GSM5575029: Colonic LP macrophages sample 1; Mus musculus; RNA-Seq
Animals were euthanized in a CO2 chamber, Colonic lamina propria cells were isolated. Briefly, colons were dissected and epithelial cells were stripped by agitation in 10mM EDTA for 30 min at 37°C. Remaining tissue was transferred to 50 ml conical tubes and digested using Collagenase VIII (Sigma-Aldrich, C2139-5G) in HBSS (with Ca /Mg) for 30-45 min at 37°C.  Cells were then filtered through 70µm cell strainers and washed twice in 1xPBS.  Cells viability and numbers were determined using automated cell counter (TECAN) and stained with the antibodies cocktails as indicated and analyzed using a LSRFortessa (BioLegend, San Diego, CA, USA ). Live CD45+, Ly6G-, CD103-, CD64+, CD11b+, MHCII+, Ly6C- scells were directly sorted into RLT buffer (Qiagen) and RNA prepared. cDNA was sythesized using Takara SmartSeq v4 reagents from 1 ng of RNA. Full length cDNA was fragmented to a mean size of 200bp with Covaris M220 ultrasonicator and Illumina sequencing were prepared from 2ng of sheared cDNA using Swift 2S Acel reagents on a Beckman Coulter Biomek i7 according to manufacturer's protocol. The finished dsDNA libraries were quantified by Qubit fluorometer, Agilent TapeStation 2200 and RT-qPCR using the Roche Kapa library qualification kit. Uniquely dual indexed libraries were pooled in equimolar ratios and evaluated for cluster efficiency and pool balance with shallow seqencing n an Illumina MiSeq. Final sequencing was performed on an Illumina NovaSeq6000 with paired-end 150bp reads at the Dana-Farber Cancer Institute Molecular Biology Core Facilities.