GSM8387291: AIG Patient 3; Homo sapiens; RNA-Seq
Corpus tissues were obtained from 2-7 mice per group and 3 AIG patients. Tissues were rinsed in PBS and placed in a 50mL conical with 10mL gland digest buffer: Advanced DMEM (Thermo Fisher), 20mM HEPES (Thermo Fisher), 0.2% BSA (MilliporeSigma, Burlington, MA), 50μg/mL gentamicin (MilliporeSigma), 1% penicillin/streptomycin (MilliporeSigma), DNaseI (100U/mL; MilliporeSigma). 1mg/mL collagenase 1A (MilliporeSigma) was added to each tube prior to 37C incubation with constant shaking for 20-45 minutes. Samples were then checked for presence of gastric glands, rinsed, and spun down (50RCF for 10 minutes). Samples were resuspended in 1mL/stomach prewarmed Trypsin-EDTA (MilliporeSigma) and incubated for ~25 minutes at 37C, agitating every 5-10 minutes. Digestion was considered complete when single cells were observed. Cells were then washed twice with DMEM plus 10% fetal bovine serum. Cells were filtered twice, counted, and resuspended to a final cell concentration of ~1,000 cells/uL. Viability of each sample was confirmed to be greater than 70% by trypan blue staining (Thermo Fisher). Library construction of mouse tissues was performed at Saint Louis University and construction of AIG human libraries was performed at the Washington University in Saint Louis Genome Technology Access Center. To generate libraries, the Chromium Controller (10X Genomics, Pleasanton, CA) was used following manufacturer protocols. Libraries were prepared using the Chromium Next GEM Single Cell 5' Kit v2 (1000263, 10X Genomics). Each library was generated to have a maximum recovery of 10,000 cells. PCR reactions were conducted in the T100 Thermal Cycler (BioRad, Hercules, CA). Final quality control was performed on the 2100 Bioanalyzer Instrument (Agilent, Santa Clara, CA) and libraries were sequenced on the NovaSeq 6000 (Illumina, San Diego, CA).