title: scRNAseq of P9.5 brain and dura ILCs: Dura  1
design: Isolation protocol: Brain and dura samples from C57BL/6J P9.5 mice were isolated from newborns under a dissecting microscope. ILCs were collected in two biological replicates with a total of 10-13 pups per replicate. 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 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. FACS sort staining: Cells sorted as viable Lineage(CD19, B220, Cd11b, Cd11c, F4/80, FceRIa, Gr-1)-CD45+CD3-CD5-CD122+ and/or CD127+ into 384-well cell-capture plates containing barcoded primers and mineral oil and, immediately stored at -80°C. Library construction protocol: FACS-sorted cells were stored in 384-well cell-capture plates containing barcoded primers and mineral oil and, immediately stored at -80C. Plates were shipped on dry ice to Single Cell Discoveries, where single-cell RNA sequencing was performed according to an adapted version of the SORT-seq protocol (Muraro et al.,2016) with primers described in van den Brinket al.,2017.Cells were heat-lysed at 65C followed by cDNA synthesis.After second-strand cDNA synthesis, all the barcoded material from one plate was pooled into one library and amplified using in vitro transcription (IVT). Following amplification, library preparation was done following the CEL-Seq2 protocol (Hashimshony et al., 2016) to prepare a cDNA library for sequencing using TruSeq small RNA primers (Illumina).The DNA library was paired-end sequenced on an Illumina Nextseq 500, high output, with a 175 bp Illumina kit(read 1: 26 cycles, index read: 6 cycles, read 2: 60 cycles).