title: Single-cell transcriptomics of mouse gut
design: Single-cell transcriptomics of mouse gut
library_construction_protocol: gut tissue sections were collected following transcardial perfusion in cold D-PBS (02-023-1A, Biological Industries). Intestine was sectioned such that the region we took samples from included the lower part of the upper third intestinal tract and washed in cold D-PBS. Samples were cleaned and then sliced longitudinally, cleaned and again washed with D-PBS. Next, samples were minced and 0.15-0.2g of the minced tissue was transferred into the digestion mix (1.07 wU/ml Liberase DH (Roche, 5401054001), (70 U/ml) Hyaluronidase (Sigma-Aldrich 385931-25KU), (70 ug/ml) DNAse (11284932001 ,Roche) I in HBSS buffer (H6648 ,Sigma-Aldrich). The samples were incubated for 25 minutes on a shaking platform at 150rpm, and gently mixed every 10 min. Following incubation, the cells were passed through a 40mM filter into a 50ml falcon and added with 12ml of Neutralization media (DMEM, Rhenium (41965039), 10% Heat inactivated FBS (Rhenium, 10270106)). The cells were pelleted using centrifugation (400 G, 4C for 5 minutes), resuspended in DMEM and counted. Depending on red blood cell (RBC) presence in the pellet, samples were then treated with RBC cell lysis (Sigma, R7757, according to the manufacturer's protocol), quenched with 10% FBS in D-PBS,and again pelleted and resuspended. Dead-cell removal protocol (Biotec Miltenyi, 130-090-101) was applied according to the manufacturer's protocol in cases when cell viability was lower than 75%. The flowthrough was then pelleted at 400G and resuspended in PBS with 0.05% BSA. Cells were counted and loaded on the 10X Genomics Chromium instrument for single-cell library preparation, aiming to capture 10,000 cells, using 3' v2 chemistry according to the manufacturer's protocol cDNA library preparation was were carried out according to the manufacturer's protocol.