title: GSM8728965: Retina from rd1 mouse sample 2; Mus musculus; RNA-Seq
library_name: GSM8728965
library_construction_protocol: Following CO2 euthanasia and cervical dislocation, six eyes from three rd10 mice were enucleated and transferred to CO2-independent medium (Thermo Fisher Scientific, Ref. 18045088) for dissection. The retinas were isolated, chopped into small fragments, and washed twice in warm homemade Ringer solution without Ca2+. The fragments were then incubated in 1 ml of activated papain (4 units/ml, Worthington Biochemical Corp., Ref. LSO 3124) at 37°C for 20 minutes. Digestion was halted by adding 1 ml of Neurobasal-A medium supplemented with L-Glutamine (1%) and fetal bovine serum (5%). DNase I (0.02 mg/ml, Sigma-Aldrich, Ref. D4263) was then added, and the tissue was gently dissociated by trituration using a 1000 µl pipette tips. A brief centrifugation (30 seconds, 115 g) was performed to separate the dissociated cells (supernatant) from aggregates. This process was repeated 4 to 6 times until the retinas were fully dissociated. The total cell suspension was then centrifuged for 10 minutes at 115 g. The resulting pellet was resuspended in a small volume of 1X PBS (calcium and magnesium-free) containing non-acetylated bovine serum albumin (400 µg/ml), then viability and cell concentration was determined using a Malassez counting chamber. The barcoded cDNAs were prepared from dissociated cells using a 10 × genomics chromium and were used to construct the indexed scRNA-sequencing libraries according to Chromium Next GEM Single Cell 3′ Library v3.1 and dual Index Kit TT according to manufacturer's protocol.
identifiers.namespace: GEO
identifiers.id: GSM8728965_r1