title: GSM9244343: Mouse ENS 39; Mus musculus; RNA-Seq
library_construction_protocol: Snap frozen myenteric tissue was pulverized and then dounce homogenized with in CST buffer, filtered through a 40µm filter, spun down at 800g for 8 minutes at 4°C, and blocked in ST-SB buffer for 10 minutes. Nuclei were inubated in directly conjugated HuC/D antibody (1:200) for 30 minutes at 4°C in the dark. Homogenates were then washed with additional ST buffer, spun at 800g for 8 min at 4°C, supernatant was removed, and nuclei were resuspended in ST-SB and filtered through a 40µm Flowmi filter. Single nuclei were sorted on a Sony SH800 as AF488+ directly into a tube with 10X Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 reverse transcription master mix without RT Enzyme C. RT Enzyme C was added when sorting was complete and the nuclei in master mix was then loaded into a 10X Chip G for barcoding and droplet generation. The sample protocol was done for submucosal tissue but some buffers were changed. TSP was used in the dounce homogenization step, blocking and staining was performed in SP-SB buffer, and washes were done in SPMB buffer. Used 10x Genomics 3' v3.1 kit. Enzyme C was added to the RT reaction after sorting and before GEM generation. Manufacturer instructions were followed for reverse transcription, cDNA amplification, fragmentation, ligation and sample single indexing PCR.
identifiers.namespace: GEO
identifiers.id: GSM9244343_r1