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| Status |
Public on Feb 13, 2020 |
| Title |
DP-Rag2ko-CBEko-TRAV17-4C-rep2 |
| Sample type |
SRA |
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| Source name |
DP-Rag2ko-CBEko-TRAV17-4C
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| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6
genotype/variation: EACBE-/-Rag2-/-
age: 4-8 weeks
tissue: CD3-stimulated thymus
cell type: thymocytes
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| Treatment protocol |
To isolate DP thymocytes from Rag1-/- or Rag2-/- mice, mice were injected i.p. with 150 μg of anti-CD3ε antibody (2C11; Biolegend) at 3 weeks of age and thymi were harvested ten days later.
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| Growth protocol |
Mice were housed in a specific-pathogen-free facility managed by the Southern Medical University Division of Laboratory animal center. All mice were handled in accordance with protocols approved by the Southern Medical University Institutional Animal Care and Use Committee.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For Repertoire-seq, a mixture 107 cell equivalents of RNA and 1 μM oligo(dT) primer in 8μl nuclease-free water was heated to 65°C for 5 min and cooled down on ice for at least five minutes to snap-anneal the oligo(dT) primer. The reaction was then adjusted to 250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2, 0.5 mM dNTPs and 5 mM dithiothreitol, before addition of 2μl Superscript II (ThermoFisher), add 1μl 25uM 5′RACE adapter sequence (5′-GTCGCACGGTCCATCGCAGCAGTCACArGrGrG-3′) and 1μl in a final volume of 20 μl. The reaction was incubated for two hours at 50°C to synthesize cDNA and add RACE adapter by template switching. Reverse transcriptase was then inactivated by incubation at 85°C for 2 minutes.
For ChIP-seq, CD3-stimulated thymus was removed,cells were filtered through nylon mech. Red cells were lysed in AcK buffer.Cells were perforated and dealed with MNase, chromatin were isolated by immunoprecipitation.
For Hi-C, libraries were prepared using in-situ HiC protocol described in (Rao et al., 2014) with small modification.
For 4C, 3C sample preparation followed a previously described protocol(Hagège et al., 2007) with small modification, 3C samples were digested overnight at 37°C with 10 U of NlaIII.After purification,ligation and PCR amplification,the library preparation followed the protocol described in ChIP libary construction.
For ChIP-seq, ChIP product through end repair, dA-tailing and linker ligation, barcodes and Illumina adapters were then added by PCR amplification. The libraries were purified with QiaQuick PCR purification reagents (Qiagen) and size selection by 0.7× and 0.2×Ampure XP beads (Beckman, A63880).
For Repertoire-seq, the library preparation followed the protocol described in ChIP libary preperation after PCR amplification.
For 4C, the library preparation followed the protocol described in ChIP libary construction after PCR amplification.
Hi-C libraries were prepared using in-situ HiC protocol described in (Rao et al., 2014) with small modification.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
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| Data processing |
library strategy: 4C-seq
1) ChIP-seq data process pipline.Clean reads were obtained following quality filtering and adaptor trimming using trim_glore (cutadapt version 0.4.4_dev) with parameter ‘-paired -q 20’, and reads were mapped to the mm10 genome assembly using bowtie2 (version 2.20.8) with parameters (-X 2000, --sensitive);RPKM normalized BigWig files were generated by BEDtools (version 2.26.0).
2)4C-seq data process pipline. Clean reads were obtained following quality filtering and adaptor trimming using trim_glore (cutadapt version 0.4.4_dev) with parameter ‘-paired -q 20; The first MboI enzyme fragment behind the viewpoint was extracted and mapped to mm10 by bowtie2 version 2.3.4.3 with parameter : --very-sensitive. Reads numbers were counted and normalized by the total mapped reads per sample after self-ligation remove, and then differential interactions were identified by 4C-ker.
3)5' RACE data process pipline. Reads were obtained following quality filtering and adaptor trimming using trim_glore (cutadapt version 0.4.4_dev) with parameter ‘-paired -q 20’. Individual reads were analyzed with MIXCR v2.1.10 to derive unique clonotypes based on CDR3 homology. Reads that comprise the clonotypes of interest (with matching VJ genes to Mus Musculus library antibody) were extracted for further analysis. The corresponding germline sequences were retrieved from IMGT, the international ImMunoGeneTics database.
4)HiC data process pipline. Reads were obtained following quality filtering and adaptor trimming using trim_glore (cutadapt version 0.4.4_dev) with parameter ‘-paired -q 20’. Hi-C mapping, filtering, correction, and binning were performed with the HiC-Pro software v2.11.1. The reads were mapped to the mm10 mouse reference genome. TADs analysis was performed with the “Insulation Method”. A publicly available script (matrix2insulation.pl) was used to detect the TAD boundaries, with the following options: “--is 2000000 --ids 800000 --nt 0.3”. The script can be accessed through GitHub (https://github.com/dekkerlab/cworld-dekker).Hic file were transfer from HiCPro result by using Juicertools (version 1.13.02).
Genome_build: mm10
Supplementary_files_format_and_content: csv(clonetype counts matrix); hic(generated by juicertools)
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| Submission date |
Feb 12, 2020 |
| Last update date |
Feb 13, 2020 |
| Contact name |
Hao bingtao |
| E-mail(s) |
haobingtao@gmail.com
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| Phone |
18578669173
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| Organization name |
Southern Medical University
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| Street address |
ShataiNan Road, Baiyun District, Guangzhou, Guangdong Province
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| City |
Guangzhou |
| ZIP/Postal code |
haobingtao@gmail.com |
| Country |
China |
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| Platform ID |
GPL21273 |
| Series (1) |
| GSE145147 |
A role of the CTCF binding site at enhancer Ea in Tcra rearrangement |
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| Relations |
| BioSample |
SAMN14087232 |
| SRA |
SRX7711473 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4307220_DP-Rag2ko-CBEko-TRAV17-4C-rep2.bedGraph.gz |
218.9 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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