hennig et al., 2018).
Experimental Factor: individual
16cell09093
Experimental Factor: developmental stage
cleavage 16-cell
Experimental Factor: single cell identifier
cell 199