GSM5351829: Shield_dnmt1_mKD_scRNA_seq_rep2; Danio rerio; RNA-Seq
Cell dissociation protocol was based on a previously described method (Farrell et al., 2018) with modifications to adapt it for 10x Genomics platform. Briefly, dnmt1 mKD and control embryos were collected 20 min after fertilization and cultured as mentioned before. Then 15 embryos at dome or shield stages for each sample were transferred into plastic Petri dishes that had previously been coated with 2% agarose and soaked with DMEM/F12 media (Gibco/Life Technologies, Cat 11330032) at least 2hrs. Next, embryos at desired stages were dechorionated and deyolked manually by forceps and transferred to a 1.5 ml Eppendorf tube with 50 µl DMEM/F12 media. Dissections were performed for up 15 min. The volume of DMEM/F12 meida containing embryos was adjusted to 200 µl, then cells were mechanically dissociated by flicking the tube 30 times and pipetting mixture 10 times through a 200 µl tip. The volume was adjusted to 1 ml with PBS containing 1.0% BSA, and spun to pellet cells at 300 g for 30s. The supernatant was removed and cells were resuspended in 80 µl of PBS containing 0.1% BSA and 20% Optiprep (StemCell, Cat 07820), aiming for a concentration above 100 cells/µl. Cells were then passed through a cell sieve (100 µm for dome stage and 70 µm for shield stage). Single cell RNA-seq library was performed with Chromium Next GEM Single Cell kit (10x Genomics, Cat PN-1000121) based on standard protocol. All libraries were sequenced by Illumina Hi-Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.