Adult 10-12 wo_Dura mater ILCs_replicate 1
Protocols: scRNAseq of sorted ILCs from whole brain from embryos at embryonal day (E16) was performed as followed:   Isolation protocol:  Whole brains were mechanically homogenized and passed through a 70 µm cell strainer with complete medium [RPMI-1640 supplemented with 2 mM L-glutamine (Gibco), 100 U/mL penicillin (Seromed), 100 μg/mL streptomycin (Seromed), 10% FCS (Sigma–Aldrich) and 1% HEPES (Gibco)]. Lymphocytes were enriched by density gradient separation (40% Percoll) and erythrocyte lysis was then performed in between the two following washing steps done with PBS/BSA.   ILCs from whole brain from n=35 female and male embryos obtained in 2 technical replicates from 5 pregnant mice.   Cells were sorted as CD45+LinLD[Live Dead dye, CD19, B220, CD11c, Gr-1, F4/80, FcεRI]-CD11b-CD3-CD5-CD127+ and/or CD122+ into 384-well cell-capture plates containing barcoded primers and mineral oil and, immediately stored at -80°C.  scRNAseq of sorted ILCs from whole brain and dura mater from newborns at postnatal day 9.5 (P9.5) was performed as followed:   Isolation protocol:  Whole brains were mechanically homogenized and passed through a 70 µm cell strainer with complete medium [RPMI-1640 supplemented with 2 mM L-glutamine (Gibco), 100 U/mL penicillin (Seromed), 100 μg/mL streptomycin (Seromed), 10% FCS (Sigma–Aldrich) and 1% HEPES (Gibco)]. Lymphocytes were enriched by density gradient separation (40% Percoll) and erythrocyte lysis was then performed in between the two following washing steps done with PBS/BSA.  Dura meninges were peeled off from the interior side of the skull cap or isolated from the brain ventricles and digested in 500 μL PBS containing 2.5 mg/ml collagenase Type VIII from Clostridium Histolyticum (Sigma) and 1 mg/ml DNAse I (Sigma) for 20 min at 37°C with 400 rpm. After digestion, cell suspension was passed through a 70 μm cell strainer (Corning) and centrifuge at 400 g for 10 min. Cells were subsequently surface stained for FACS and sorted on a BD Aria III machine.   ILCs from whole brain and dura were collected in two biological replicates from n=10-13 newborns at P9.5.   Cells were sorted as CD45+LinLD[Live Dead dye, CD19, B220, CD11b, CD11c, Gr-1, F4/80, FcεRI]-CD3-CD5-CD127+ and/or CD122+ into 384-well cell-capture plates containing barcoded primers and mineral oil and, immediately stored at -80°C.  FACS-sorted single cells were deposited into 384-well hard-shell plates (Bio-Rad) containing 5 μl Vapor-Lock (QIAGEN), 100–200 nl reverse transcription primers, dNTPs, and ERCC synthetic mRNA spike-ins, centrifuged briefly, and stored at −80°C. Cells were heat-lysed for 5 minutes at 65°C. Reverse transcription and second-strand synthesis mixes were dispensed using the Nanodrop II liquid handling system (GC Biotech), generating double-stranded cDNA containing a T7 promoter sequence. Following second-strand cDNA synthesis, all barcoded cDNA from each plate was pooled. The aqueous nucleic acid phase was separated from the oil phase and subjected to in vitro transcription (IVT) for linear RNA amplification. Amplified RNA was used as input for library preparation following the CEL-Seq2 protocol (Hashimshony et al., 2016). Reverse transcription primers included a 24 bp poly(T) stretch, a 4 bp UMI, an 8 bp cell-specific barcode, the 5′ Illumina TruSeq Small RNA adapter, and a T7 promoter. Libraries were generated using TruSeq Small RNA kit adapters (Illumina).
strain
C57BL/6J
experimental variable
Adult_Dura mater_ILCs_1
organism
Mus musculus
organism
Mus musculus
collection date
not collected
scientific_name
Mus musculus
cell type
Innate lymphoid cell
common name
house mouse
organism part
immune system
isolate
not applicable
compartment
Dura mater
age
P70-84
geographic location (country and/or sea)
not collected