GSM2507389: Emb1 Rep1; Ciona intestinalis; RNA-Seq
Cell sorting was performed using a Biorad S. For each timepoint, an unelectroporated cell sample was first analyzed to determine the levels of background autofluorescence and gates on forward scatter and GFP fluorescence were conservatively selected to enrich for single cells with or without strong GFP signal. The electroporated sample was then sorted directly into RNA lysis buffer (Zymogen Quick RNA miniprep kit R1054). For GFP positive notochord enriched cells, 2000-15000 cell were purified into 500 ul buffer, while 100,000 GFP negative (notochord depleted) cells were simultaneously sorted into 2 mL of lysis buffer. Samples were stored at -80C until RNA purification.RNA was purified with the Zymogen Quick-RNA miniprep kit (R1054) and eluted in 50 ul elution buffer.10-60 ng of RNA per sample was treated with DNase (Roche) and concentrated to 10 ul with the Zymogen Clean and Concentrator kit (R1015). Final purified RNA concentration was 0.5 – 4.0 ng/ul. Amplifed cDNA was prepared using the NuGEN Ovation RNA-Seq System V2 (7102) .  2.5 ul of purified RNA was used in half-reactions following their standard protocol. cDNA samples were fragmented with the Covaris S220 Ultrasonicator using the recommended settings to obtain average fragment length of 300bp and the NEBNext DNA Library Prep Master Mix set for Illumina from New England BioLabs (NEB ) was used for library construction. Obtained libraries were checked with the Agilent 2100 Bioanalyzer