GSM6735068: Organoid, replicate 1, scRNAseq; Homo sapiens; RNA-Seq
The central region of the human corneas were cut into small pieces and digested in 2 ml of digestion mix: 2 mg/ml collagenase type 1 (#SCR103; EMD Millipore) in DMEM-F12 supplemented with 5% FBS (#10438026; Gibco), 1% antibiotic-antimycotic (#15240062; Gibco), 1mM L-ascorbic acid using rotatory nutator at 37oC for 6h at 5% CO2. The supernatant was collected by allowing the undigested tissue to settle under gravity every 2 hours (h) and this was repeated 2-3 times. The collected supernatant samples were stored on ice at all stages and pooled after a final 6h of digestion, and centrifuged at 500 g for 5 min.  The supernatant was discarded and the pellet resuspended in 5 ml of 1x Accutase and incubated at 37oC for 20 min in a 5% CO2 incubator.  Fresh 5 ml of DMEM-F12 with 5% FBS was added and passed through a 40 µm cell strainer and centrifuged at 500 g for 5 min/4oC. Supernatant was discarded and the pellet resuspended in PBS with 0.04% BSA or complete DMEM-F12 to make a single cell suspension. Live cells were counted by trypan-blue exclusion in a Countess® II automated cell counter. A total 6 individual (3 organoids and 3 human corneas) cDNA libraries were prepared using the Chromium Single Cell 3' Library & Gel Bead Kit v2 (10x Genomics) and sequenced using an Illumina HiSeq 4000 as 150-bp paired-end reads.