RNA Isolation, qPCR, and RNA-seq analyses of FACS-isolated cells

Total RNA from the sorted cell was isolated using the Direct-zol™ RNA MiniPrep Kit (Zymo Research Corporation. Irvine, CA, USA). For verifying the Dcx+ or Nestin+ cell population enrichment, the isolated RNA was reverse transcript by MessageBOOSTER™ Whole Transcriptome cDNA Synthesis Kit (MBWT80510, Illumina) for qPCR. Quality, size, and concentration of the RNA during library preparation were analyzed using Bioanalyser 2100 (RNA Pico Kit, Agilent). For RNA-seq, Strand-specific, poly(A) selected cDNA libraries were generated using Nugen Ovation® Ultralow Library Systems (Illumina) according to the manufacturer’s protocol. Library validation and normalization were performed using RT-PCR and Quant-iT PicoGreen (Invitrogen). Cluster generation and high-throughput sequencing were performed on a HiSeq 2500 (Illumina), using the paired-end 100 bp protocol. Reads were aligned to the mouse genome GRCm38 with annotation from Gencode (gencode.vM15.primary_assembly.annotation.gtf)using STAR (v2.5.3a) with options: --runMode alignReads --clip3pAdapterSeq AGATCGGAAG --outSAMunmapped None --outFilterMultimapNmax 5 --outFilterMultimapScoreRange 1 --outFilterMismatchNoverLmax 0.06 --alignIntronMin 20 --alignIntronMax 1000000 -alignSJDBoverhangMin 1 --outFilterIntronMotifs RemoveNoncanonicalUnannotated --outSAMtype BAM SortedByCoordinate --limitBAMsortRAM 5000000000 --quantMode GeneCounts _ --outSAMattributes All --outFilterType BySJout --outFilterScoreMin 10 --outSAMattrRGline ID:foo --alignEndsType EndToEnd. Reads mapped to the “+” strand of annotated genes were counted by STAR using the above option “--quantMode GeneCounts”.