GSM3703664: cLP_p2mono_ko_hh_S3; Mus musculus; RNA-Seq
Colons and/or caeca were harvested from mice, washed in PBS/BSA and content flushed with forceps. Intestines were then opened longitudinally and washed once more before blotting to remove mucus. Gut tissue was then cut into 1 cm long pieces and placed in 50 mL centrifuge tube (Greiner) in ice cold PBS + 0.1% BSA. Colons were incubated 2 times at 200 rpm in 40 mL HBSS + 0.1% BSA + 1% Penicillin-Streptomycin (PS, Lonza) + 5mM EDTA (Sigma-Aldrich) at 37 °C for 10 min before the supernatant was aspirated. Tissue was placed in 40 mL PBS + 0.1% BSA + 1% PS for 5 min. Intestines were then incubated with 20 mL RPMI + 10% FCS +1% PS + 2.5 U/mL Collagenase VIII (Sigma-Aldrich) + 2 U/mL DNAse I (Roche), shaking at 200 rpm for 45 mins - 1 hour at 37 °C. Supernatant was filtered through a 70 μm cell strainer to which 30 mL of ice cold PBS + 0.1% BSA + 1% PS + 5 mM EDTA was added to ablate collagenase/DNase activity. Cells were washed in 30 mL PBS/BSA before filtering once more through a 40 μm cell strainer. The cells were then pelleted by centrifugation at 400 rcf for 10 minutes at 4 °C.  LPLs were isolated by resuspending cells in 4 mL P80 (80% P100 (9:1 percoll:10X PBS) + 20% RPMI) percoll in a 15 mL centrifuge tube (Greiner) before overlaying 4 mL P40 (40% P100 + 60% 1X PBS) layer. Cells were spun at 2000 rcf for 20 min at room temperature, slow acceleration, no brake. Mucus and cellular debris were aspirated from the surface of the P40 layer with a Pasteur pipette and pipetted into 40 mL of ice cold PBS + 0.1% BSA. Cells were pelleted by centrifugation at 400 rcf for 10 min and resuspended in 1 mL RPMI + 10% FCS + 1% PS before counting. Cells were sorted on a FACS ARIA II cell sorter (BD) using a 100 μm nozzle. For Bulk samples, cells were sorted directly into SmartSeq II lysis buffer (Picelli et al., 2013). For 10X samples submission, cells were sorted into 1.5 mL microcentrifuge tubes coated with FCS. For each sample 100 cells were FACs sorted into 2ul of Smart-seq2 lysis buffer and cDNA prepared according to the Smart-seq2 protocol. RNA-seq libraries were prepared using Nextera XT kits (Illumina).