GSM8515115: Vil1Cre negative, Gsdmc1-4 fl/fl, Day 10 DSS colon, Hashtag counts; Mus musculus; RNA-Seq
After collecting the colons, the colonic content was squeezed out and the colons flushed with 1ml of ice-cold HBSS. The colons were first cut longitudinally and then into 0.5-1cm fragments. These fragments were washed three times with 30ml of ice-cold HBSS. After the final wash, the fragments were resuspended in 10ml of epithelial dissociation media (1X HBSS supplemented with 15 mM EDTA). Colonic fragments were shaken at 220 RPM and 37°C for 15 minutes and then placed on ice for 5 minutes. The colonic fragments were shaken vigorously 30 times, vortexed for 2 seconds, and then placed into 30 ml of ice-cold HBSS and gently inverted. Colonic fragments were then finely minced and incubated in 5 ml of pre-warmed tissue digestion media [tissue media (RPMI supplemented with 5% FBS, 10mM HEPES, 1mM Sodium Pyruvate, 5mM L-Glutamine, 1X NEAA, 1X Pen/Strep) supplemented with 100µg/ml Liberase™ TM Research Grade (Roche Life Science) and 100µg/ml DNase I (Sigma)] per 4 cm of colon tissue and incubated at 220 RPM, 37°C for 45 minutes. After digestion 50µl of 0.5M EDTA and 50µl of FBS were added to quench the reaction, gently swirled to mix, and pipetted 40 times using a wide bore P1000 pipet tip. This suspension was filtered through a 40µm cell strainer (Falcon). Cells were pelleted at 400xg for 10 minutes at 4°C. The pellet was resuspended in ACK buffer to lyse red blood cells and incubated on ice for 3 minutes. ACK was quenched using 20 ml of tissue base media and pelleted again at 400xg for 10 minutes at 4°C. The cells were resuspended in tissue media, counted and 2x106 cells were transferred to 5 ml FACS tubes (Falcon) for downstream analyses. In an effort to reduce technical batch effects, cells isolated from individual mice were labelled with unique oligos and pooled prior to loading the 10X controller.  Briefly, 2x106  cells were pelleted at 500xg for 5 minutes and incubated with 0.5 µg TruStain FcX (anti-mouse CD16/32) (Biolegend) in 50 µL of FACS buffer (PBS supplemented with 5% FBS, 2mM EDTA) for 10 minutes at 4°C. As commercially available hashtag antibodies target CD45 and MHC class I, we opted to reduce hashtag bias towards immune cells by utilizing a custom cocktail of antibodies with markers of major subsets. Following Fc receptor blockade, cells were stained with a cocktail of antibodies consisting of anti-CD45-biotin (clone 30-F11; 103104), anti-H-2K-biotin (clone 28-8-6, 114603), anti-CD31-biotin (clone IM7, 103004), anti-CD140a-biotin (clone APA5, 135910), anti-CD140b-biotin (clone APB5, 136010), anti-PDPN-biotin (clone 8.1.1, 127404) and, anti-EpCAM-biotin (clone G8.8, 118204), anti-CD31 (clone MEC13.3, 102504) (all Biolegend) resuspended at 0.5µg per antibody in 50µL of FACS buffer for 20 minutes at 4°C (Biolegend). Following antibody staining, cells from each individual animal were washed 3 times and incubated with a unique TotalSeqTm-B-0952, -B-0953, -B-0954 Streptavidin-PE (cat. # 405287, 405289, and 405291 respectively, all Biolegend) at 4°C for 20 minutes. Finally, cells were washed 3 times in 3mL of 1% BSA in PBS prior to resuspending the cells in 200µl 1% BSA in PBS. Cells were counted, and individual mice across the same condition were pooled at equal cellular ratios, for a total of 4 pools each representing one condition. Libraries were constructed as outlined in the 10x Genomics guide for Dual-indexed 3' transcriptomics v3.1 with Feature Barcoding.