title: GSM3855908: 72 hpf, nonskin2, replicate B; Danio rerio; RNA-Seq
library_construction_protocol: Dechorionated embryos were transferred into 1.5 ml tubes and rinsed with Ca2+-free Ringer's solution for 15 min. During this incubation, yolk was removed by gently pipetting embryos through a 200 µl tip (with the end cut off) three to five times. Yolk-free embryos were transferred into a 35 mm petri dish with 5 ml of 0.25% Trypsin-EDTA. Embryos were incubated at 28.5 °C and homogenized with a 200 µl tip every 10 min until most cells were dissociated (20 to 50 min). 55 µl 100 mM CaCl2 and 550 µl FCS were added to stop digestion. Dissociated cells were transferred into a 15 ml tube and centrifuged at 300 g for 5 min at 15 °C. Cells were rinsed once with 10 ml suspension solution (colorless Leibovitz medium L-15 with 0.3 g/L glutamine, 0.8 mM CaCl2, Pen 50U/ml, Strep 0.05 mg/ml, and 1% FCS). Dissociated cells were resuspended in suspension solution to ~10^7 cells/ml and immediately proceeded to cell sorting at the UCLA Broad Stem Cell Research Center Flow Cytometry Core. BD FACS ARIA II SORP instruments sorted cells, using a 488 nm laser for GFP detection and a 561 nm laser for RFP (dsRed) detection. Sorted cells in suspension solution were immediately lysed using the Qiagen RNeasy kit. Lysis was completed within two hours of dissociating cells. Total RNA was isolated following the Qiagen RNeasy kit and stored at –80 °C. Quality of all samples was assayed with an Agilent Bioanalyzer, and only samples with RNA Integrity Number > 8 were used for creating sequencing libraries. Poly-A bead-purified RNA-Seq libraries were prepared with the Illumina TruSeq RNA Library Prep Kit.
attributes.tag: GEO Accession
attributes.value: GSM3855908