GSM8684550: GF_HET_midterm; Mus musculus; RNA-Seq
Large intestine tissue was cut into 2 cm segments and placed in a 50 ml Falcon tube with 10 ml cold 1X HBSS, then shaken by hand and incubated in a bacterial shaker at 37°C and 225 rpm for 15 minutes to separate intraepithelial and lamina propria lymphocytes. The tissue was then transferred to a new tube containing EDTA-only mix and shaken under similar conditions. Next, the contents were moved to a tube with FACS buffer, gently mixed, and transferred to a 6-well plate for mincing. Digestion buffer was added, and the plate was shaken at 37°C until digestion was complete. The digested tissue was filtered, washed with FACS buffer, centrifuged to discard the supernatant, resuspended in FACS buffer, and finally transferred to FACS tubes for analysis. The collected cells were then stained with CD45  (Biolegend, Cat# 103122) and Live/Dead (L/D) Blue markers (Thermo Fisher, Cat# L34962), and the CD45-positive, L/D Blue-negative cells were sorted for single-cell library preparation. Single cellswere processed through throgh the GemCode Single Cell Platform using the Chromium Next GEM Single cell 3' Kit v3.1 (10X Genonmics, Pleasanton) according to the manufacturer's protocol. In brief, Cell suspensions were loaded onto a Chromium Single-Cell Chip G along with the ereerse transcription (RT) master mix and single cell 3' gel beads. Following generation of single-cell gel bead-in-emulsions (GEMs), reverse transcription was performed using a Applied Biosystems™ Veriti™ 96-Well Thermal Cycler (Thermo Fisher Scientific, US). Amplified cDNA was purified using SPRIselect beads and converted to the sequencing library according to the manufacturer's protocol. Library was sesquenced on an Ilumina NovaSeq platform following read length: 150 bp Read 1 (16bp single cell barcode, 10x barcod; 12bp Unique molecular Identifier, UMI), 150 bp Read 2 (transcipt insert), 10 bp i7 Index (sample index), 10 bp i5 index (sample index)