RNA was extracted from LGR5+ ISC, Sec-Pro, Ent-Pro, GOB and ENT with Trizol (Invitrogen) and RNeasy Mini Kits (Qiagen), processed, and hybridized to Mouse Genome 430A 2.0 microarrays (Affymetrix). Data quality was verified using Affy QC Report in R bio-conductor. mRNA profiles were analyzed by RMA to normalize expression indices39 (link), and differential expression across cell types was identified using LIMMA40 by F test with p <0.002. Values in the expression heatmap (Extended Data Fig. 3 ) were normalized by magnitude normalization and cell-type specificity was determined by k-means clustering of differentially expressed genes. ATOH1 binding at enhancers was used as a function to determine if ATOH1 activates Sec-Pro-specific genes. Each ATOH1 enhancer (>2 kb upstream or <1 kb downstream from a TSS) was assigned to a nearby gene (TSS <20 kb from an ATOH1 summit). After normalization of tags for sequence depth and peak width, the sum of tag densities for each gene was used to measure its correlation with differential expression.
Mouse genome 430a 2.0 microarrays
Manufactured by Thermo Fisher Scientific
The Mouse Genome 430A 2.0 microarray is a lab equipment product designed for gene expression analysis in mice. It contains probes for more than 39,000 transcripts, providing comprehensive coverage of the mouse genome. The microarray is intended for use in a variety of research applications, such as studies of gene regulation, disease pathways, and genetic responses to experimental conditions.
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3 protocols using mouse genome 430a 2.0 microarrays
1
Analysis of Transcriptional Profiles Across Intestinal Cell Types
2
Analysis of Transcriptional Profiles Across Intestinal Cell Types
3
Transcriptional Profiling of Megakaryocyte Maturation
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RNA isolated from purified MK using RNeasy Mini kits (Qiagen, catalog# 74104) was processed and hybridized to Mouse Genome 430A 2.0 microarrays (Affymetrix) according to the manufacturer’s instructions. Microarray experiments were done in triplicate. Data were processed using robust multiarray analysis (RMA) to normalize expression indices47 . Genes with a unique RefSeq ID assigned to the probe set and called as “present” in at least 1 sample were retained for analyses. Differentially expressed genes between MKImm and MKMat were identified using LIMMA48 (link), with false discovery rate (FDR) <0.05 and fold-change ≥1.5. GO analysis was performed using DAVID tools49 (link) with default parameters, and GO terms with FDR <0.001 were selected. cDNA for RT-PCR analysis was synthesized using the QuantiTect reverse transcription kit (Qiagen, Catalog# 205311). Down- or up-regulated genes in Nfe2−/− MK34 (link) were chosen on the basis of ≥2-fold change on the microarray expression indices between wild-type and Nfe2−/− cells.
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