GSM7432137: Photoconverted brain endothelial cells, rep2; Danio rerio; RNA-Seq
Photoconversion ofTg(fli1:Gal4)ubs3;(UAS:Kaede)rk8PHBC or CtAs ECs was performed using a Zeiss LSM 710 confocal microscope (Carl Zeiss, objective lenses: Plan-Apochromat ×20/0.8 M27). Briefly, anesthetised embryos were mounted laterally in 1% low-melting agarose and the fluorescent Kaede protein was photoswitched by scanning the selected region of interest with a 405 nm laser (five iterations of 50 s). After isolation from the agarose, embryos were washed in Ca2+/Mg2+-free HBSS (Hank's Balanced Salt Solution, Gibco) and dissociated at 28.5 °C for 30 min in TrypLE select (Thermo Fischer Scientific, 12563011). Dissociation was stopped by the addition of FBS and centrifugation. The cell pellet was resuspended in HBSS containing Ca2+/Mg2+and 5% FBS, filtered, and submitted to FACS (BD Biosciences FACSAria III). Single-cell cDNA libraries were prepared according to the established protocol for Smart-Seq2. In brief, poly-adenylated mRNA was transcribed to cDNA using oligo(dT) primer and SuperScript II reverse transcriptase (ThermoFisher Scientific). Synthesis of second strand cDNA was achieved using a template switching oligo, and the double stranded cDNA was then amplified using polymerase chain reaction (PCR) for 23 – 26 cycles. After bead purification, overall cDNA quality was controlled (QC) by analyzing randomly selected single cell samples (wells) on a 2100 Bioanalyzer with a DNA High sensitivity chip (Agilent Biotechnologies). When the sample (plate) passed the QC, the cDNA was fragmented and tagged (tagmented) using Tn5 transposase, and each single cell sample (well) was uniquely indexed using Illumina Nextera XT index kits (set A-D). Thereafter, the indexed single-cell samples from one plate were pooled to be sequenced together on one lane of a HiSeq3000/4000 sequencer (Illumina), using dual indexing and 50 base read-length. ScRNA-Seq with Smart-seq2