title: GSM4718658: scRNAseq_olig2_eGFP_48hpf; Danio rerio; RNA-Seq
library_construction_protocol: 24, 36, and 48 hpf Tg(olig2:EGFP) euthanized embryos were collected in 1.7 ml microcentrifuge tubes and deyolked in 100 μl of pre-chilled Ca free Ringers solution (116 mM NaCl, 2.6 mM KCl, 5 mM HEPES, pH 7.0) on ice. Embryos were pipetted intermittently with a p200 micropipettor for 15 minutes and left for 5 min. 500 μl of protease solution (10 mg/ml BI protease, 125 U/ml DNase, 2.5 mM EDTA, 1X PBS) was added  to microcentrifuge tubes on ice for 15 min and embryos were homogenized every 3 min with a p100 micropipettor for 15 min. 200 μl of STOP solution (30% FBS, 0.8 mM CaCl2, 1X PBS) was then mixed into the tubes. Samples were then spun down at 400g for 5 min at 4°C and supernatant was removed. On ice, 1 ml of chilled suspension media (1% FBS, 0.8 mM CaCl2, 50 U/ml Penicillin, 0.05 mg/ml Streptomycin) was added to samples and then spun down again at 400g for 5 min at 4°C. Supernatant was removed, and 400 μl of chilled suspension media was added and solution was filtered through a 35 μm strainer into a collection tube. Cells were FAC sorted to distinguish EGFP+ cells using a MoFlo XDP100 cell sorter at the CU-SOM Cancer Center Flow Cytometry Shared Resource and collected in 1.7 ml FBS coated microcentrifuge tubes in 200 μl of 1X PBS. The Chromium Box from 10X Genomics was used to capture cells using Chromium Single Cell 3' Reagent Kit part no. PN-1000075.  Libraries were sequenced on the Illumina NovaSEQ6000 Instrument. 
attributes.tag: GEO Accession
attributes.value: GSM4718658