GSM6503212: Il10ra-/-, CD45.2+ P3/P4 macrophages 2006; Mus musculus; RNA-Seq
Animals were euthanized by CO2 and colonic lamina propria cells were isolated. Briefly, colons and cecums were dissected and epithelial cells were stripped by 10mM EDTA in HDMI for 30 min at 37°C. Remaining tissue was transferred to 50 ml conical tubes minced 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 strainer and washed twice in 1xPBS. Viability and cell numbers were determined using an automated cell counter (TECAN) and stained with the antibody cocktail and analyzed using a LSRFortessa (BioLegend, San Diego, CA, USA ). 5000-25000 P3/P4 LP macrophages from bone marrow chimeras 8 weeks following reconstitution were FACS-sorted (gated on: CD103-, Ly6G-, CD11b+, CD11cint, CD64+, Ly6C-, CD45.1+ or CD45.2+) directly into RLT buffer (Qiagen) on a FACS Aria II (BD Life Sciences) and mRNA was extracted using the RNeasy Micro kit (Qiagen). Libraries were prepared using Roche Kapa mRNA HyperPrep strand specific sample preparation kits from 200ng of purified total RNA according to the manufacturer's protocol on a Beckman Coulter Biomek i7. The finished dsDNA libraries were quantified by Qubit fluorometer and Agilent TapeStation 4200. Uniquely dual indexed libraries were pooled in an equimolar ratio and shallowly sequenced on an Illumina MiSeq to further evaluate library quality and pool balance. The final pool was sequenced on an Illumina NovaSeq 6000 targeting 40 million 50bp read pairs per library at the Dana-Farber Cancer Institute Molecular Biology Core Facilities.