GSM8758976: P21 Mouse retina snRNA-seq Tet 1/2/3 tcKO; Mus musculus; RNA-Seq
Dissected retina tissue was dissociated on ice in 500ul cold Homogenization Buffer (25mM KCl, 5mM MgCl2, 10mM Tris-HCl, 1mM DTT, cOmplete Mini Protease Inhibitor Cocktail (Roche)) with RNase inhibitors (200U/ml RNasin (Promega), 200U/ml SUPERaseIn (Invitrogen)) using a Dounce homogenizer, with 15 strokes each of pestles A and B. The lysate was strained through a 40um cell strainer, which was then rinsed with 9ml additional Homogenization Buffer. The strained lysate was centrifuged for 5 min at 500xg in a 4°C swinging-bucket centrifuge. After discarding the supernatant, the nuclei pellet was gently resuspended in 1ml cold Resuspension Buffer (25mM KCl, 3mM MgCl2, 50mM Tris-HCl, 1mM DTT) with RNase inhibitors (200U/ml RNasin (Promega), 200U/ml SUPERaseIn (Invitrogen)) using a wide-bore pipet tip. Nuclear concentration was quantified by Countess. Appropriate numbers of nuclei were then processed according to the PIPseq T20 3' Single Cell RNA Kit v4.0 (Fluent BioSciences). Completed libraries were sequenced on a NovaSeq X Plus (Illumina Inc) and reads processed using pipseeker (v3.3.0) and aligned to the GRCm39 reference genome.