title: GSM6206476: colon_S031; Mus musculus; RNA-Seq
library_construction_protocol: Heart MF: The heart of Cx3cr1Gfp/+ mouse was removed, the organ was chopped into small pieces and digested for 30 min at 37°C in RPMI medium without fetal calf serum supplemented with 1 mg /ml Collagenase IV and 1 mg/ml DNase I. The digestion was stopped by addition of staining buffer (2mM EDTA, 1% FCS in PBS) and the cell suspension was minced through a 100 μm cell strainer. After centrifugation at 1,200 rpm, 4oC for 6 min, supernatant was discarded and cells were blocked with anti-CD16/32 antibodies for 10 min, before anti-F4/80-biotin antibodies were added for 25 min on ice. After washing, the cell pellet was incubated with anti-biotin beads (Miltenyi) for 15min on ice. Cells were washed again (1,200 rpm, 4oC for 6 min) and MACS was performed with LS columns (Miltenyi). After elution and washing of cells, lung cells were stained for CD45, streptavidin, dump (Ly6C, Ly6G), CD11b, MerTK, CD64 and MHCII Colon MF: Cells from Cx3cr1Gfp/+ mice were isolated as previously described 48. In brief, the intestine was removed and feces were flushed with cold PBS without calcium and magnesium. The intestine was longitudinally opened and cut into 0,5 cm pieces. Intestinal epithelial cells were removed by incubation with the HBSS containing 1mM DTT, 2mM EDTA and 5% of fetal calf serum. The cell suspension was incubated at 37 °C, 125 rpm for 40 min. The cell suspension was shortly vortexed and passed through a 100μm mesh. The intestinal tissue pieces were transferred to a 50 ml Falcon tube containing 5 ml PBS with 5% FBS, 1 mg/ml of collagenase VIII (Sigma) and 0.1 mg/ml DNase I. Samples were incubated at 37 °C, 250 rpm for 40 min. Digested colon tissue was vortexed for 40 sec, passed through a 80μm mesh and cells were collected by centrifugation at 1,200 rpm for 10 min at 4 °C. Cells were stained after CD16/32 block with antibodies against CD45, CD11b, lineage (Ly6C, Ly6G, B220) and MHCII. Lung AMs: The lung was removed and cut into small pieces. Tissue was collected in RPMI medium without fetal calf serum supplemented with 1 mg/ml Collagenase A and 1 mg/ml DNase I. Tissue was digested for 30 min at 37°C. The suspension was minced and filtered through a 100μm cell strainer. The cell suspension was centrifuged at 1,200 rpm, 4oC for 6 min. The supernatant was removed and cells were blocked with anti-CD16/32 antibodies for 10 min, before anti-CD45-biotin antibodies were added for 25 min on ice. Afterwards, cells were washed with staining buffer (2mM EDTA, 1% FCS in PBS) at 1,200 rpm, 4oC for 6 min, supernatant was discarded and anti-biotin beads (Miltenyi) were added for 15min. Cells were washed again (1,200 rpm, 4oC for 6 min) and MACS was performed with LS columns (Miltenyi). After elution and washing of cells, lung cells were stained for lineage (Ly6C, Ly6G), streptavidin, CD11b, CD64, CD11c and SiglecF. Spleen RPM: The spleens were removed from Bl6 mice and minced through a 100 μm cell strainer. After washing with staining buffer (2mM EDTA, 1% FCS in PBS) at 1,200 rpm, 4oC for 6 min, supernatant was discarded and cells were blocked with anti-CD16/32 antibodies for 10 min, before anti-F4/80-biotin antibodies were added for 25 min on ice. After washing, the cell pellet was incubated with anti-biotin beads (Miltenyi) for 15 min on ice. Cells were washed again (1,200 rpm, 4oC for 6 min) and MACS was performed with LS columns (Miltenyi). After elution and washing of cells, lung cells were stained for CD45, streptavidin, lineage (Ly6C, Ly6G, B220), CD11b, CD11c and MHCII. Tongue Langerhans cells: To separate the epithelium from the rest of the tongue and to dislodge Langerhans cells, extracted tongues were injected with 5 U/ml of Dispase II (Sigma) in HEPES buffered saline until completely distended. They were then incubated for 15 min at 37oC and the epithelium layer was peeled off using forceps. The epithelium was minced in 500 μL of PBS with 0.2 mg of DNAse I (Roche), 4.8 mg Collagenase IV (Gibco) and 0.15 mg (60U) hyaluronidase I (Sigma) and incubated for 15 min at 37oC. 10 ml sorting buffer were added to the crude tissue