GSM7596846: Human_056316OD_macula_NeuNPos; Homo sapiens; RNA-Seq
Frozen retinal tissues were homogenized in a Dounce homogenizer in 1ml Tris-based lysis buffer with 0.1% NP-40. Nuclei were stained with NEUN/RBFOX3 and/or CHX10/VSX2, washed once, pelleted at 500g for 5min, resuspended in PBS/BSA and stained with DAPI. NEUN+ and/or CHX10+ DAPI+ single nuclei were collected using a flow cytometer. The sorted nuclei were pelleted again, resuspended in PBS/BSA solution, and adjusted to a concentration of 1000 nuclei/µL. ~12µL of the nuclei suspension was loaded into a 10X Chromium Single Cell Chip (10X Genomics, Pleasanton, CA) with a targeted recovery of 8000 nuclei. Single nuclei libraries were generated using the Chromium 3' V3.1 platform (10X Genomics, Pleasanton, CA) according to the manufacturer's protocol. Briefly, single nuclei were partitioned into Gel-beads-in-EMulsion (GEMs) where nuclear lysis and barcoded reverse transcription of RNA would take place to yield full-length cDNA; this was followed by amplification, enzymatic fragmentation, and 5' adaptor and sample index attachment to yield the final libraries. Single nucleus RNA sequecning (10X Genomics)