 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 27, 2024 |
| Title |
EACBEko-Rag1ko-DN-DpnII-HiC-rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Thymus
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Thymus
strain: C57BL/6
cell type: DN thymocytes
genotype: EACBE-/-Rag1-/-
|
| Growth protocol |
All experiments involving mice were performed using protocols approved by Southern Medical University Animal Studies Committee. Mice were on a C57Bl/6 genetic background . Animals were housed and bred in a specific pathogen-free animal facility.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For Hi-C, We performed with DN thymocytes from 3-4 Rag1-/- or EACBE-/- Rag1-/- mice per experiment or 10 million CD3-stimulated DP thymocytes from Rag1-/- mice per experiment. In brief, cells were crosslinked with a final concentration of 1% formaldehyde at room temperature for 10 min and quenched with glycine (final concentration 0.125 M) on ice for 5 min. Crosslinked cells were lysed and followed by adding 150U DpnII(New England Biolabs,R0543L) to digest the chromatin overnight at 37℃ with gently shaking. DpnII was inactivated at 65℃ for 20 min, DNA fragment ends were biotinylated with biotin-dATP, and the sample was diluted, and proximity ligated for 4-6 hours at room temperature. Crosslinked DNA was reversed by addition of SDS, proteinase K, and NaCl, and allowed to incubate overnight at 68℃. DNA was purified by phenol/chloroform, followed by isopropanol precipitation, and resuspended in 100ul 10Mm Tris-HCl,pH:8.0. DNA was treated with T4 DNA polymerase to remove unligated biotinylated ends and sheared to 300-500bp by sonication subsequently, the ligated junctions were pulled down with Dynabeads MyOne Streptavidin C1 magnetic beads. End-repair, dA-tailing, adapter-ligation, and PCR amplification with P5 and P7 prime were performed on biotinylated DNA fragments bound to beads. After purification, libraries were sequenced on an Illumina NovaSeq 6000 platform to obtain 150 bp paired-end reads.
For 3C-HTGTS, mouse thymus and liver were removed, cells were filtered through nylon mech.10 million cells were cross-linked with 1% formaldehyde. Cell were lysed and digest with 200U MboI overnight at 37°C. The sample was ligated with T4 DNA ligase overnight at 16°C. Crosslinks were reversed by Proteinase K and RNase A prior to DNA extraction with 1:1 phenol-chloroform and precipitation with ethanol. The 10ug 3C DNA was sonicated to 300-500bp and was linearly amplified with a biotinylated primer. The biotin-labeled single-stranded DNA products were enriched with streptavidin C1 beads, and followed by 3’ ends ligation with the bridge adaptor. The adaptor-ligated products were amplified via nested PCR using a nested primer and an adaptor-complementary primer.The detailed primers used in this study are also listed in Supplementary information, Table S1. And a final PCR for another 5-10 cycles with P5 and P7 was performed. After purification of the PCR products, the final libraries were sequenced on an Illumina NovaSeq 6000 platform to obtain 150 bp paired-end reads.
For ATAC-Seq, a total of 5 × 104 cell pellets were washed once with cold PBS, cells lysed on ice for 3 min in 50 μL ice-cold Lysis Buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% TWEEN 20, and 0.01% Digitonin in DEPC H2O), resuspended in 1 mL ice-cold RBS-Wash (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.1% TWEEN 20) and pelleted at 4 °C at 500 × g for 5min. Tagmentation was performed in 1 × Tagmentation Buffer (10 mM Tris pH 7.4, 5 mM MgCl2, 10% DMF, 33% PBS, 0.1%TWEEN 20, and 0.01% Digitonin) using 100 nM Tn5 Transposase for 30 min at 37 °C. Immediately, Free DNA was purified using QIAquick PCR Purification Kit (QIAGEN, 28106, GERMANY) according to the manufacturer’s instructions. And a final PCR for 10-15 cycles of amplification with P5 and P7 primers was performed. After purification, libraries were sequenced on an Illumina NovaSeq 6000 platform to obtain 150 bp paired-end reads.
For HTGTS-VDJ, DNA was extracted by DNA Isolation Mini Kit (Vazyme, DC102) and sonicated to about 500bp on a Qsonica Bioruptor Sonicator. Sonicated DNA was linearly amplified with a biotinylated primer that anneals to sites of interest. Biotin-labeled single stranded DNA products were enriched with streptavidin C1 beads (65001, Thermo Fisher Scientific), and followed by 3' end ligation with the bridge adapter. The adapter-ligated products were amplified through nested PCR using a nested primer and an adapter-complementary primer . The detailed primers used in this study are also listed in Supplementary information, Table S2. And a final PCR for another 10-12 cycles of amplification with P5 and P7 primers was performed. After purification, libraries were sequenced on an Illumina NovaSeq 6000 platform to obtain 150 bp paired-end reads.
