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| Status |
Public on Mar 01, 2024 |
| Title |
ChIP-seq_Old_p300_rep2 |
| Sample type |
SRA |
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| Source name |
progenitor B cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: progenitor B cells
tissue: Bone marrow
genotype: Rag2-/-
treatment: Primary pro-B cells by CD19+ selection
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| Treatment protocol |
Retrovirus was produced by transfection of Plat-E cells with retroviral plasmids containing Ebf1 cDNA and a selective GFP marker (pMYs-Ebf1-IRES-EGFP, as described in Treiber et al., 2010), or with GFP-only plasmids as the control (pMSCV-IRES-GFP, Addgene Cat# 20672). Supernatants were collected at both 48 and 72 hours post-transfection and subsequently concentrated using the Retro-X Concentrator (Takara, Cat#631456), following the manufacturer's instructions. Young and old Rag2-/- CD19+ cells were cultured with 2 ng/mL IL-7 for three days and then exposed to the resuspended virus with the supplement of 10 μg/ml polybrene. The cells were cultured for an additional 72 hours and subsequently sorted based on GFP expression before subsequent analysis. Lentivirus was produced by transfection of 293T cells with the pHIV-Wapl-IRES-BFP and pHIV-IRES-BFP plasmids. To make these plasmids, the BFP DNA fragment was amplified from plasmid pEJS614_pTetR-P2A-BFPnls/sgNS (Addgene, #108650), and then integrated into pHIV-IRES-Zsgreen (Addgene, #18121) using the NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs, #E2621S) as per the manufacturer's instructions, resulting in the generation of pHIV-IRES-BFP plasmids. Subsequently, Wapl cDNA was introduced into pHIV-IRES-BFP, creating the pHIV-Wapl-IRES-BFP. Supernatants were collected at both 48 and 72 hr post-transfection and subsequently concentrated using the Lenti-X Concentrator (Takara, Cat#631232). D345 cells were infected with the resuspended lentivirus with the addition of 8 μg/ml polybrene. After infection, the cells were cultured for 72 hr and sorted based on BFP expression. The sorted cells were then cultured for 2-3 additional days to obtain an increased number of BFP-positive cells for experimental purpose.
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| Growth protocol |
Rag2-/-Ebf1+/+Pax5+/+, Rag2-/-Ebf1+/-Pax5+/+ and Rag2-/-Ebf1+/−Pax5+/− pro-B cell lines are cultured as previous described (Ramamoorthy et al., 2020). The D345 cell line was maintained in RPMI1640 (Gibco) supplemented with 10% fetal bovine serum (FBS, Gemini) and a 1ⅹ penicillin-streptomycin solution (Gibco). 293T human embryonic kidney cells used for lentiviral expression was purchased from ATCC (Cat# CRL-3216) and cultured in DMEM (Gibco) with 10% FBS (Gemini) and 1ⅹ penicillin-streptomycin solution (Gibco).
The Platinum-E (Plat-E) cell line used for retroviral expression was purchased from Cell Biolabs (Cat# RV-101) and cultured in DMEM (Gibco) with 10% FBS (Gemini), 1ⅹpenicillin-streptomycin solution (Gibco), 10mM Hepes and 0.3 μg/mL L-glutamine. All cells were cultured at 37C in a humidified atmosphere with 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction 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.
For C57BL/6J mice, total bone marrow was extracted from tibia and femurs and erythrocytes are lysed. Cells were pre-purified using CD19+ selective beads (Stem Cell Technology, Cat#18954) and sorted with IgM-B220+CD43+ markers. Flow cytometry experiments were performed on BD FACSAria II Cell Sorter. Flowjo software was used for data analysis.
For the Hi-C libraries. Genome-wide in situ Hi-C was performed with Rag2-/- young and old primary pro-B cells, and Rag2-/-Ebf1+/+Pax5+/+, Rag2-/-Ebf1+/-Pax5+/+, Rag2-/-Ebf1+/−Pax5+/−, D345-BFP and D345-Wapl-BFP 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, 1-2×106 cells were 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 at the Single Cell & Transcriptomics Core at Johns Hopkins University.
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 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.
For the ChIP-seq libraries.For the chromatin immunoprecipitation (ChIP) of histone modifications, CTCF, and Rad21, both young and old primary Rag2-/- pro-B cells underwent crosslinking with 1% formaldehyde (Sigma) for 10 minutes at room temperature. The reaction was subsequently quenched with 125 mM glycine, and cell lysis was initiated in a buffer containing 1% SDS. Chromatin was then sheared using a Bioruptor (Diagenode) in cycles of 30 seconds on and 30 seconds off, totaling 15 minutes of shearing time. Immunoprecipitation was performed using specific antibodies, with 1×106 cells used for each assay, and antibody dilutions followed established protocols.
As for Brg1 and p300 ChIPs, we followed the protocol by Bossen et al., 2015, with some modifications. In this case,2×106 young and old primary Rag2-/- pro-B cells were first crosslinked with 1.5 mM EGS at room temperature with rotation for 15 minutes. Subsequently, the cells were fixed with 1% formaldehyde (Sigma) for 15 minutes at room temperature with rotation and quenched with 200 mM glycine. After two PBS washes, the cells were lysed in a buffer containing 1% SDS. Notably, we extended the sonication time to a total of 20 minutes of shearing time, while the subsequent steps of the protocol mirrored the previously mentioned procedure.
