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GEO help: Mouse over screen elements for information. |
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Status |
Public on Apr 13, 2022 |
Title |
RNA WT_DE 1 |
Sample type |
SRA |
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Source name |
Normal definitive endoderm
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Organism |
Homo sapiens |
Characteristics |
cell type: Normal definitive endoderm
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated using the Trizol reagent following the manufacturer’s instructions. RNA integrity was determined using Agilent 2100 Bioanalyzer (Agilent Technologies). Subsequently, polyA tailed RNA were selected using the Dynabeads oligo(dT) (Thermo Fisher Scientific). Total gnomic DNA was isolated using the DNeasy Blood & Tissue kit (Qiagen). RNA libraries were prepared using the NEBNext® Ultra™ RNA Library Prep kit for Illumina (New England Biolabs). ATAC-seq libraries were prepared as previously described60. In brief, cells were enzymatically dissociated and lysed in lysis buffer (10 mM Tris-HCl, PH7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). Immediately after centrifugation, transposition reactions were carried out by adding Tn5 Transposes from the Illumina Nextera DNA library preparation kit to the isolated nuclei and incubated at 37 oC for 30 min. DNA fragments were purified using the MinElute PCR Purification kit (Qiagen) and amplified using the KAPA real-time library amplification kit. Libraries were purified using the PCRClean DXTM beads (Aline Biosciences). CMS-IP-seq libraries were performed as previously described with small modifications (Huang et al., 2012). Purified genomic DNA (with 5% of mouse DNA and 0.5% lambda DNA spike-in) was sheared to 200–500 bp fragments using the M220 Focused-ultrasonicator (Covaris). Bisulfite converted DNA libraries with methylated adapters were enriched using an in-house anti-CMS antibody bound to protein A/G dynabeads. Amplified libraries were purified by AmpuXP beads (Beckman Coulter) and then sequenced using the Illumina NextSeq (75 and 40 cycle, single-end) systems. Genomic DNA was isolated using the DNeasy Blood & Tissue kit (Qiagen). WGBS library preparation and sequencing were conducted by BGI (Shengzhen, China). In brief, purified genomic DNA (with 1% of unmethylated lambda DNA spike-in, Promega) was sheared to a fragment size of 100–700 bp (main size 250bp) using Covaris focused ultrasonicator according to the manufacturer’s instructions. DNA fragments were subjected to bisulfited conversion using the EZ DNA Methylation-gold kit (Zymo Research). Bisulfite converted DNA was amplified and finally subjected to sequence on a MGISEQ-2000 system (100-cycle, paired-end) at BGI (Shengzhen, China). ChIP-seq was performed for TET1 (WT_PP cells), H3K4me1 (WT_PP and TKO_PP cells), and H3K27ac (WT_PP and TKO_PP cells). For TET1 ChIP-seq, approximately 5 x 107 cells were crosslinked, washed and snap frozen following the Cell Fixation protocol from Active Motif. TET1 ChIP-seq library preparation and sequencing were conducted by Active Motif. Chromatin immunoprecipitation of H3K4me1 and H3K27ac were performed as previously described28. WT_PP and TKO_PP cells were fixed, washed, and lysed in nuclear lysis buffer (50 mM Trish HCl, pH 8.0, 5mM EDTA, 1% SDS, and 1 protease inhibitor cocktail). Chromatin was sheared to 200-500 bp fragments using Bioruptor® (Diagenode), and the DNA fragments were precipitated with appropriate antibodies. ChIP-seq libraries were prepared using the NEBNext® Ultra IITM DNA Library Prep kit (New England Biolabs) following the manufacture’s instruction and subjected to high throughput sequencing on a Illumina HiSeq 2500 system (150 bp, paired-end) at Novogene (Tianjin, China).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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Data processing |