Construction of libraries were described as the above extract protocol
|
| |
|
| Library strategy |
Hi-C |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
EACBEko-Rag1ko-DN-DpnII-HiC.allValidPairs
EACBEko-Rag1ko-DN-DpnII-HiC.allValidPairs.hic
bed, bedgraph, bw files containing data for all replicates are available on rep 1 sample.
|
| Data processing |
Illumina Casava1.7 software used for basecalling.
Hi-C data process pipline. For each sample, reads were obtained following quality filtering and adapter trimming using fastp (v0.20.0). Hi-C mapping, filtering, correction, and binning were performed with Hi-C-Pro (v2.11.1). The paired-end reads were mapped to the mouse mm10 genome. Self-circle ligation, dangling ends, re-ligation, and the other dumped types were filtered out with Hi-C-Pro after mapping. We generated raw contact matrices at 10 kb, 20 kb, 50 kb, 100 kb, 500 kb, 1 Mb resolutions. For raw contact matrix correction, we used the iterative correction method (ICE). The hicpro2juicebox.sh utility was used to convert the allValidPairs output of the pipeline into Juicebox hic format at fragment resolution. Heatmap, differential analysis and visualization of local interactions from Hi-C data were obtained using HiTC (v1.34.0) R package. A/B compartment analysis was performed at 100 kb resolution using a publicly available script runHiCpca.pl that performs PCA analysis with HOMER (v4.10.4). Then, we used the first principal component (PC1) to predict regions of active (A compartments) and inactive chromatin (B compartments). The TAD structure (insulation) was defined by the insulation score. The matrices which were used to calculate the insulation score were normalized by ICE method for discarding the bias of raw matrices. And then, the insulation score was computed at 10 kb resolution. The script matrix2insulation.pl was used to detect TAD boundaries, with the following options: ‘--is 100001 --ids 20001 --nt 0.3 -ss 0 --im mean’. TADs were called using normalized Hi-C matrices at 10 kb resolution with insulation2tads.pl. Chromatin loops were called using Fit-Hi-C (v2.0.7). Input files of Fit-Hi-C were created by using a publicly available script hicpro2fithic.py from Hi-C-Pro. Next, loops were called using fixed-size bin resolutions from 10 kb in all cell types. Briefly, significant interaction loops (P < 0.0001 and contact frequencies > = 5) were identified through jointly modeling the contact probability using raw contact frequencies and ICE normalization vectors with the Fit-Hi-C algorithm.
3C-HTGTS data processing for pairwise chromatin interactions. Paired-end Illumina sequencing fastq data were filtered by removing adapters and low-quality reads with using fastp (v0.20.0). Trimmed reads again were extracted from the sequence file after quality control with Cutadapt (v1.18). Paired-end reads containing nested primer or adapter primer were merged manually using restriction enzyme recognition sequences into single reads with Pear (v0.9.6), then the first digested fragment behind the bait was obtained by splitting the single reads into fragments according to restriction enzyme recognition sequences. The remaining single-end reads were aligned to the enzyme-digested mm10 reference genome with Bowtie2 (v2.4.5, parameter: -p 8 --sensitive), and mouse genome sequence (mm10) was retrieved from the UCSC and we extracted concordantly exact alignments using SAMtools (v1.9). Self-ligation reads and off-targeted reads were filtered after mapping. For visualization, we converted the final bam files into bedGraph files using Bedtools (v2.29.2). The signal peak bedGraph file was obtained by post-comparison filtering, signal statistics, and standardization. We normalized bedGraph files using the CPM (Counts Per Million in cis) normalization method and visualized them on the IGV genome browser. Differential pair-wise interactions were identified by the R package R.4Cker (v1.0.0, k=30) with the function nearBaitAnalysis called to define domains of interaction with the bait and DESeq2 (v1.34.0, p < 0.05). Finally, we organized the results report and visualized it with the Bioconductor package ggplot2 (v3.3.6).
