GSM5169170: Embryonic stage13 Hand-GFP+ cells from wild type Drosophila [Hrt20_21hr]; Drosophila melanogaster; RNA-Seq
Collected embryos were washed thoroughly with ice-cold 1X phosphate buffered saline (PBS) and then dechorionated in fresh 4-5% bleach (sodium hypochlorite solution; Sigma) for 3 min at room temperature. Dechorionated embryos were extensively rinsed with H2O. Then embryos (~150 mg) were transferred to a Dounce homogenizer (15 ml Tissue Grinder; Dounce). A 5 ml suspension of embryos (Schneider medium) was subjected to mechanical disruption using the Dounce homogenizer, achieved by five strokes using a pestle. Homogenized samples were transferred to 15 ml centrifuge tubes. Collagenase (Type I) (20 U/ml; Worthington), 100 U total, was added to the Schneider medium with the embryos and incubated at room temperature for 10 min. Next, 10 μl of ethylenediaminetetraacetid acid (EDTA) (0.5 M; final concentration of 1 mM) and 0.5 ml 2.5% Trypsin were added to the 5 ml cell solution to digest the samples for 20 min at room temperature. The trypsin reaction was stop by adding 20% fetal bovine serum (FBS) and cells were filtered out using a 70 μm mesh. The cells were then pelleted by centrifugation (1000xg, 5 min, 4°C) and resuspended in 2 ml pre-chilled artificial hemolymph. Isolated embryonic cells were sorted by flow cytometry (BD Influx cell sorter). The library preparation (Single Cell 3' Reagent Kits v3, 10x Genomics, CA, USA) and sequencing were performed using Illumina HiSeq by Novogene Corporation Inc (Sacramento, CA). Single-cell RNA-Seq (Single Cell 3' Reagent Kits v3, 10x Genomics, CA, USA)
GEO
GSM5169170