 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 01, 2024 |
| Title |
ChIP-seq_Old_CTCF_rep1 |
| 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: old
chip antibody: CTCF
|
| 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 |
SAMN31093689 |
| SRA |
SRX17749772 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6605809_ChIP-seq_Old_CTCF_rep1.bed.gz |
455.7 Mb |
(ftp)(http) |
BED |
| GSM6605809_ChIP-seq_Old_CTCF_rep1_peaks.bed.gz |
1.5 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
