title: NextSeq 500 sequencing; scRNA-Seq of Phallusia mammillata from 32-cell to 64-cell stage
design: scRNA-Seq of Phallusia mammillata from 32-cell to 64-cell stage
library_construction_protocol: Adult P. mammillata animals were procured from the Roscoff Marine Station (France). P. mammillata em- bryos 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).
attributes.tag: Experimental Factor: individual
attributes.value: 64cell08071
attributes.tag: Experimental Factor: developmental stage
attributes.value: cleavage 64-cell
attributes.tag: Experimental Factor: single cell identifier
attributes.value: cell 556