GSM8647244: Batch2_Replicate_2_Hash; Mus musculus; RNA-Seq
Mice colon was harvested and immediately preserved in ice-cold HBSS. Shortly after, tissue was incubated for 20min at 37°C in PBS containing 2% FCS and 5 mM DTT. Supernatant was discarded, colon was cut into small pieces and again incubated at 37°C for 30min in RPMI 1640 containing Liberase (2.5mg/ml) and DNase (50 U/ml) with shaking. Supernatant was then collected and centrifuged for 10min at 4 °C and 400g. The resulting pellet was washed three times with PBS containing 10%FCS. After the last washing step, pellet was resuspended in 1ml PBS containing 10 % FCS. Per experimental condition, single cells were tagged using TotalSeq-B anti-mouse Hashtags (Biolegend; #155831, #155833, #155835, #155837) at 25 µg/ml for 30 min at 4°C following the manufacturer's instructions. The different experimental conditions were tagged as follows: wildtype mice, TotalSeq-B0301 anti-mouse Hashtag 1 antibody; JUP-KO mice, TotalSeq-B0302 anti-mouse Hashtag 2 antibody; DSS-treated WT mice, TotalSeq-B0303 anti-mouse Hashtag 3 antibody; DSS-treated JUP-KO mice, TotalSeq-B0304 anti-mouse Hashtag 4 antibody. For each batch, single cells from the different experimental conditions were pooled and flow-sorted on a BD FACS Aria II to purify viable single cells. The subsequent processing of cells was performed using the 10x Chromium Next GEM Single Cell 3' v3.1 with cell surface barcode technology (dual index). Approximately 23,000 – 25,000 pooled cells were loaded to capture about 14,000 cells per batch. The DNA libraries for gene expression analysis and barcodes were pooled and sequenced paired-end on an Illumina NextSeq 2000 instrument with a P3 flow cell and 100 cycles, generating approximately 1,200 million reads. scRNA-seq was performed using the 10x Chromium Next GEM Single Cell 3' v3.1 with cell surface barcode technology (dual index).