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| Status |
Public on Mar 06, 2020 |
| Title |
4C_dCas9_CBSg_af |
| Sample type |
SRA |
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| Source name |
Neuro-2A cell
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| Organism |
Mus musculus |
| Characteristics |
cell type: Neuro-2A cells
genotype: CBS af blocked
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| Treatment protocol |
Neuro-2A cell at ~80% confluency is used for transfection. Briely, for each well in the 6-well plate, 1.25ug of dCas9 plasmid and 1.25ug of sgRNA plasmid (For multiple sgRNA combination, all sgRNA share the 1.25ug) is transfected using the Lipofectamine 3000 transfection reagent. 48 hr after transfection, puromycin (Sigma) was added to the culture medium at a final concentration of 2ug/ml. The culture medium was replaced every day with puromycin for a total of 3 days. Puromycin was then removed and cells were cultured in normal culture medium for 2 days.
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| Growth protocol |
Mouse Neuro-2A cells were cultured in DMEM medium (Hyclone) supplemented with 10% (v/v) FBS and 1× penicillin-streptomycin. Cells were maintained at 37 °C in a humidified incubator containing 5% (v/v) of CO2, and were passaged every three days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The cells were centrifuged at 500 g for 5 min and the pellets were used for QHR-4C experiments. The cell pellets were suspended for crosslinking in 900 ml 2% formaldehyde at room temperature for 10 min. The crosslinking reaction was stopped by adding and mixing with 100 ml of 2M glycine for a final concentration of 200 mM. The fixed cells were spin down at 800 g at 4 °C for 5 min and washed twice by suspending briefly in 1 ml ice-cold PBS. Cells were then permeabilized twice with 200 ml ice-cold 4C permeabilization buffer each for 10 min (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, and 1× protease inhibitors).
The cells were then digested overnight at 37 °C with DpnII while shaking at 900 rpm. After inactivation of DpnII at 65 °C for 20 min, proximity ligation was carried out for 24 hr at 16 °C with T4 DNA ligase in 0.1 ml 1 × T4 ligation buffer. The ligated product was then reverse cross-linked and DNA was extracted using phenol-chloroform. Glycogen was added to facilitate DNA precipitation. The precipitated DNA was then dissolved in 50 ml water, and sonicated using the Biorupter system (with low energy setting at a train of 30-second sonication with 30 second intervals for 12 cycles) to obtain DNA fragments ranging from 200 to 600 bp.The captured fragments were linear-amplified using a 5’ biotin-tagged primer complementary to the viewpoint fragment in 100ul of PCR system for a total of 60 cycles. This primer should be neither too close to the DpnII site to facilitate the nested PCR at the final amplification step, nor too far away from the DpnII site to maximize the product amount. The amplification products were denatured by incubating at 95 °C for 5 min, and immediately chilled on ice to obtain ssDNA. The ssDNA was then enriched and purified with Streptavidin Magnetic Beads (Invitrogen) according to the manufacturer’s instructions. The ssDNA on-beads was then ligated in 15 µl with 0.1 µM of adapters (Supplementary Table 1) at 16 °C for 24 hr. The adapters were generated by annealing two complementary primers matching Illumina P7 sequences. After ligation, free adapters were removed by washing the beads twice with the B/W Buffer (5 mM Tris-HCl, 1 M NaCl, 0.5 mM EDTA, pH 7.5). Finally, the QHR-4C libraries were generated by one-step PCR amplification (94 °C, 2 min; 94 °C, 10 sec, 60 °C, 15 sec, 72 °C, 1 min for 19 cycles; and a final extension at 72 °C, 5 min) with captured ssDNA on beads as the template and a pair of PCR primers. The forward primer matches the Illumina P5 and the viewpoint sequence adjacent to the viewpoint DpnII site with barcodes, and the reverse primer matches Illumina P7 with indexes. The PCR products were purified with a PCR purification kit (Qiagen).
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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
Duplicated paired-end reads were removed by FastUniq (version 1.1) program, and only the unique reads were used for analyses using the Bowtie and r3Cseq program.
Reads were aligned to the reference mouse genome (NCBI37/mm9) using the Bowtie program (version 1.2.2)
File format is transformed to BAM using samtools version 1.9
The reads per million (RPM) value was calculated using the r3Cseq program (version 1.20) in the R package (version 3.3.3)
Genome_build: NCBI37/mm9
Supplementary_files_format_and_content: zipped bedGraph files include RPM values for each Sample
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| Submission date |
Oct 15, 2019 |
| Last update date |
Mar 07, 2020 |
| Contact name |
Xiao Ge |
| Organization name |
Shanghai JiaoTong University
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| Street address |
No. 800 Dongchuan Road
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| City |
Shanghai |
| ZIP/Postal code |
200240 |
| Country |
China |
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| Platform ID |
GPL21273 |
| Series (2) |
| GSE138646 |
Tandem CTCF sites function as insulators to balance spatial chromatin contacts and topological enhancer-promoter selection |
| GSE138919 |
Tandem CTCF sites function as insulators to balance spatial chromatin contacts and topological enhancer-promoter selection [IV] |
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| Relations |
| BioSample |
SAMN13035914 |
| SRA |
SRX7000732 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4122972_4C_dCas9_CBSg_af.RPMs.bedGraph.gz |
242.5 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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