suspension as it was passed through a 100μm cell strainer and centrifuged for 6 min at 1,200 rpm and 4oC. The pellet (enriched for tongue Langerhans cells) was then further processed accordingly. Tongue macrophages: Tongues were extracted, minced in 500 μL of PBS with 0.2 mg of DNAse I (Roche), 2.4 mg Collagenase IV (Gibco) and 0.15 mg (60U) hyaluronidase I (Sigma) and incubated for 45 min at 37oC. 10 ml sorting buffer were added to the crude tissue suspension as it was passed through a 100 μm cell strainer and centrifuged for 6 min at 1,200 rpm and 14oC. Pellet was resuspended in 3 ml PBS, layered over 3 ml of Ficoll-PaqueTM and centrifuged for 17 min at 2,000 rpm and 14oC with no acceleration nor break. The interface containing leukocytes was collected, 5 ml sorting buffer were added centrifuged for 6 min at 1,200 rpm and 4oC. Leukocyte-containing cell pellets were further processed. All tongue preparations were performed according to this protocol, if not otherwise stated. Skin Langerhans cells: Ears were dissected and placed over 0.05% Trypsin with EDTA in PBS for 1.5-2 hours at 37oC, until the epidermis could be peeled off using forceps. The epidermis was minced and the crude tissue suspension in sorting buffer was passed through a 100 μm cell strainer and centrifuged for 6 min at 1,200 rpm and 4oC. Cell-pellets were further processed. Microglia: Brains were dissected, and the olfactory bulb and cerebellum were removed. The remaining brain was minced and filtered through a 70μm cell strainer in ice-cold high-glucose DMEM (Sigma). The cell suspension was centrifuged at 1,200 rpm at 4oC for 6 minutes and the pellet was resuspended in 5 ml of 40% Percoll (GE Healthcare) in PBS and transferred to a 15 ml tube. Tubes were centrifuged at 2,000 rpm for 25 minutes (min) at 14oC with no acceleration nor break. Pellet was resuspended in 10 ml sorting buffer and centrifuged for 6 min at 1,200 rpm and 4oC. Leukocyte-containing cell pellets were further processed. 500-20,000 FACS sorted cells were lysed with 100μl of lysis/binding buffer (Life Technologies), snap-frozen on dry ice and stored at -80°C until further use. mRNA purification was performed with the DynabeadsTM mRNA DIRECTTM Purification Kit (Life Technologies) according to the manufacturer's guidelines. MARS-seq barcoded RT primers were used for .CC-BY 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.09.479699doi: bioRxiv preprint https://doi.org/10.1101/2022.02.09.479699http://creativecommons.org/licenses/by/4.0/19reverse transcription with the Affinity Script cDNA Synthesis Kit (Agilent) in a 10μl reaction volume The MARS-seq protocol was used for bulk RNA sequencing (Jaitin et al., 2014) After reverse transcription, samples were analyzed by qPCR and samples with similar Ct values were pooled. Samples were treated with Exonuclease I (New England BioLabs (NEB)) for 30min at 37°C and for 10min at 80°C followed by a 1.2X AMPure XP beads (Beckman Coulter) cleanup. The second strand synthesis kit (NEB) at 16°C for 2hours was used for cDNA synthesis followed by a 1.4X AMPure XP bead cleanup. In vitro transcription (IVT) was performed at 37°C for 13-16h with the HiScribe T7 RNA Polymerase kit (NEB). The remaining DNA was digested by Turbo DNase I (Life Technologies) treatment at 37°C for 15min followed by a 1.2X AMPure XP bead cleanup. RNA fragmentation (Invitrogen) was performed at 70°C and the reaction was stopped after 3min with Stop buffer (Invitrogen) followed by a 2X AMPure XP bead cleanup. Ligation of the fragmented RNA to the MARS-seq adapter was performed at 22°C for 2h with T4 RNA ligase (NEB) followed by a 1.5X AMPure XP bead cleanup. A second reverse transcription reaction was performed with MARS-seq RT2 primer and the Affinity Script cDNA Synthesis Kit (Agilent Technologies) followed by 1.5X AMPure XP bead cleanup. Finally, the library was amplified using P5_Rd1 and P7_Rd2 primers and the Kapa HiFi Hotstart ready mix (Kapa Biosystems) followed by a 0.7X AMPure XP bead cleanup. Fragment size was measured using a TapeStation (Agilent Technologies) and library concentrations were measured with a Qubit fluorometer (Life Technologies).