GSM4907340: E18_SI; Mus musculus; RNA-Seq
isolation protocol: Small intestines from fetal mice were isolated from embryos under a dissecting microscope. Surrounding mesenteric tissue was removed under further magnification and cut open longitudinally. Tissue was incubated for 30 min at 37°C under agitation in HBSS containing calcium and magnesium (HBSS+/+), 2% FCS, 10 mM HEPES, 0.2 U/mL Liberase DL (Sigma) and 50 µg/mL DNaseI (Sigma). Leftover tissue was dissociated using a 22G needle and cell suspension was passed through a 70 µm strainer and washed in PBS/BSA. Cells were subsequently surface stained for FACS and in the second step with Total-Seq antibodies (BioLegend) according to manufacturer's instructions. Cells were sorted on a BD Aria II machine. FACS sort and CITE-Seq staining: Cells sorted according to viable Lineage(CD19, CD3, CD5, F4/80,FceRIa, Gr-1)-CD45+CD122+ and/or CD127+. Cells were furthermore stained with the following TotalSeq A antibodies (BioLegend) according to the manufacturer's instructions: CD4, CD49b, NK11, DNAM1, CD3, CCR6, NKp46, cKit, Nrp1, NKG2D, TCRgd, TCRb, CD49a, CXCR5, CD27, CD200R1, PD1, CD127, a4b7, ICOS, Sca1, ST2, CD25 and KLRG1 antibodies. Genotypes were hashtagged using TotalSeq A hashtags 1-3. library construction protocol: A total of 8000 cells were loaded in the ChromiumTM Controller for partitioning single cells into nanoliter-scale Gel Bead-In-Emulsions (GEMs). Single Cell 3′ reagent kit v3.1 was used for reverse transcription, cDNA amplification and library construction of gene expression libraries (10x Genomics) according to the manufacturer's instructions. TotalSeqTM-A libraries were prepared following the protocol for 10x Single Cell 3′ Reagent Kit v3.1 provided by BioLegend, including primer sequences and reagent specifications. All libraries were quantified using a QubitTM 2.0 Fluorometer (ThermoFisher) and quality was tested on a 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent). Sequencing was performed using the NextSeq500/550 HighOutput Kit v2.5 (150 cycles) on a NextSeq500 sequencer (Illumina). Antibody and hashtag libraries were amplified using the KAPA HiFi ready mix with primers containing Illumina Small RNA RPIx sequences and Truseq D70x_s, respectively. Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.1 