For the 150 bp paired-end raw data of RNA-seq, low quality bases and adaptor were trimmed using TrimGalore v.0.5.0. Clean reads were aligned to hg38 reference genome using STAR v.2.5.354 with default parameters and only uniquely mapped reads were used for downstream analysis. Count matrix for each gene were generated using htseq-count (HTSeq package55). DESeq2 was used to identify significantly differentially expressed genes (DEGs) between groups (WT and TKO ; WT and TET1KO) at the ES, DE and PP stages (fold change ≥ 2; FDR < 0.05). Hierarchical cluster analysis of the union DEGs was used to determine the signature genes and groups. The ‘ClusterProfilter’ package in R was performed for the functional enrichment analysis of DEGs in KEGG pathways. Adaptor trimming of raw reads were performed by TrimGalore v0.5.0, and high-quality reads were uniquely aligned to hg38 reference genome using Bowtie2 with ‘--very-sensitive’ option. Reads mapped to Mitochondrial DNA and PCR duplicate reads were removed and those uniquely mapped reads were finally extracted for downstream analysis. Genrich v.0.5 with ATAC-seq mode (option: -j, -q 0.01) was applied for each sample (with two biological replicates) to call ATAC peaks. Bedtools intersect was used to count the reads fall into peak regions, and the significantly differential accessible regions (DARs) were detected using DESeq2. Volcano plots were plotted using R package ggplot2. Motif annotation of DARs was performed using HOMER software. The analysis of CMS-IP data was performed by in house software ‘CMSIP’ v0.1.1. Briefly, raw reads were mapped to hg38 and mm10 genome references using bsmap v.2.89 (options: -n 1 -q 3 -r 0). After removing the PCR duplicates and reads mapped to both human and spike-in mouse genome, species specific reads were used to do the normalization for each sample according to the spike-in size factors. The mean wigsum for every 200 bp windows of the whole human genome was used to call the hydroxymethylation enriched peak regions for each group agaist to input, respectively. Differentially hydroxymethylation regions (DHMRs) in between WT_PP and TKO_PP were identified (g-test; FDR < 0.05). GREAT analysis with single-nearest genes option was used to perform the functional annotation of hypo-DHMRs. For WGBS data analysis, paired-end 100 bp reads were mapped against hg38 using bsmap v.2.89 with paired mode (options: -n 1 -q 3 -r 0), and only uniquely mapped reads were retained. More than 26.6 million CpG sites with coverage ≥ 5 reads were detected in both WT_PP and TKO_PP samples, which were used for downstream analyses. BSeQC and mcall module in MOABS30 was applied to do the quality control and calculate the methylation ratio for each CpG site (options: --trimWGBSEndRepairPE2Seq 40). Bisulfite conversion efficiencies were estimated using spike-in unmethylated lambda phage DNA. Mcomp module was used to call significantly differentially methylation CpG sites (DMCs) and differentially methylation regions (DMRs) (absolute credible difference of DNA methylation ratio of at least 20% and adjust p value of less than 0.05). The output bedGraph files from mcall include single base resolution DNA methylation ratios, which were transformed to a bigwig file format. The bigwig files were uploaded to the UCSC genome browser for visualization. Motif enrichment analysis of DMRs was performed using HOMER software and the functional annotation was performed by using GREAT with default settings. The quality control and alignment for raw reads of ChIP-seq data were similar to ATAC-seq data analysis above, TrimGalore (options: --quality 20 and --length 50) was used to remove adaptor and only uniquely mapped reads were retained. Bam2wig.py was used to transform the bam file to normalized bigWig files (option: -t 2000000000). For the WT_PP TET1 ChIP-seq, Macs262 was used to call ChIP-seq enriched peak regions with default parameters. Genome_build: hg38
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Submission date |
Mar 05, 2020 |
Last update date |
Apr 13, 2022 |
Contact name |
Jianfang Li |
E-mail(s) |
jianfanglee003@gmail.com
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Organization name |
Texas A&M University
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Street address |
2121 W. Holcombe Boulevard
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City |
Houston |
ZIP/Postal code |
77030 |
Country |
USA |
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Platform ID |
GPL16791 |
Series (1) |
GSE146486 |
TET1 dioxygenase is required for FOXA2-associated chromatin remodeling in pancreatic beta-cell differentiation |
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Relations |
BioSample |
SAMN14308806 |
SRA |
SRX7858848 |
Supplementary file |
Size |
Download |
File type/resource |
GSM4387068_RNA_WT_DE1.bw |
601.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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