title: GSM8704924: Cx3cr1-GFP+ cells from host retina upon transplantation - Sample 2; Mus musculus; RNA-Seq
library_construction_protocol: Three days following RGC transplantation, mice were euthanized, eyes enucleated and retinal tissue isolated and transferred into a tube containing PBS with 0.04% BSA on ice. Eight mouse retinas were pooled for each sample. The tissue was dissociated by mechanical and enzymatic methods using Gentle MACS protocols. Briefly, retinas were allowed to settle to the bottom of the tube and excess PBS carefully removed. Tissues were then rinsed with HBSS and transferred to a MACS M-tube and let to settle to the bottom of the tube before again removing excess HBSS. Tissues were then resuspended in 2ml of dissociation solution containing DNAse 1 (1mg/ml). The mixture was incubated at 37°C for 5min in a heating bath. The M-tube was then inverted ensuring that all retina tissues remained in solution, and the tube placed into the MACS tissue dissociator and the brain 1 program run. At the end of brain 1 program, the M-tube was removed from the MACS dissociator and incubated at 37°C for 5mins in a heating bath. The tube was then inverted again and placed back into the MACS dissociator and program brain 2 initiated, after which 10mL of isolation buffer was added to the dissociate and the mixture triturated 7-10 times with a 1000µl pipette tip. Then 12ml of the homogenate was transferred into a 15ml tube and cells pelleted at 200g for 5 mins at 4°C. The cell pellet was resuspended in 500µl of isolation buffer and filtered using a 70um cell strainer into a FACS tube for sorting. All tubes for this procedure were pre-blocked with 2% BSA in HBSS. Separate single stain samples for TdTomato+ and GFP+ were prepared and used to set the gates. The harvested cells were sorted separately for TdTomato +ve, and GFP +ve, and collected into separate collection tubes each containing 200μl of PBS with 0.04% BSA. The sorted cells were taken for single cell RNA sequencing library preparation. Of the sorted cells, up to 10,000 viable cells per sample were used for single cell RNA library preparation following the 10x genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 protocol. Briefly, the droplet-based encapsulation method was used to encapsulate single cells with gel beads, partitioning oil, and a reverse transcription master mix generating GEMs. The generated GEMs were cleaned, gene expression libraries constructed and sequenced at a sequencing depth of 20,000 read pairs per cell, paired-end dual indexing (See manufactures instructions for detail).