GSM6070167: Colon, anti-GFP_48hr, replicate 3, scRNA-seq; Mus musculus; RNA-Seq
Transverse colon was isolated from each animal and feces were removed. After a brief wash in cold PBS, the colon was cut longitudinally, and the tissue was cut into 3-4 mm length fragments. Tissue fragments were incubated in pre-warmed (37 °C) PBS with 5 mM EDTA in a shaker at 37 °C at 150 rpm for 15 minutes. After 15 minutes, the tubes containing the samples were vigorously shaken for 10 seconds to release more epithelial cells. The epithelial cells floating in suspension were removed to a new tube and centrifuged at 200 rcf for two minutes. The residual tissue containing the remaining epithelia and stroma/lamina propria was then incubated in 8-12.5 mL of lamina propria dissociation buffer (AdvDMEM/F12 with 10 mM HEPES, 0.2 % FBS, DNAse1 (80 U/mL), Liberase TM (0.2 mg/mL), and 1 % antibiotic/antimycotic) at 37 °C for 30 minutes with horizontal shaking at 150 rpm. After pelleting, the epithelial cells were resuspended in 1 mL of TrypLE with DNase1, incubated at 37 °C for five minutes, and then triturated with a P1000 pipette for 30 seconds. After trituration, 10 mL of PBS plus 50 U/mL DNAse1 were added to the epithelial cells, and they were centrifuged at 500 rcf, 4 °C, and the supernatant was removed. Epithelial cells were then washed one time in FACS buffer (HBSS, 2% FBS, 10 mM HEPES, 1 mM sodium pyruvate, and 1% Pen-strep or antibiotic/antimycotic solution) before another round of centrifugation and final resuspension in 0.5 mL of FACS buffer. Following 30 minutes of dissociation in LP dissociation buffer, the remaining tissue fragments and suspension were centrifuged at 500 rcf for five minutes. Supernatant was removed down to 1 mL, and the sample was triturated with a P1000 until the solution was homogeneous and all tissue fragments had dissociated. After trituration, the sample was centrifuged at 500 rcf for 5 minutes at 4 °C and washed in FACS buffer prior to resuspension in 1 mL of FACS buffer in preparation for FACS. All cells were passed through a 40-micron filter prior to FACS. DAPI was used to assess viability, and only DAPI-negative cells were collected. Cells were collected from the epithelial fraction and then from the epithelial/lamina propria fraction and combined (1:5 ratio) and counted on a hemocytometer prior to cell capture. Standard 10x Genomics Chromium 3' v3 scRNA-seq reagents (PN1000075) were used. Approximately 4000-4500 cells were loaded per channel. Cells from one animal replicate were captured per channel. Standard 10x Genomics Chromium 3' v3 scRNA-seq RT, cDNA amplification, and sequencing library preparation protocols were followed. Multiplexed sequencing libraries were sequenced on Illumina Nova Seq 6000 S1 lanes, averaging about 50,000 reads per cell.