GSM8572047: G4_PM_mRNA; Homo sapiens; RNA-Seq
Tissue samples were collected into ice-cold MACS tissue storage solution (Miltenyi Biotech). After washing with DPBS (Thermo Fisher Scientific), mucosal tissues were minced into small pieces by dissecting scissors.  The minced tissues were digested in 5 ml TrypLE Express (Thermo Fisher Scientific) in gentleMACS C tubes (Miltenyi Biotech) using GentleMACS Octo Dissociator (Miltenyi Biotech) at 37oC for 30 min.  The digested cells were passed through a 40 micromete strainer (Greiner Bio-One), collected into 1% BSA DPBS-treated 50 ml conical tube.  After washing twice with ice-cold DPBS, the cells were incubated with anti-FcR antibody (Miltenyi Biotech) and then stained with PE-conjugated anti-EpCAM antibody (Novus Biological), Alexa Fluor 647-conjugated anti- CD45 antibody (BioLegend) and DAPI (Kirkegaard & Perry Laboratories) in 400 microliter DPBS.  After washing with DPBS twice, cells were resuspended in phenol-free DMEM (Thermo Fisher Scientific).  Live DAPI-negative EpCAM+ cells were sorted by FACS using a BD FACSAriaTM Fusion (Becton Dickinson) and collected into 1% BSA DPBS-treated microfuge tube containing 800 microliter phenol-free DMEM. Single cell RNA-seq libraries were produced using Chromium 3' v3 kit (10X Genomics) according to the standard manufacturer's protocol and sequenced on Illumina NovaSeq6000.