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Sample GSM6605824 Query DataSets for GSM6605824
Status Public on Mar 01, 2024
Title ChIP-seq_Young_Rad21_rep2
Sample type SRA
 
Source name progenitor B cells
Organism Mus musculus
Characteristics strain: C57BL/6
cell type: progenitor B cells
tissue: Bone marrow
genotype: Rag2-/-
age: young
chip antibody: Rad21
Growth protocol For Rag2-/- mice, total bone marrow was extracted from tibia and femurs and erythrocytes are lysed. Pro-B cells were purified by combining positive selection using CD19+ selective beads (Stem Cell Technology, Cat #18954) and sorting by CD19+ and B220+ markers.
Rag2-/-Ebf1+/+Pax5+/+, Rag2-/-Ebf1+/-Pax5+/+ and Rag2-/-Ebf1+/−Pax5+/− pro-B cell lines are cultured as previous described (Ramamoorthy et al., 2020).
Extracted molecule genomic DNA
Extraction protocol For the capture Hi-C libraries, whole genome Hi-C libraries were first generated as previously described. To enrich the IgH locus (mm10, chr12: 113,201,001 - 116,030,000), SureSelect Target Enrichment probes (Table S3) with 2× tiling density were designed and manufactured by Agilent (Agilent Technologies Inc.). Hi-C libraries were hybridized to probes as specified by the manufacturer. Enriched libraries were sequenced using Illumina NextSeq sequencer to generate paired-end 150-bp reads.
Genome-wide in situ Hi-C was performed with Rag2-/- young and old primary pro-B cells, and Rag2-/-Ebf1+/+Pax5+/+, Rag2-/-Ebf1+/-Pax5+/+ and Rag2-/-Ebf1+/−Pax5+/− pro-B cell lines using the Arima Hi-C Kit (Arima Genomics), including KAPA Hyper Prep indexing and library amplification (catalog no. KK8500, Roche Molecular Systems Inc) according to the manufacturer’s instructions. For each assay, 1X10^6 cells to be used as the input materials and two biological replicates were performed for each group. Samples were sequenced 2×150 bp on an Illumina NovaSeq instrument.
For the ChIP-seq libraries, Young and old primary Rag2-/- pro-B cells were crosslinked with 1% formaldehyde (Sigma) for 10 min, quenched with 125 mM of glycine and lysed in lysis buffer containing 1% SDS. Chromatin was sheared by Bioruptor (Diagenode) and followed by immunoprecipitation with specific antibodies. For each immunoprecipitation assay, 1×10^6 cells was used. Antibody information anti-H3K27ac (Active Motif, # 39133; 1:500), anti-H3K27me3 (Diagonode, #C1541005; 1:500), anti-CTCF (Abcam, # ab70303; 1:250), anti-Rad21 (Abcam, # ab992; 1:500).
Total RNA was prepared using the Direct-zol RNA Miniprep Kit (# R2051, Zymo Research) according to manufacturer’s instructions. RNA libraries for Rag2-/-Ebf1+/+Pax5+/+, Rag2-/-Ebf1+/-Pax5+/+ and Rag2-/-Ebf1+/−Pax5+/− pro-B cell lines (two replicates for each group) were prepared using SMARTer Stranded Total RNA-Seq Kit v2 (# 634412, Takara Bio USA, Inc.) and sequenced 1×100 bp on an Illumina NovaSeq instrument.
Total RNA was prepared using the Direct-zol RNA Miniprep Kit (# R2051, Zymo Research) according to manufacturer’s instructions. The RNA libraries for young and old Rag2-/- primary pro-B cells (four biological replicates for each group) were prepared using the NEXTFLEX Rapid Directional RNA-Seq Kit (NOVA-5138-07, PerkinElmer) and sequenced 2×75 bp on an Illumina NovaSeq instrument.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NovaSeq 6000
 
Description Primary pro-B cells by CD19+ selection
Data processing Hi-C and capture Hi-C raw reads were processed to the mouse reference genome mm10 by HiCUP (v0.7.2) with the settings of Arima. High-quality and unique paired-end tags (PETs) from HiCUP were further processed to the HIC file through Juicer (v1.6.0) for visualization with Juicebox. These PETs were also processed with cLoops2 (v0.0.2) for quantifications. Chromosome X was excluded from the analysis. Compartment analysis of eigenvectors (PC1) were obtained by hicPCA in HiCExplorer3 package (v3.6) with parameters of -noe 1 at the resolution of 100 kb. Hi-C TADs were called by Juicer arrowhead with parameters of -r 25000 -k KR and quantified by cLoops2. Hi-C or capture Hi-C virtual 4C tracks were generated by the cLoops2 dump module.
CTCF and Rad21 ChIP-seq raw reads were mapped to the mouse reference genome mm10 by Bowtie2 (v2.3.5). Only non-redundant reads with MAPQ >=10 were used for the following analysis. BigWig tracks were generated by bamCoverage in deepTools (v3.3.0) with parameters of --ignoreDuplicates --minMappingQuality 10 --normalizeUsing CPM for visualization and quantification aggregation analysis.
RNA-seq raw reads were mapped to mm10 by STAR (v2.7.3a) and quantified into RPKM with Cufflinks (v2.2.1). BigWig tracks were also generated by STAR as signal quantified as RPKM.
Assembly: mm10
Supplementary files format and content: BEDPE files for Hi-C and capture Hi-C samples were provided for valid interactions.
Supplementary files format and content: BED files for single-end ChIP-seq samples were provided for processed high quality unique reads.
Supplementary files format and content: TEXT files for processed gene expression in FPKM from RNA-seq data were also provided.
 
Submission date Sep 29, 2022
Last update date Mar 18, 2024
Contact name Yaqiang Cao
E-mail(s) caoyaqiang0410@gmail.com
Organization name NHLBI
Department System Biology Center
Lab Laboratory of Epigenome Biology
Street address 9000 Rockville Pike
City Bethesda
State/province Maryland
ZIP/Postal code 20892
Country USA
 
Platform ID GPL24247
Series (1)
GSE214438 Age-associated chromatin re-organization in progenitor B cells
Relations
BioSample SAMN31093674
SRA SRX17749795

Supplementary file Size Download File type/resource
GSM6605824_ChIP-seq_Young_Rad21_rep2.bed.gz 559.0 Mb (ftp)(http) BED
GSM6605824_ChIP-seq_Young_Rad21_rep2_peaks.bed.gz 2.5 Mb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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