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| Status |
Public on Jun 26, 2020 |
| Title |
MCM2-Y90A-chase-30m, replicate |
| Sample type |
SRA |
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| Source name |
EdU labeled MCM2-Y90A HeLa cells with chasing 30 min, replicate
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| Organism |
Homo sapiens |
| Characteristics |
strain background: Adult female
cell line: HeLa-S3 with MCM2-Y90A
cell type: epithelial adenocarcinoma
genotype/variation: MCM2-Y90A
tissue: cervix
developmental stage: 31 years
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| Treatment protocol |
HeLa-S3 cells were synchronized at G1/S entrance with thymidine/aphidicolin double block (2 mM thymidine (Sigma, T1895) for 18 h; remove thymidine and continue to culture for 9 h; 1ug/ml aphidicolin (Abcam, 38966-21-1) for another 14 h). Remove aphidicolin. Pulse labeling with 20 uM EdU (Thermo Fisher, E10187) for 4 min. Remove EdU and chase with 20 uM thymidine for a series of desired time length (0 min to 24 h).
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| Growth protocol |
HeLa-S3 Cells were cultured at 37 °C with 5% CO2. Culture medium contains DMEM High Glucose (HyClone, SH30022), 10% FBS (BI, 04-010-1ACS) and 1X Penicillin-Streptomycin (Gibco, 15140122). Cells were passed every two days. Detached from plates with 0.25% Trypsin at 37 °C for 2 to 3 min. The sub-cultivation is about 1:5 to 1:6.
Mouse embryonic stem cells were cultured on gelatin coated dishes in ES culture medium containing DMEM (ThermoFisher 11995), supplemented with 15% fetal bovine serum (Hyclone 30070.03), 1% None essential amino acids (MerckM7145), 1% L-glutamine (BBI Life SciencesE607004), 0.1% β-mercaptoethanol (Gibco 21985023), 0.01 % LIF (107 U/ml), and 1% Penicillin-Streptomycin (Gibco, 15140122).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Harvest cells and extract DNA with phenol-chloroform.
Fragment DNA with sonication (Covaris M200) or restrictive enzyme AluI (NEB, R0137). Size select DNA fragments ranging from 100 bp to 200 bp with AMPure beads (1.05X to 2.8X) (Beckman, A63881). Click it reaction to conjugate biotin to EdU labeled DNA: (Tris pH7.5 buffer 15 mM; Biotin azide (Sigma, 762024) 400 uM; CuSO4 (Sigma, 209198)/THPTA (Sigma, 762342) mix (1:5) (100 uM: 500 uM); Sodium ascorbate (freshly prepared, Sigma, A4034) 5 mM). Reaction at 34 °C for 20 min. DNA was purified with MinElute PCR purification kit (Qiagen, 28004). The click it reaction system is modified from (1) (2). End repair (NEB, E6050), dA-tailing (NEB, E6053) and adaptor ligation (NEB, E6056). Hairpin adapter and methylated sequencing adapter were pre-mix at the molar ratio of 3:1 before adapter ligation. After ligation, 1.4X AMPure beads were added to remove un-ligated adapters. Dissolve adapter ligated DNA in 1X TE buffer. Incubate DNA with M280 streptavidin beads (Thermo Fisher, 11205D) in 1X Binding & Washing buffer (5 mM Tris-Cl (pH 7.5), 1M NaCl, 0.5 mM EDTA (pH 8.0)) for 20 min. Wash with 1X Binding & Washing buffer containing 0.5% IGEPAL CA-630 (Sigma, I3021) for 6 times, and then wash with ddH2O once. Dissolve beads in 40 ul ddH2O. Perform bisulfite conversion (Qiagen, 59104) with DNA on beads. PCR amplification (KAPA KR0413) with bisulfite converted DNA. PCR product was running on a 2% agarose-gel and the larger band ranging from 300 bp to 400 bp was hairpin-ligated sample. Recover this band with gel-extraction kit (TIANGEN, DP208). Clean up with AMPure beads (0.9X). Dissolve DNA in 0.1X TE buffer. The HAMMER-seq library is ready for Next Generation Sequencing.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Enrich EdU-labeled DNA, add hairpin, and BS-seq
MCM2-c30m-rep
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| Data processing |
Basecalls performed by Annoroad Gene Technology Co. Ltd.
Raw reads from Illumina platforms were processed by HBS-Tools. In brief, preprocessing of raw reads, including bad quality bases trimming, sequencing adaptor, and hairpin adaptor removal were performed with the module of ‘hbs_process’. And then each preprocessed read pair was aligned pairwise using Smith-Waterman algorithm and only aligned parts were kept to recover the original genomic sequence. The recovered original sequences were then mapped to the genome assembly, with which the methylation information was extracted into the result SAM output. The original sequence recovery and mapping was performed with the module ‘hbs_mapper’ of HBS-Tools. The aligned SAM files, that contain both strands of the hairpin read, were then de-duplicated based on the following criteria: the two strands were firstly concatenated with the one with higher-methylation as head and the lower as tail, and strand pairs with the same concatenated sequence were regarded as duplication and only one copy was retained. Based on the de-duplicated aligned reads (in SAM file), reads of each pair were discriminated as parent (higher) or daughter (lower) strand according to the No. of methylated CpGs, with equally methylated reads randomly distributed to parent or daughter. Read pairs with no methylated CpGs on both strands were discarded. Then, methylation maintenance events were called based on parent/daughter read pairs, among which maintained methylation, un-maintained methylation, maintained un-methylation and de novo methylation were designated as MM, MU, UU, UM, respectively. All events were summed up for each genomic CpG site by chromosome coordinates, so now we have 4 numbers indicating 4 kinds of events of MM/MU/UU/UM for each CpG site.
Genome_build: hg19 or mm10
Supplementary_files_format_and_content: Processed data files are provided as gzipped text files, that contain methylation mainenance events called for covered CpG sites. All events were summed up for each genomic CpG site by chromosome coordinates, so we have 4 numbers indicating 4 kinds of events of MM/MU/UU/UM for each CpG site. Maintenance events are also asigned into individual arm of the replication fork. Upper arm indicates parental Waston and daughter Crick strand, and lower arm indicates parental Crick and daughter Watson strand. All coloumns are indicated by the first row of the files.
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| Submission date |
Jun 23, 2020 |
| Last update date |
Jun 09, 2023 |
| Contact name |
Zhuqiang Zhang |
| E-mail(s) |
zhangzhuqiang@ibp.ac.cn
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| Organization name |
Institute of Biophysics, CAS
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| Street address |
15 Datun Road, Chaoyang District.
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| City |
Beijing |
| State/province |
--- |
| ZIP/Postal code |
100101 |
| Country |
China |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE131098 |
Hammer-seq to measure kinetics of DNA methylation maintenance in HeLa cells |
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| Relations |
| BioSample |
SAMN15351814 |
| SRA |
SRX8601109 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4633242_Mcm2-c30m.rep.events.txt.gz |
64.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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