For the RNA-seq libraries.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 (PerkinElmer, Cat#NOVA-5138-07,) and sequenced 2×75 bp on an Illumina NovaSeq instrument at the Single Cell & Transcriptomics Core at Johns Hopkins University. RNA libraries for Rag2-/-Ebf1+/+Pax5+/+, Rag2-/-Ebf1+/-Pax5+/+, Rag2-/-Ebf1+/−Pax5+/− and D345-BFP and D345-Wapl-BFP pro-B cell lines (two replicates for each group) were prepared using SMARTer Stranded Total RNASeq Kit v2 (Takara, Cat# 634412) and sequenced 1×100 bp on an Illumina NovaSeq instrument at the Single Cell & Transcriptomics Core at Johns Hopkins University. RNA libraries for pro-B cells infected with Ebf1-GFP or GFP containing retrovirus were prepared using the SMART-Seq mRNA kit (Takara, Cat#634773) and sequenced 2×100 bp on an Illumina NovaSeq instrument at the Computational Biology and Genomics Core in NIA.
For the HiChIP libraries. The H3K27ac HiChIP procedure was executed employing the Arima Hi-C+ Kit (Arima Genetics Inc. Cat# A101020), strictly adhering to the manufacturer's guidelines outlined in the Arima-HiC+ documents A160168 v00 (HiChIP) and A160169 v00 (library preparation). In each sample, a total of 5×106 cells were utilized. These libraries were individually barcoded and then combined for sequencing on an Illumina NovaSeq instrument.
For the VDJ-Seq. It was performed following the HTGTS-Rep-Seq protocol by Hu et al., 2016. Briefly, 200 ng – 2 μg of genomic DNA isolated from primary pro-B cells sorted from young and old C57BL/6J mice was sonicated with Covaris using the setting of Durations 10s, Peak power 50, Duty Factor 20%, Cycles/ Burst 200. Sheared DNA was linearly amplified using a biotinylated JH1 primer and enriched with streptavidin C1 beads (Thermo Fisher Scientific). A bridge adaptor was ligated to enriched single stranded DNA to the 3’ end and then amplified by a nested primer set. Products were further amplified using P5-I5 and P7-I7 primers to be final libraries and sequenced by Illumina MiSeq sequencer to generate paired-end 250-bp reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Hi-C, H3K27ac HiChIP,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.Replicates from H3K27ac HiChIP samples were combined and down-sampling equally to 39 million PETs for all following analysis.
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 saved as BED files 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.Visualization of ChIP-Seq tracks were performed by IGV or the cLoops2 plot module (v0.0.2) .ChIP-Seq peaks were identified using the cLoops2 callPeaks module. For transcription factor data, the key parameters -eps 150 -minPts 20,50 were employed. For H3K27ac data, the parameters -eps 150,300 -minPts 20,50 -sen were used. Peak identification was performed on each replicate individually, followed by the compilation of a union set encompassing both conditions (young and old) and overlapping replicates. Differential peaks were identified for young and old conditions using a Poisson test. The test utilized average counts from replicates for each peak and considered normalized total reads of 30 million. A p-value cutoff of <1e-5 and a fold change cutoff of >=2 were used to identify significant differential peaks. ChIP-seq peak or domain regions aggregation analyses, including heatmaps and average profile plots, were generated using the computeMatrix and plotHeatmap commands from the deepTools (v3.3.0) package.Quantification of ChIP-seq signals in Hi-C compartments was performed by the cLoops2 quant module. H3K27ac peak annotations were performed by the anoPeaks.py script in cLoops2 package. H3K27ac peaks within a 2 kb range upstream or downstream of the transcription start site (TSS) are annotated as promoters, while those outside this range are labeled as enhancers for the downstream analysis.
RNA-seq raw reads were mapped to mm10 by STAR (v2.7.3a) and quantified into RPKM with Cufflinks (v2.2.1).
VDJ-Seq analysis was performed using HTGTS-Rep.
Assembly: mm10
Supplementary files format and content: BEDPE files for capture Hi-C, Hi-C, HiChIP samples were provided for valid interactions.BED (BEDPE) files for single-end (paired-end) ChIP-seq samples were provided for processed high quality unqiue reads. TEXT files for processed VDJ-seq were provided. TEXT files for processed gene expression in FPKM from RNA-seq data were also provided.
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| Submission date |
Nov 29, 2023 |
| Last update date |
Mar 18, 2024 |
| Contact name |
Yaqiang Cao |
| E-mail(s) |
caoyaqiang0410@gmail.com
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| Organization name |
NHLBI
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| Department |
System Biology Center
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| Lab |
Laboratory of Epigenome Biology
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| Street address |
9000 Rockville Pike
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| City |
Bethesda |
| State/province |
Maryland |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE214438 |
Age-associated chromatin re-organization in progenitor B cells |
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| Relations |
| BioSample |
SAMN38493562 |
| SRA |
SRX22673814 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7921747_ChIP-seq_Old_p300_rep2.bedpe.gz |
859.0 Mb |
(ftp)(http) |
BEDPE |
| GSM7921747_ChIP-seq_Old_p300_rep2_peaks.bed.gz |
309.7 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
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