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| Status |
Public on Jun 01, 2024 |
| Title |
KO_NPC_Hi-C_Rep3 |
| Sample type |
SRA |
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| Source name |
Neural progenitor cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Neural progenitor cells from ganglionic eminence
strain: C57BL/6
restriction enzyme: MboI
genotype: Setdb1-KO
age: Embryonic day 15.5
fetus id: 3000-6KO
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| Growth protocol |
Ganglionic eminences of fetal brains were harvested, dissociated with Accutase solution (Sigma A6964), and cultured in ultra-low attachment plates (Corning 3471) with advanced DMEM/F-12 (Gibco 12634-010) supplemented with N-2 (Gibco 17502048), B-27 (Gibco 12587010), GlutaMAX (Gibco 35050061), P/S (Sigma V900929), heparin (5 ug/ml), EGF (20 ng/ml) and FGF (20 ng/ml). Fresh medium was added every 3 days, and neurospheres were dissociated with Accutase and passed every 5 days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
3 million cells were lysed in NP40 lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM Mg(Ace)2, 0.1 mM EDTA, 10 mM Tris pH8, 0.1% NP40) , fixed in 1% formaldehyde (Sigma 252549) at RT for 5 minutes and quenched with 125 mM glycine at RT for 10 minutes. Nuclei were incubated in 0.5% SDS at 62 ℃ for 10 minutes, and quenched with 1.25% Triton X-100 (Sigma 93443) at 37 ℃ for 15 minutes. Chromatin was digested with 100 U of MboI restriction enzyme (NEB R0147) in 1X NEBuffer2 (NEB B7002S) at 37 ℃ overnight with rotation. Enzyme digestion was inactivated with incubation at 65 ℃ for 20 minutes, and nuclei were collected with centrifugation. Restriction fragment overhangs were filled and DNA ends were marked with biotin using End-it DNA Repair Kit (Epicentre ER0720) supplemented with 0.256 mM biotin-14-dATP (Life Technologies 19524-016), 0.4 mM dCTP, 0.4 mM dGTP and 0.4 mM dTTP (NEB N0446S). For proximity ligation, DNA fragments were ligated with 2000 U of T4 DNA ligase (NEB M0202) in 1200 μl of ligation master mix (T4 DNA ligase buffer (NEB B0202), 0.83% Triton X-100, 0.1 mg/ml bovine serum albumin (NEB B9200S)) at RT for 4 hours with slow rotation. After ligase inactivation at 65 ℃ for 5 minutes, nuclei were collected with centrifugation, and the chromatin was reverse-crosslinked in PK lysis buffer (1 M Tris pH8, 50 mM EDTA, 2% SDS, 2 M NaCl) at 65 ℃ for 4 hours, followed by RNase A (Sigma R6513) and proteinase K (Sigma P2308) digestion. DNA was further purified by phenol/chloroform (1:1) and ethanol precipitation, dissolved in buffer EB (Qiagen 19086) and sonicated to ~ 300 base pairs by Covaris E220 ultrasonicator system. For biotin pull-down, 100 μl per sample of 10 mg/ml Dynabeads MyOne Streptavidin T1 beads (Life technologies 65602) was washed with 400 μl per sample of 1 X TWB (5 mM Tris-HCl pH7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20), resuspended in 100 μl per sample of 2X binding buffer (10 mM Tris-HCl pH7.5, 1 mM EDTA, 2 M NaCl) and incubated with samples at RT for 15 minutes with rotation. The beads were separated on magnetic stand, washed three times with 1 X TWB (pre-warmed to 55 ℃) and once in 100 μl buffer EB (Qiagen 19086).
The beads were collected for DNA end repair with End-it DNA Repair Kit, A-tailing with Klenow Exo-minus (Epicentre KL06041K), adapter (Vazyme N802-01) ligation with Fast-Link kit (Epicentre LK11025) and PCR amplification with VAHTS HiFi Universal Amplification Mix for Illumina (Vazyme N618-02). Supernatant was taken as the library, which was further size-selected with SPRIselect beads (Beckman B23318) and sent for deep sequencing on Illumina NovaSeq 6000 (paired-end, 150 bp).
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| Library strategy |
Hi-C |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Quality control was conducted by FastQC (version 0.11.9), and reads were trimmed with Trim Galore (version 0.6.7, --quality 20 --phred33 --stringency 1 --length 20 --fastqc --paired).
Restriction sites of MboI on mm10 were generated with “hiccup_digester” function of HiCUP (version 0.8.0).
High quality clean reads were aligned to mm10 with Bowtie2 program (version 2.4.1) in HiCUP (max di-tag length: 700, min di-tag length: 50).
“ValidPairs” were generated with the “mapped_2hic_fragments.py” script of HiC-Pro (version 2.11.4) in default settings.
“.hic” files were generated with “hicpro2juicebox.sh” utility of HiC-Pro and JuicerTools (version 1.22.01).
Assembly: mm10
Supplementary files format and content: HIC: binary contact matrix for visualization in Juicebox
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| Submission date |
Nov 13, 2023 |
| Last update date |
Jun 01, 2024 |
| Contact name |
Yan Jiang |
| E-mail(s) |
yan_jiang@fudan.edu.cn
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| Organization name |
Fudan University
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| Department |
Institutes of Brain Science
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| Street address |
131 Dongan rd
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| City |
Shanghai |
| ZIP/Postal code |
200032 |
| Country |
China |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE247617 |
Histone methyltransferase SETDB1 silenced SINE B2 retrotransposons and maintained mitotic cell cycle of neural progenitor cells (Hi-C) |
| GSE247620 |
Histone methyltransferase SETDB1 silenced SINE B2 retrotransposons and maintained mitotic cell cycle of neural progenitor cells |
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| Relations |
| BioSample |
SAMN38231008 |
| SRA |
SRX22507767 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7898072_KO_NPC_Hi-C_Rep3.validPairs.hic |
7.0 Gb |
(ftp)(http) |
HIC |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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