GSM4904705: Plate number LC396; Mus musculus; RNA-Seq
Retinas were removed at P6, dissociated into single cells using Liberase TM and Dnase I and stained for PECAM1. PECAM1+EGFP+ cells were flow sorted into a chilled 384-well PCR plate (Greiner, 785290) containing 1.2 μl of primer/lysis mix [20 nM indexed polydT primer (custom made, IDT), 1:6.000.000 dilution of ERCC RNA spike-in mix (Ambion, 4456740), 1 mM dNTPs (NEB, N0446S), 1.2 units SUPERaseIN Rnase Inhibitor (Thermo Fisher, AM2696), DEPC water (Thermo Fisher, AM9920)] using a BD FACSAria III flow cytometer (BD Biosciences, San Jose, CA, USA). Prepared RNA mixture plates were sealed and immediately frozen upside down at -80C. Single cell transcriptome libraries were generated using the CelSeq2 protocol (Hashimshony et al., 2016 Genome Biol.) with the following adaptations: Second strand synthesis was performed using NEBNext Second Strand Synthesis module (NEB, #E6111S) in a final reaction volume of 8 µl and NucleoMag NGS Clean-up and Size select magnetic beads (Macherey-Nagel, 7449970.5) were used for all DNA purification and size selection steps.