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Sample GSM5952745 Query DataSets for GSM5952745
Status Public on Aug 03, 2022
Title mTEC3_rep2+B36
Sample type SRA
 
Source name thymus
Organism Mus musculus
Characteristics strain: C57BL/12
tissue: thymus
cell type: thymus epithelial cell type 3
batch: 2
treatment: none
Extracted molecule genomic DNA
Extraction protocol DNA and RNA were isolated using TRIzol reagent (Sigma Aldrich, Darmstadt, Germany) and further AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) purification, following the manufacturer’s instructions. For RNA isolation (RNase-free DNAse set, Qiagen) DNAse I digestion was performed on the column. Proteinase K digestion was applied for RNA and DNA isolation (Puregene Proteinase K, Qiagen). Prior to library preparation, DNA and RNA quality was checked by Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and quantified by Qubit fluorimetry (Thermo Scientific, Wilmington, USA). All RNA samples reached an RNA integrity number (RIN) > 8.5.
Generation of tWGBS libraries was performed as previously published(Wang et al., 2013). Briefly, 20-30ng DNA from sorted adult mTEC populations was tagmented in a 20µl reaction mix at 55°C for 8min, followed by addition of 15µl 5M guanidinium thiocyanate and purification with HighPrep beads (Gaithersburg, MD, United States). Oligonucleotide replacement and gap repair were performed at 37°C for 30min in a 20µl mix, followed by another bead-mediated purification. Bisulfite treatment was done with the EZ DNA Methylation kit (Zymo, Irvine, CA, U.S.A.) using 16 cycles (60min at 50°C, 15sec at 95°C). Four differently barcoded tWGBS libraries per sample were generated under real-time conditions to monitor amplification using a program of initial melting at 95°C for 3min followed by cycling with 95°C for 20sec, 62°C for 15sec and 72°C for 40sec. After bead-mediated purification, libraries were quantified and pooled per sample in equimolar amounts with a final concentration of 10nM. Each pool was sequenced at the DKFZ Genomics and Proteomics Core Facility using paired-end, 125 bp, on one lane of the Illumina HiSeq2000 v4 platform.
 
Library strategy Bisulfite-Seq
Library source genomic
Library selection RANDOM
Instrument model Illumina HiSeq 2000
 
Data processing The Omics IT and Data Management Core Facility (ODCF; DKFZ, Heidelberg) processed the tWGBS data. Reads alignment was executed using an updated version of the previously published pipeline(Wang et al., 2013), implemented as a Roddy Workflow (https://github.com/DKFZ-ODCF/AlignmentAndQCWorkflows) in the automated One Touch Pipeline(Reisinger et al., 2017). Briefly, Trimmomatic v.0.30(Bolger et al., 2014) was used for trimming of adaptor sequences of raw reads, followed by in-silico bisulfite-convertion(Li, 2013) (C>T for the first read, G>A for the second). Converted reads were aligned to the in-silico bisulfite-converted reference genome mm10, which was augmented with the PhiX and lambda phage sequences, using the BWA-MEM v0.7.8(Li, 2013) software with default parameters. After alignment, reads with a mapping quality ≥25 and nucleotides with a Phred-scaled quality score ≥25 were converted back to their original state. Sambamba v0.4.6( https://lomereiter.github.io/sambamba/ ) was used for PCR duplication removal per library. Methylation calling and M-bias QC was executed using MethylDackel (Ryan, 2014) v0.4.0.
Methylation calls were imported into R statistical environment for further analysis and smoothing using BSSeq package v1.20.0(Hansen et al., 2012). BigWig files were exported via rtracklayer v1.46.0(Lawrence et al., 2009) and visualized using the IGV software.
Assembly: mm10
Supplementary files format and content: bigwig coverage tracks
 
Submission date Mar 14, 2022
Last update date Aug 03, 2022
Contact name Jakub Abramson
E-mail(s) jakub.abramson@weizmann.ac.il
Organization name Weizmann Institute of Science
Department Department of Immunology
Street address 234 Herzl st.
City Rehovot
ZIP/Postal code 7610001
Country Israel
 
Platform ID GPL13112
Series (2)
GSE192875 Transcriptional regulation of the thymus master regulator Foxn1
GSE198591 Transcriptional regulation of the thymus master regulator Foxn1 [WGBS]
Relations
BioSample SAMN26657927
SRA SRX14462291

Supplementary file Size Download File type/resource
GSM5952745_mTEC3_rep2.bw 146.0 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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