GSM8658428: control; Mus musculus; RNA-Seq
Samples were dissociated into single cells using the 10x Genomics Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit #1000414, according to the manufacturer's instructions. For this, frozen samples were placed on a chilled glass petri dish on ice macerated in fixation buffer contraining formaldehyde using a razor blade before transfering into a 2ml low protein binding centrifuge tube, titurated using a wide bore 1ml pipette, and fixed for 20 hrs at 16-24C. The fixation buffer was then quenched and the tissue pellet dissociated using dissociation solution containing 0.2mg/ml Liberase LT for 20 minutes at 37C, titurated vigorously using a 1ml pipette before straining, and storing in quenching buffer. Cell concentration and dispension was then determined using the Countess III FL Automated Cell Counter (VANTAGE core) and propidium iodide staining solution, Invitrogen #P3566. Dissociated cells from each of the pooled samples were individually hybridized with bar coded genome-wide probe sets from 10X Genomics Flex kits according to the manufacturer's instructions. After probe hybridization and construction of libraries, samples were sequenced using an Illumina Novoseq 6000 PE150 sequencer, and bar codes deconvoluted to provide individual sample datasets.