GSM2694019
GSM2694019: OPC2; Danio rerio; RNA-Seq
Spinal cord dissection, tissue dissociation and nuclear labelling:  Adult zebrafish were terminally anesthetized, their spinal cords exposed and the spinal cord tissue was carefully removed. Up to 5 spinal cords were dissected at once and placed in 1ml of Hanks´ Buffered Salt Solution (HBSS, Gibco)). For the dissociation of spinal cords into a single cell suspension at room temperature, the tissue was incubated for 3 min with 100 µL 0.25% Trypsin (Sigma; T4549) / 1mM EDTA and triturated using a 200 µL pipette during the whole incubation time avoiding the formation of air bubbles. Then, tissue digestion was stopped by adding 100 µL of 20% fetal calf serum (Sigma; F0804) in 2 mM CaCl2 and 300 µL HBSS. Afterwards the cell suspension was allowed to sit for 5’ at on ice, before it was applied to a 20 µm (OPCs, Miltenyi Biotec, # 130-101-812) or a 35 µm (OL, Corning, # 352235) cell strainer, respectively. After washing with 9 ml of HBSS, the cells were pelleted by centrifugation for 5’ at RT at 300g. The supernatant was removed, cells were resuspended in 1 ml HBSS including 1 µl Vybrant™ DyeCycle™ Violet Stain (Molecular Probes, Invitrogen, V35003) and incubated for 30´ at 28 °C to stain cellular nuclei.  FACsorting of myelin rich spinal cord tissue:  Cell suspensions were sorted directly into 96 well plates, filled with culture media only, or pre-seeded with MN using a BD FACSAria sorter. For the detection of GFP a 488 nm excitation laser and a 530/30 bandpass filter were used. DsRed was detected with a 582/15 bandpass filter after excitation with a 561 nm laser. Vybrant™ DyeCycle™ Violet Stain was detected after 405 nm excitation and a 450/40 bandpass filter. Cellular events were defined by forward and side scatter profile and by the dsRed or GFP signal of the corresponding transgenic line. From this selection, all events that showed incorporation of the Vybrant™ DyeCycle™ Violet Stain were gated because this proved to be an ideal selection to separate the small zebrafish CNS cells from remaining cellular and myelin debris. For the RNAseq experiment, the SMARTer Ultra Low Input RNA for Illumina Sequencing Kit - HV from Clontech was used to reverse transcribe the RNA and amplify full-length cDNA according to the user manual. Therefore, 2000 cells from five biological replicates of both lines Tg(olig2:eGFP) and Tg(mbp:GFP) where directly FACsorted in 5ul reaction buffer containing RNAse Inhibitor. After amplification of cDNA using 12 cycles, the cDNA was sheared to 200bp fragment length with 8-micro TUBE strip in the ultrasonicator Covaris LE220, followed by a library prep using NEBNext Ultra DNA Library Prep Kit for Illumina (NEB).
GEO
GSM2694019
GEO Accession
GSM2694019