RNA-seq analysis dataset: FACS-sorted Ptf1a-lineage cells from E14.5 hypothalamus of Ptf1a hetero and homozygous mice
Description
RNA-seq analysis on hypothalamic Ptf1a-lineage cells Because Ptf1a is a transcription factor, this protein should regulate transcription of various genes in Ptf1a-expressing cells of the hypothalamus. To detect those downstream genes, we purified cells in the Ptf1a-lineage from the hypothalamus at E14.5 by FACS sorting, subsequently performed RNA-seq analysis and obtained 706 differentially expressed genes. 386 genes had lower and 320 had higher expression in Ptf1a-lineage cells from homozygous Ptf1a deficient mice. The down-regulated genes included Prdm13, Corl2 and Lhx5 that were known downstream targets of Ptf1a, in addition, Sox3 and Nkx2-1, which are known to be required for hypothalamic development. Interestingly, among those affected genes, we detected several genes encoding transmembrane and/or secreted proteins, such as Jag2 (ligand for Notch receptors), Sema6b, Sema4g (ligands for Plexin family proteins). These genes may have a non-cell autonomous role in the sexual differentiation of hypothalamic cells adjacent to Ptf1a-lineage cells. Kiss1 expression was not altered, as was expected by the finding that most Kiss1-expressing cells were not in the Ptf1a lineage.
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Fluorescence-Based FACS Hypothalamic tissues were dissected from E14.5 Ptf1acre/+; Ai9 and Ptf1acre/cre; Ai9 brains and incubated with 100 µl of Papain solution (12 units/ml in 0.2 mg/ml Cystein, 5 mg/ml D-Glucose, 0.02% BSA, 0.5 mM EDTA, and 1 mg/ml DNase I) at 37ºC for 20 min. After cell dissociation with gentle pipetting, 400 µl of stop solution (2.5% FBS, 0.5% BSA in PBS) was added and the samples were strained through a 40 µm cell strainer. Fluorescence-activated cell sorting (FACS) for RFP-positive cells was performed by using FACSAria II SORP flow cytometer (BD Bioscience) and FACS Diva software. Dead cells were stained and excluded by using Propidium Iodide (Immunostep). Isolated cells were directly collected into Trizol (Invtrogen) to prevent RNA degradation. RNA-Seq RNA-seq was used for transcriptome analysis. RNA was extracted from 4 control and 4 KO FACS-sorted Ptf1a-lineage cells. Briefly, total RNA was isolated using Trizol reagent (Invitrogen) and RNA quality examined by RNA 6000 Pico kit (Agilent). 10 ng total RNA was used for RNA-seq library preparation with SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian (TaKaRa); 2×36 base paired-end sequencing was performed with NextSeq500 (Illumina) by i-Laboratory LLP (Tsukuba). FASTQ files were analyzed using CLC Genomics Workbench (Version 10.1.1; Qiagen). Sequences were mapped to mouse genome (mm10) and quantified for annotated genes. Expression values were estimated by total tag counts at each gene region and analyzed by Empirical Analysis of DGE tool (Qiagen) (Morito et al., 2018). Heatmaps were generated by MeV (http://mev.tm4.org/).