GSM7734029: Retina, EAU, biol rep1; Mus musculus; RNA-Seq
The retinas were carefully dissected out and placed into Hanks' Balanced Salt Solution (HBSS) containing 10 mM HEPES on ice for transport. After all retinas were collected, they were dissociated using the Worthington Papain Dissociation system. Retinas were placed in a solution of 20 U/ml papain, 0.005% DNase I with 1 mM L-cysteine and 5 mM EDTA in Earle's Balanced Salt Solution (EBSS), for 10 minutes at 37°C with frequent, gentle agitation. Samples were then diluted by addition of 500 μl of EBSS to inactivate the papain and centrifuged at 300 x g for 5 min at room temperature. Pellets were resuspended in 525 μl of a solution containing 1 mg/ml ovomucoid and BSA and 100 U/ml DNase I in EBSS. The resulting suspension was carefully layered over 500 μl ovomucoid/BSA solution and centrifuged at 70 x g for 6 min. Supernatant was discarded and cells were resuspended in a solution of PBS containing 0.04% BSA. scRNAseq transcriptome processing was performed using the Chromium 10X system involving GEM generation, post GEM-generation clean-up, cDNA amplification and DNA quantification. Chromium Single Cell Reagent Kits solution (10X SC RNA CITE-TSC, 10X SC RNA 5pr v2.0 Chemistry) was used to deliver a scalable microfluidic platform for digital scRNA-seq by profiling 500-10,000 individual cells per sample. A pool of ~3,500,000 10x Barcodes were sampled separately to index each cell's transcriptome. Libraries were generated and sequenced from the cDNAs and 10x Barcodes were used to associate individual reads back to the individual partitions. The library was sequenced using the Illumina NovaSeq platform.