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Sample GSM5133392 Query DataSets for GSM5133392
Status Public on Jun 17, 2021
Title 120min_with_auxin_rep2
Sample type SRA
Source name G1E-ER4 cells
Organism Mus musculus
Characteristics cell type: erythroid
treatment: auxin
time point: 120min
Treatment protocol Cells were synchronized to pro-metaphase with 200ng/ml nocodazole for 7-8.5h. For post-mitotic enrichment, cells were released from nocodazole for 0, 25min, 60min, 120min and 240min for pro-meta, ana/telo, early-G1, mid-G1 and late-G1 respectively.
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).
Extracted molecule genomic DNA
Extraction protocol In-situ Hi-C experiments were performed as previously described 1. Briefly, sorted cells (5 million for prometaphase and ana/telophase and 10million for early- and mid-G1 phase) were lysed in 1ml cold Cell Lysis Buffer (10mM Tris pH 8, 10mM NaCl, 0.2% NP-40/Igepal) for 10min on ice. Cells were pelleted at 4℃ and washed with 1.2 x DpnII buffer. Nuclei were permeabilized with 0.3% SDS for 1h at 37℃ and quenched with 1.8% Triton X-100 for 1h at 37℃. Chromatin was digested with 300U DpnII restriction enzyme (NEB, R0543M) in-situ at 37℃ over night with shaking. 300U DpnII restriction enzyme was added for an additional 4h at 37℃ with shaking. Nuclei were incubated at 65℃ for 20min to inactivate DpnII. After cool down, digested chromatin fragments were blunted with pCTP, pGTP, pTTP and Biotin-14-dATP (Thermal Fisher Scientific, 19524016) using 40U DNA Polymerase I, Large (Klenow) fragment (NEB, M0210). DNA was ligated in-situ with 4000U T4 DNA ligase (NEB, M0202M) for 4h at 16℃ followed by further incubation for 2h at RT. Nuclei were then incubated in 10% SDS containing proteinase K (3115879 BMB) at 65℃ overnight to reverse crosslinking. RNA was then digested with DNase-free RNase at 37℃ for 30min. DNA was then extracted by phenol-chloroform extraction, precipitated, and dissolved in nuclease free water. DNA was sonicated to 200-300bp fragments (Epishear, Active Motif, 100% amplitude, 30s ON and 30s OFF, 25-30min) and purified with AMPure XP beads (Beckman Coulter).
For hic library construction, biotin-labeled DNA after extraction was purified by incubation with 100μl Dynabeads MyOne Streptavidin C1 beads (Thermal Fisher Scientific, 65002) at RT for 15min. DNA libraries were constructed using the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB E6040, M0543L, E7335S). To elute DNA, streptavidin bead- bound DNA was incubated in 0.1% SDS at 98℃ for 10min. DNA was purified with AMPure XP beads and index labeled with NEBNext multiplex oligos for 6 cycles on a thermal cycler, using the NEBNext Q5 Hot Start HIFI PCR master mix. Index labeled PCR products were then purified with AMPure XP beads and sequenced on an Illumina NextSeq 500 sequencer.
Library strategy Hi-C
Library source genomic
Library selection other
Instrument model Illumina NextSeq 500
Data processing For each biological replicate, paired end reads were aligned to the mouse reference genome mm9 using bowtie2 (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) through the Hi-C Pro software 41. PCR duplicates were removed and uniquely mapped reads were paired to generate a validPair file. The output validPair file was converted into “.hic” file using the hicpro2juicebox utility. For merged samples, similar steps were taken on reads merged from each biological replicate.
Genome_build: mm9
Supplementary_files_format_and_content: .hic formated files of each individual biological replicate of hic data are provided
Submission date Mar 03, 2021
Last update date Jun 17, 2021
Contact name Yemin Lan
Organization name University of Pennsylvania
Street address 3400 Civic Center Blvd
City Philadelphia
State/province PA
ZIP/Postal code 19104
Country USA
Platform ID GPL19057
Series (2)
GSE168176 CTCF and transcription orchestrate chromatin structure re-configuration after mitosis [Hi-C]
GSE168251 CTCF and transcription orchestrate chromatin structure re-configuration after mitosis
BioSample SAMN18140101
SRA SRX10239397

Supplementary file Size Download File type/resource 118.6 Mb (ftp)(http) COOL
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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