title: GSM6792278: Wild type  F. nucleatum; Mus musculus; RNA-Seq
library_construction_protocol: This protocol was adapted from Haber et al 2017. To generated single-cell suspensions, ApcMin/+ and wild type mice were euthanized at 11 weeks of age, colons and small intestines were excised, rinsed with ice cold sterile 1X Ca2+ and Mg2+ free PBS (Gibco, 14190144) and flushed of fecal contents using a blunt 1.5 inch 22G needle filled with ice cold sterile 1X Ca2+ and Mg2+ free PBS (Gibco, 14190144). The tissue was opened longitudinally and sliced into small fragments roughly 1 cm in length. The tissue was incubated in RPMI supplemented with L-glutamine (Corning, 45000-396), 1 mM EDTA (Neta Scientific, QB-A611-E177-10), and 10% FBS (Avantor, 97068-085) for 90 minutes, shaking every 30 minutes. The tissue was then incubated at 37°C for 15 minutes and continuously shaken. The supernatant was passed through a 100 m cell strainer and held on ice until loading the cells on 10X Chromium. The remaining tissue was resuspended in RPMI (Corning, 45000-396) supplemented with 20% FBS (Avantor, 97068-085), 0.1 mg/ml DNase I (Thermo Scientific, 90083), and 0.5 mg/ml collagenase A (Millipore Sigma, 10103586001) and incubated at 37°C on a shaker for 30 minutes. The tissue was then gently mechanically dissociated using a rubber plunger of a syringe. The tissue and the dissociated contents were passed through a 100 m cell strainer. The single cell suspension was then pelleted via centrifugation (400 x g for 10 minutes at 4°C). The cell suspension was resuspended in 1X Ca2+ and Mg2+ free PBS (Gibco, 14190144) containing 0.04% weight/volume BSA (VWR, 97061-420) and combined with earlier collected fraction and placed on ice. Sample viability was determined before loading the cells on 10X Chromium using the Countess II Automated Cell Counter (ThermoFisher). The desired number of transcriptomes from viable cells for each sample was 5000-6000 cells per sample. Libraries targeting 5000-6000 viable cells per sample (for colon and small intestine tissues) were generated using a 10X Genomics Chromium Controller and the Chromium Next GEM Single Cell 3' Kit v3.1 . Per manufacturer description, briefly, cells were separated into GEMs along with beads coated in oligos that capture mRNAs using a poly-dT sequences. This was followed by cell lysis and barcoded reverse transcription of mRNA, followed by amplification, enzymatic fragmentation, and 5′ adaptor and sample index attachment. The libraries were sequenced using an Illumina NextSeq 2000 run with the 100 bp P2 kit.