GSM7596425: Myenteric cells, tdTomato-positive; Mus musculus; RNA-Seq
The mice were euthanized by isoflurane followed by CO2 asphyxiation. Age-matched wild type C57Bl6/J mice were also used as control for tdTomato expression. After isolation of the colon, fecal material was flushed with an 18-gauge needle and adipose tissue was removed. Isolated colon was then cut longitudinally and pinned with the intestinal lumen facing down on a 4% agarose pad. The longitudinal muscle and myenteric plexus (LMMP) was then microdissected and cut into 1-2 cm pieces. The tissues were then enzymatically dissociated to release myenteric neurons using 1 mg/mL collagenase IV and 0.05% trypsin. Dissociated cells were filtered through 40 m cell strainer and stained with eFluor506 fixable viability dye Live single DAT-tdTomato-positive cells were collected by fluorescence-activated cell sorting (FACS Aria). All cells were processed according to 10X Genomics Chromium Single Cell 3' Reagent Guidelines (https://support.10xgenomics.com/single-cell-gene-expression). Briefly, cells were partitioned into nanoliter-scale Gel Bead-In-EMulsions (GEMs) using 10X GemCode Technology. Primers containing (i) an Illumina R1 sequence, (ii) a 16 bp 10x barcode, (iii) a 10 bp Unique Molecular Identifier (UMI) and (iv) a poly-dT primer sequence were incubated with partitioned cells resulting in barcoded, full-length cDNA from poly-adenylated mRNA. Silane magnetic beads were used to remove leftover biochemical reagents/primers, then cDNA was amplified by PCR. Enzymatic fragmentation and size selection was used to optimize cDNA amplicon size prior to library construction. R1 (read 1 primer sequence) were added during GEM incubation, whereas P5, P7, a sample index (i7), and R2 (read 2 primer sequence) were added during library construction via end repair, A-tailing, adaptor ligation and PCR. 10X Genomics Chromium