GSM8827604: Perturb-seq lib 12, GEX; Danio rerio; OTHER
Two zebrafish lines wild type and Gt(foxd3-Citrine)ct110 were crossed in late afternoons. Embryos were collected the next morning and incubated until desired stages. Embryos were dissociated with Liberase TM (Roche) for 25 min at 32°C. The cell suspension was filtered by 40-µm mini strainer (PluriSelect) and stained with 1-3 µg/ml DAPI. Citrine-positive live cells were sorted into 100µl of 0.1% UltraPure BSA (Invitrogen) in PBS, then centrifuged at 600× g for 5 minutes at 4°C. Care was taken to remove the supernatant without disturbing the cell pellet, leaving approximately 40 µl of supernatant. The cell pellet was reconstituted in the remaining supernatant. Cell count and quality were assessed using Trypan Blue and C-Chips (NanoEnTek). The protocol CG000510 Chromium NextGEM Single Cell 5' v2 CRISPR User Guide (Rev B, 10x Genomics) was adhered to for the preparation of single-cell gene expression and CRISPR libraries. Sequencing of the libraries was carried out on NextSeq and NovaSeq platforms (Illumina). CG000510 Chromium NextGEM Single Cell 5' v2 CRISPR User Guide (Rev B, 10x Genomics)