GSM8389436: CT2, control mice, pool of 4 mice, biological replicate 2; Mus musculus; RNA-Seq
Cell suspensions from fetal mouse skin were obtained through mechanical dissociation and enzymatic digestion. Briefly, fetal skin from E18.5 embryos was harvested, finely minced and digested in RPMI medium containing 0.1 mg/ml DNAse I (Sigma), 400U/ml Collagenase I (Sigma), 0.1 mg/ml Liberase DL (Roche) and 1 mg/ml Dispase II (Roche) for 45 minutes on a rotating plate at 37°C. Digested tissue samples were filtered and single cell suspensions were used for flow cytometry. Cell suspensions from mouse skin were blocked with anti-mouse CD16/32 (S17011E, Biolegend) for 15 min at 4°C. Surface staining using CD45, F4/80, CD64, CD117 and ST2 was performed in FACS buffer for 1 hour at 4°C and mast cells (CD45+ F4/80- CD64- CD117+ ST2+) were sorted using FACS. Samples quality control and libraries preparation were performed at the GeT-Santé facility (Toulouse, France, www.get.genotoul.fr). RNA concentration and purity were determined using a ND-2000 Spectrophotometer (ThermoFisher Scientific). Integrity of RNA was checked with a Fragment Analyzer (Agilent Technologies), using the RNA Standard Sensitivity Kit. 260/280 purity ratios were all > 1.8. Integrity indices revealed acceptable and homogenous values for the RNA Integrity Number (8.2-8.6) and 28S/18S ratios (0.8-1.2). RNA-seq paired-end libraries were prepared at the GeT-Santé facility according to Illumina's protocol with some adjustments, using the TruSeq Stranded mRNA library prep Kit (Illumina). Briefly, mRNAs were first selected from either 3000 ng (DRG sequencing) or 500 ng (sorted fetal MC sequencing) of total RNA using poly-T beads. Then, RNAs were fragmented for 2' and retrotranscribed to generate double stranded cDNA. Compatible adaptors were ligated, allowing the barcoding of the samples with unique single indices. 11 cycles of PCR were applied to amplify libraries, and an additional double-sized purification step allowed to obtain 280-1000 pb fragments. Libraries quality was assessed using the HS NGS kit on the Fragment Analyzer (Agilent Technologies).Libraries quantification and sequencing were performed at the GeT-PlaGe core facility (INRAE, Toulouse, France). Libraries were quantified by qPCR using the KAPA Library Quantification Kit (Roche) to obtain an accurate quantification. Libraries were equimolarly pooled and RNA sequencing was then performed on one SP lane of the Illumina NovaSeq 6000 instrument (Illumina), using the NovaSeq 6000 SP v1 Reagent Kit (300 cycles), and a paired-end 2 x 150 pb strategy.