3C-HTGTS data processing for three-way chromatin interactions. Paired-end Illumina sequencing reads were filtered by removing adapters and low-quality reads with using fastp (v0.20.0). Trimmed reads were extracted from the file after quality control with Cutadapt (v1.18). Paired-end reads containing nested primer or adapter primer were merged at restriction enzyme cut sites into single reads with Pear (v0.9.6), then fragments with multiple MboI cut sites were split and each fragment was aligned to the enzyme-digested mm10 reference genome by Bowtie2 (v2.4.5, parameter: -p 8 --sensitive). We extracted concordantly exact alignments using SAMtools (v1.9). We converted the final bam files into bed files using Bedtools (v2.29.2). Self-ligation and off-targeted fragments were filtered. Subsequently, we put all fragments retrieved from the same read according to the unique ID of each read on one line, then removed the continuous fragments. To create contact matrices, we extracted the first two digested fragments after the bait fragment, or a variety of combinations of three fragments were obtained by arranging all fragments from the same read. Raw contact matrices were generated at 3 kb, 5 kb, and 10 kb resolutions. For raw contact matrices correction, these interaction counts were normalized for a total of 1,000,000 interactions at the same resolutions. Similar to a Hi-C matrix, coverage was represented in a 2-dimensional matrix where each point represented the number of interactions found between two bins meaning a specific resolution. Differential analysis and visualization of local interactions from three-way interactions were obtained using the R package GENOVA (v1.0.0). Loops seen on the IGV genome browser were called using fixed-size bin resolutions from 3 kb to 10 kb. Briefly, interaction loops (contact frequencies >= 5) were identified by using raw contact frequencies.
ATAC-Seq data process pipline. Raw sequence reads were initially processed for removing adapter sequences and poor quality reads by fastp (v0.20.0). Subsequently, the remaining reads were mapped to the human genome hg38 using Bowtie2 (v2.4.5, parameter: -p 8 --sensitive). PCR duplicated fragments were filtered by Picard (v2.22.8). Then, we filtered the unmapped, multi-mapped reads and mapping to the reads on chrM. FRiP (Fragments Ratio in Peaks) value was calculated by using Bedtools (v2.29.2) and awk (v4.0.2). We used deepTools (v3.5.0) to generate bigWig file with CPM normalization, and these files can be visualized in IGV. SAM files were converted to BAM format using SAMtools (v1.9) and used for peak calling. MACS2 (v2.2.4) with parameters (--nomodel --shift -100 --extsize 200 -B --keep-dup all --broad --broad-cutoff 0.1) was used to call peaks.
HTGTS-VDJ data process pipline. The raw data were filtered by fastp (v0.20.0). Trimmed reads again that nested primer and adapter primer were removed were extracted from the sequence file after quality control with Cutadapt (v1.18). Contamination, and low-quality reads were also removed. T-cell receptor beta chain V, D, and J gene identification, CDR3 sequence extraction in clean reads were performed using MiXCR (v3.0.11). The corresponding germline sequences were mapped to the reference sequences derived from international ImMunoGeneTics (IMGT) information.
Assembly: mm10
Supplementary files format and content: The hicpro2juicebox.sh utility was used to convert the allValidPairs output of the pipeline into Juicebox .hic format.
Supplementary files format and content: BedGraph files were generated using Bedtools (v2.29.2) with normalized values for each sample.
Supplementary files format and content: BigWig files were generated using bamCoverage with normalized values for each sample.
|
| |
|
| Submission date |
Apr 25, 2024 |
| Last update date |
Apr 27, 2024 |
| Contact name |
Hao bingtao |
| E-mail(s) |
haobingtao@gmail.com
|
| Phone |
18578669173
|
| Organization name |
Southern Medical University
|
| Street address |
ShataiNan Road, Baiyun District, Guangzhou, Guangdong Province
|
| City |
Guangzhou |
| ZIP/Postal code |
haobingtao@gmail.com |
| Country |
China |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE265888 |
Dynamic Chromatin Boundaries of TCR Loci Coincide V(D)J recombination During T Cell Development |
|
| Relations |
| BioSample |
SAMN41086374 |
| SRA |
SRX24374024 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8230922_EACBEko-Rag1ko-DN-DpnII-HiC_10kb.loop.cis.bedpe.gz |
835.1 Kb |
(ftp)(http) |
BEDPE |
| GSM8230922_EACBEko-Rag1ko-DN_chr14_10kb.insulation.bedGraph.gz |
168.4 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM8230922_EACBEko-Rag1ko-DN_chr6_10kb.insulation.bedGraph.gz |
203.7 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
