Nucleospin rna xs

Total RNA was isolated from mast cells by using RNAiso Plus reagent (Takara) and NucleoSpin RNAXS (Takara). The cDNA was synthesized using SuperScript VILO cDNA synthesis kit (Life Technologies), and semiquantitative PCR was then performed using TAKARA Ex Taq HS DNA polymerase (Takara). The level of HDC, MCP5, CPA, Axin2, and Tcf7 mRNA was quantified using the SYBR Green detection system (Applied Biosystems). Results were normalized by expression of the GAPDH transcripts. The sequences of the primers used in this study are listed in Tables 1 and 2.

Prior to FACS sorting, ILCs were enriched using Dynabeads^™ Untouched^™ Human NK Cells Kit (Invitrogen), allowing for the depletion of T, B, DCs, and macrophages. Enriched ILCs were labeled with flourochrome-conjugated monoclonal antibodies, listed in Supplementary Table 2, and C10 (CD3⁻CD14⁻CD19⁻CD34⁻CD56^BrightCD94⁺CD16⁻CD127⁻ CD49a⁻), C2 (CD3⁻CD14⁻CD19⁻CD34⁻CD56^BrightCD94⁺ CD16⁻CD127⁻CD49a⁺), and cNK (CD3⁻CD14⁻CD19⁻ CD34⁻ CD56^DimCD94⁺CD16⁺), were sorted using the BD FACS Aria II (BD Biosciences) into fetal bovine serum for culture and activation or lysing buffer (NucleoSpin^® RNA XS, Takara Bio USA Inc, Mountain View, CA) for RNA isolation.

Total RNA was isolated from sorted microglia using an RNeasy Micro Kit (Qiagen, Hilden, Germany) and further purified using NucleoSpin RNA XS (Takara Bio Inc., Kusatsu, Japan/Macherey-Nagel, Düren, Germany). RNA-seq libraries were generated with the Ovation SoLo RNA-Seq System, Mouse kit (NuGEN, Redwood City, CA, USA) using 5 ng of total RNA. The cDNA libraries were sequenced by 50-base single-read sequencing on an Illumina HiSeq 2500 sequencer (Illumina, San Diego, CA, USA). The sequencing run and the base call analysis were performed according to the HiSeq 2500 System Guide with TruSeq SBS kit v3-HS. After sequencing, raw sequence data were generated with processing by CASAVA-1.8.4 with version RTA 1.17.20.0. Reads were mapped to the mm10 genome with tophat2. Two base mismatches were allowed for the mapping. Normalized FPKM values and differential gene expression analyses were generated with cuffdiff2. Q values (Benjamini-Hochberg correction) lower than 0.05 were considered significant. Gene ontology enrichment analysis was performed using GOseq. Results were visualized using CateGOrizer [23 ]. Heatmaps were generated using heatmap.2 in the gplots package of R.

Cells were harvested from 20 ml synchronized culture by centrifugation and stored at −80 °C until RNA extraction. Total RNA was extracted with NucleoSpin® RNA XS (TaKaRa) according to the manufacturer’s instructions. cDNA was synthesised from 300 ng of total RNA using a mixture of a random hexamer and oligo dT primer (1:9 in ratio of concentration) with PrimeScript RTase (TaKaRa).
The quantitative PCR analyses were performed using a StepOne-Plus Real-Time PCR system (Life Technologies) and Power SYBR Green Master Mix (Life Technologies). The values of the respective genes were normalised with the data on 18S ribosomal RNA. The primer sequences are shown in Supplementary Table 2.

Fetal liver erythroblasts from E11.5 embryos were stained with Zombie NIR Fixable Viability Kit (Biolegend) to discriminate dead cells, followed by the staining with anti-CD71 and anti-Ter119 antibodies (Biolegend). Live CD71⁺Ter119⁺ and CD71⁺Ter119^- cells were sorted using an SH800Z cell sorter. The total RNA of sorted cells was isolated using NucleoSpin RNA XS (Takara Bio) according to the manufacturer’s protocol.
RNA integrity (RIN) was examined using Bioanalyzer (Agilent) with RNA 6000 Pico Kit (Agilent) to confirm the RIN > 7. Then, the RNA-seq libraries for Illumina sequencing were generated using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs) according to the manufacturer’s protocol. Sequencing was performed using NextSeq 500 Sequencer and NextSeq 500/550 High-Output v2 Kit (Illumina).
The RNA-seq analysis was performed using the Galaxy web server (use.galaxy.org). Raw reads were trimmed by cutadapt (http://journal.embnet.org/index.php/embnetjournal/article/view/200), aligned to the mm10 murine reference genome by HISAT2^{84 (link)}, and then counted via the featureCounts package^{85 (link)}. The read counts were further processed by limma^{86 (link)}.