GSM2571699: P4-1_RNA-seq; Mus musculus; RNA-Seq
A protocol from Siegert et al. (Nature Neuroscience 2012) was used to dissociate retinal cells. Retinas were dissected in HBSS and incubated in an activated papain solution for 5 min at 37 °C. After removing the papain solution, retinas were washed with HBSS containing 2% FCS and then dissociated in HBSS containing 2% by triturating the tissue with a fire-polished Pasteur pipette. The suspension was filtered before FACS sorting. Per time point (P0 till P12), we FACS-sorted three retinas, each from a different animal. If 20,000 cells could not be obtained from one retina, retinas of different animals were pooled. Cells where sorted using a BD Influx™ cell sorter (Becton, Dickinson and Company, BD Biosciences, San Jose, CA USA) with the BD FACS™ Sortware sorter software (Version 1.01.654). The sort was performed with a 100 μm nozzle tip, at a sheath pressure of 19.0 psi, and a frequency of 28.90 kHz. The following gates were set: gate 1 was forward scatter FSC-Area against side scatter SSC-Area, gate 2 was FSC-Width against FSC-Height, gate 3 was SSC-Width against SSC-Height, and gate 4 was 530/40 (488 nm) against 710/50 (561 nm). RNA isolation was done using the Pico Pure kit. Total RNA amplification was done using the NuGEn Ovation RNA-Seq System V2 followed by library preparation with the Illumian TruSeq Nano DNA Sample prep protocol. Samples were sequenced in the Hiseq in 50 cycles, producing single-end reads. RNA libraries were prepared for sequencing using standard Illumina protocols