title: GSM4996497: In vivo grafted hESC-RPE and rabbit, rabbit 1 [P13104_9002_S2_L002]; Homo sapiens; Oryctolagus cuniculus; RNA-Seq
library_construction_protocol: A rabbit eye with 30-day integrated hESC-RPE were enucleated and pigmented areas including neuroretina, choroid and RPE layer was dissected out, trimmed and mixed together in 500 uL of digestion buffer. Digestion buffer consisted of: 2mg/ml collagenase IV (ThermoFisher Scientific, 17104019), 120 U/μl DNase I (NEB, Sigma, 4536282001), and 1mg/ml papain (Sigma, 10108014001) in PBS. Eppendorfs containing the samples were rotated and incubated at 37°C on a thermocycler at 300g for 25 min until samples were homogenized. Samples were pipetted every 5 min to digest the tissue sample into single cells. Digestion was stopped by adding equivalent volume of 10% fetal bovine serum (ThermoFisher Scientific, 10082147) in PBS, the samples were filtered using a 30µm MACS Smart Strainer (Miltenyi, 130-098-458) followed by Dead Cell Removal kit (Miltenyi, 130-090-101) to remove dead cells and debris. Cells were finally resuspended to 1000 cells/uL in 1% BSA in PBS prior to scRNA-sequencing. Cells were transported at 4°C to the Eukaryotic Single Cell Genomics Facility (ESCG, SciLifeLab, Stockholm, Sweden) where a 3' cDNA library was prepared for single cell RNA sequencing (scRNA-seq) using the 10X Genomics platform and NovaSeq 6000 software.