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| Status |
Public on Nov 22, 2023 |
| Title |
HP1a_b_tir2_1h_with_a_no_d_rep2 |
| Sample type |
SRA |
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| Source name |
G1E-ER4 cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: G1E-ER4 cells
cell type: erythroid
genotype: HP1a-AID-mCherry;HP1b-FKBP(F36V)-GFP
treatment: noc 8h release 1h + 5-Ph-IAA 8h
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| Treatment protocol |
G1E-ER4:HP1a-AID-mCherry/HP1b-FKBPF36V-GFP cells were arrested to prometaphase through nocodazole treatment for 8h. During the last 4 hours of nocodazole treatment, cells were treated with one of the following combinations of drugs: (i) 5-Ph-IAA(-)/dTag13(-); (ii) 5-Ph-IAA(+)/dTag13(-); (iii) 5-Ph-IAA(-)/dTag13(+) and (iv) 5-Ph-IAA(+)/dTag13(+). Cells were then allowed to be released from nocodazole and proceed toward the next cell cycle. After 1h and 6h of nocodazole release, early-G1 and late-G1 cells under different HP1 protein configurations were collected. Note, that drug treatment was maintained during the course of nocodazole release.
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| Growth protocol |
G1E-ER4 is a subline of the GATA1-null proerythroblast cell line G1E (Weiss et al, Mol. Cell. Biol. 17, 1642–51 (1997).). Cells were cultered in IMDM medium, supplemented with 15% FBS, penicillin/streptomycin, kit ligand, monothioglycerol and Epoetin alpha at 37 degrees with 5% CO2 (as described in Weiss et al., 1997).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
FACS purified cells (0.1 million) were lysed in 1ml cold Cell Lysis Buffer (10mM Tris pH 8, 10mM NaCl, 0.2% NP-40/Igepal) for 10min on ice. Nuclei were pelleted at 4℃ and washed once with cold 1.4 × NEB buffer 3.1 (NEB Cat#B7203S). Nuclei were then re-suspended in 25ul of 1.4 × NEB buffer 3.1 and treated with 0.1% SDS for 10min at 65℃. Nuclei were then treated with 1% Triton X-100 to quench SDS for 1h at 37℃. Chromatin was digested with 25U DpnII restriction enzyme (NEB, Cat#R0543M) at 37℃ overnight with shaking (950rpm on a thermal mixer). After overnight digestion, an additional stroke of 25U DpnII was added into the system to further digest the genome for 4 hours at 37℃. After the 2nd stroke of digestion, nuclei were incubated at 65℃ for 20min to inactivate DpnII. Digested chromatin fragments were then cooled down to room temperature and blunted with dCTP, dGTP, dTTP (Diamond Cat#B110049, B110050, B110051) and Biotin-14-dATP (active motif, Catalog No: 14138) using 6.6U DNA Polymerase I, Large (Klenow) fragment (NEB, Cat#M0210). Chromatin was then ligated in-situ with 2000U T4 DNA ligase (NEB, Cat#M0202M) for 4 hours at 16℃ followed by further incubation for 2 hours at room temperature. Chromatin was then reverse-crosslinked overnight at 65℃ in the presence of 1% SDS and proteinase K (G-clone Cat#EZ0970). After reverse-crosslinking, DNase-free RNase A (Vazyme Cat#DE111-01) was added to the system for 30min at 37℃ to remove RNA. DNA was then extracted by phenol-chloroform extraction, precipitated, and dissolved in 130ul of nuclease free water.
