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Sample GSM3898215 Query DataSets for GSM3898215
Status Public on Jun 26, 2020
Title Ligase1-K126A-chase-24h
Sample type SRA
Source name EdU labeled Ligase1-K126A HeLa cells with chasing 24 hr
Organism Homo sapiens
Characteristics gender: female
developmental stage: adult
cell line: HeLa-S3 with Ligase1-K126A
cell type: epithelial adenocarcinoma
genotype/variation: Ligase1-K126A
tissue: cervix
developmental stage: 31 years
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).
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.
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.
Library strategy Bisulfite-Seq
Library source genomic
Library selection RANDOM
Instrument model HiSeq X Ten
Description Enrich EdU-labeled DNA, add hairpin, and BS-seq
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
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.
Submission date Jun 19, 2019
Last update date Jun 09, 2023
Contact name Zhuqiang Zhang
Organization name Institute of Biophysics, CAS
Street address 15 Datun Road, Chaoyang District.
City Beijing
State/province ---
ZIP/Postal code 100101
Country China
Platform ID GPL20795
Series (1)
GSE131098 Hammer-seq to measure kinetics of DNA methylation maintenance in HeLa cells
BioSample SAMN12096318
SRA SRX6095331

Supplementary file Size Download File type/resource 71.3 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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