GSM7035738: zebrafish embryo, 30% epiboly, NT, replicate A, scRNAseq; Danio rerio; RNA-Seq
Drop-seq droplet encapsulation of cells. Libraries were built according to the Drop-seq protocol version 3.1 (12/28/2015, available http://www.dropseq.org/) with the following modifications. Iodoacetamide (IAA, Sigma-Aldrich) was diluted in ethanol to a 100mM working concentration. Following breakage of droplets, beads were washed once with 300uL of 5x IAA buffer (250mM NaPO4 pH8), and incubated in IAA solution (50mM NaPO4 pH8, 10mM IAA, 20% DMSO, 6% Ficoll PM-400) for 15min at 32°C with rotation. DTT was added to a final concentration of 20mM to stop the reaction, and beads were washed twice with 6x SSC solution before continuing with reverse transcription. An estimated 50–100 STAMPs were included in each 50uL PCR reaction, and 12–13 cycles of PCR amplification were performed. Libraries were sequenced using Illumina Nextseq v2.2 Mid or High Output 150bp chemistry.