title: GSM6855742: KO mice treated with AOM-3DSS; Mus musculus; RNA-Seq
library_construction_protocol: Colonic tumor/ non-tumor tissue-infiltrating cells were prepared as follows. Briefly, tissue pieces were cut into 1 mm slices, then they were shaken for 40 min in Hanks' Balanced Salt Solution (HBSS) containing 3 mM EDTA at 37℃. Intra-epithelial lymphocytes and colonic epithelial cells in the culture supernatant were discarded, and the gut slices were washed twice with HBSS. Then, they were shaken at 37℃ for 120 min in RPMI containing 10% FBS and 1% streptomycin+penicillin, 200 U/ml collagenase (C2139; Sigma-Aldrich), and 5 U/ml Dnase 1 (Sigma-Algrich). After the incubation, the samples were vortexed for 10 sec, and single cell suspension was harvested after sterile gauze-filtration. To prepare lymphocytes, tissue-infiltrating cells were further purified on a 45%/66.6% discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient at 2200 rpm for 20 min. Colonic polyp-infiltrating myeloid cells for scRNA-seq analysis were stained with anti-mouse CD11b and CD11c antibodies for flow cytometry and were sorted by FACS Aria Fusion (BD Biosciences). After colonic polyp-infiltrating myeloid-derived cells were harvested, cell count and viability was estimated using fluorescence Cell Analyzer (Countstar® Rigel S2) with AO/PI reagent. Fresh cells were resuspended at 1×106 cells per ml in 1×PBS and 0.04% bovine serum albumin. Single-cell RNA-Seq libraries were prepared using SeekOne® MM Single Cell 3' library preparation kit (SeekGene Catalog No.SO01V3.1). Briefly, appropriate number of cells were loaded into the flow channel of SeekOne® MM chip which had 170,000 microwells and allowed to settle in microwells by gravity. After removing the unsettled cells, sufficient Cell Barcoded Magnetic Beads (CBBs) were pipetted into flow channel and were also allowed to settle in microwells with the help of a magnetic field. Next excess CBBs were rinsed out and cells in MM chip were lysed to release RNA which was captured by the CBB in the same microwell. Then all CBBs were collected and reverse transcription were performed at 37℃ for 30 minutes to label cDNA with cell barcodes on the beads. Further Exonuclease I treatment were performed to remove unused primer on CBBs. Subsequently, barcoded cDNA on the CBBs was hybridized with random primer which had reads 2 SeqPrimer sequence on the 5' end and could extend to form the second strand DNA with cell barcode on the 3' end. The resulting second strand DNA were denatured off the CBBs, purified and amplified in PCR reaction. The amplified cDNA product was then cleaned to remove unwanted fragment and added to full length sequencing adapter and sample index by indexed PCR. The indexed sequencing libraries were cleanup with SPRI beads, quantified by quantitative PCR (KAPA Biosystems KK4824) and then sequenced on illumina NovaSeq 6000 with PE150 read length.