DNA was sonicated to 200-300bp fragments with a QSonica Q800R3 sonicator (40% amplitude, 15s ON and 15s OFF, 13min sonication) and purified with VAHTS DNA Clean Beads (Vazyme Cat#N411). Biotin-labeled DNA fragments was enriched by incubation with 100μl Dynabeads MyOne Streptavidin C1 beads (Thermal Fisher Scientific, Cat#65002) at room temperature for 15min. Streptavidin C1 beads were then washed twice with 1 × B&W buffer (5mM Tris-HCl, pH 7.5; 0.5mM EDTA; 1M NaCl). Beads were re-suspended in 30ul of 1 × NEB Buffer2 (NEB Cat#7002S) containing 9U of T4 DNA polymerase (NEB, Cat#M0203S) and 40uM dATG/dGTP and incubated at 20℃ for 4 hours to remove unligated fragments. Beads were then washed twice with 1 × B&W buffer and subject to library construction. DNA end repair, dA-tailing and adaptor ligation were constructed on beads, using the AHTS Universal DNA Library Prep Kit for Illumina or MGI (Vazyme, Cat# ND627-01, NDM607-02) based on the manufacture’s protocol. To eluted DNA from streptavidin beads, the library mixture was treated with 0.1% SDS and incubated at 98℃ for 10min. Released DNA was collected from the supernatant and purified with VAHTS DNA Clean Beads. Finally, the library was amplified for 9 cycles on a thermal cycler, using the VAHTS® HiFi Amplification Mix. PCR products were then purified with VAHTS DNA Clean Beads and sequenced on an Illumina NextSeq500 or MGI DNBSEQ-T7 sequencing platform.
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| Library strategy |
Hi-C |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
DNBSEQ-T7 |
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| Description |
hic
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| Data processing |
In-situ Hi-C data from each individual clone or biological replicates were aligned to mouse reference genome mm9 using bowite2 (global parameters: --very-sensitive -L 30 --score-min L, -0.6,-0.2 --end-to-end –reorder; local parameters: --very-sensitive -L 20 --score-min L,-0.6,-0.2 --end-to-end --reorder). Reads were paired up and PCR duplicates were removed through the Hi-C Pro software (v3.0.0) to generate a valid interaction pair file. The output pair files were then converted to “.hic” files using the hicpro2juicebox utility. HiC files were also converted to “.cool” files using the Hicexplorer (v3.7). For replicate or clone-merged samples, similar steps were taken on reads merged from each clone or biological replicates.
Assembly: mm9
Supplementary files format and content: cool and hic files of each individual biological replicate of hic data are provided.
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| Submission date |
Mar 21, 2023 |
| Last update date |
Nov 22, 2023 |
| Contact name |
Haoyue Zhang |
| E-mail(s) |
zhang_adam@szbl.ac.cn
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| Organization name |
Shenzhen Bay Lab
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| Street address |
Gaoke Innovation Center, Guangqiao Road, Guangming District, Shenzhen
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| City |
Shenzhen |
| ZIP/Postal code |
518107 |
| Country |
China |
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| Platform ID |
GPL28330 |
| Series (2) |
| GSE227816 |
Selective compartmentalization in condensin-depleted mitotic chromosomes [Hi-C I] |
| GSE228402 |
Selective compartmentalization in condensin-depleted mitotic chromosomes |
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| Relations |
| BioSample |
SAMN33843583 |
| SRA |
SRX19740039 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7108994_HP1a_b_tir2_1h_with_a_no_d_rep2.hic |
1.7 Gb |
(ftp)(http) |
HIC |
| GSM7108994_HP1a_b_tir2_1h_with_a_no_d_rep2_100k.cool.gz |
40.8 Mb |
(ftp)(http) |
COOL |
| GSM7108994_HP1a_b_tir2_1h_with_a_no_d_rep2_10k.cool.gz |
225.1 Mb |
(ftp)(http) |
COOL |
| GSM7108994_HP1a_b_tir2_1h_with_a_no_d_rep2_25k.cool.gz |
142.0 Mb |
(ftp)(http) |
COOL |
| GSM7108994_HP1a_b_tir2_1h_with_a_no_d_rep2_50k.cool.gz |
76.0 Mb |
(ftp)(http) |
COOL |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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