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Status |
Public on Mar 13, 2018 |
Title |
nasBS-seq_pulse_parent_rep6 |
Sample type |
SRA |
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Source name |
H9
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Organism |
Homo sapiens |
Characteristics |
cell type: human embryonic stem cells passages: 55
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Growth protocol |
H9 human embryonic stem cells were obtained from WiCell. Cells were cultured in STEMPRO hESC SFM on cultureware coated with Geltrex Matrix at 37 °C under 5% CO2. Medium was changed every day. Cells were grown to 60-70% confluence before EdU labeling.
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Extracted molecule |
genomic DNA |
Extraction protocol |
For pulse, H9 cells were transferred to medium containing 50 µM EdU and were cultured at 37 °C for 20 min before formaldehyde crosslinking. For chase, after labeling with EdU, cells were washed with DPBS twice, and cultured in medium containing 50 µM dT for 8 hr before formaldehyde crosslinking. Crosslinking with 1% formaldehyde was done in DPBS at RT for 10 min and was quenched with 0.125 M glycine. Cells were collected and washed with DPBS, and lysed in Lysis Buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 10% glycerol). Pellets were collected and re-suspended in Sonication Buffer (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 0.1% sodium deoxycholate, 0.5% sodium lauroylsarcosine). Sonication was done on ice using Bioruptor. Triton X-100 was added to a final concentration of 1% before centrifugation at 15,000 xg. Supernatant was collected as fraction of chromatin. Elution Buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1% SDS) was added to chromatin in a 1:1 ratio. Crosslinking reversal was done by incubation at 65 °C for 8 hr. Proteinase K was added to a final concentration of 0.2 mg/ml before incubation at 55 °C for 30 min. Phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation was done sequentially to purify genomic DNA. Click chemistry was performed in a 200 µl volume (174 µl DNA in DPBS, 2 µl 1 mM biotin-azide, 4 µl 100 mM CuSO4, 20 µl 100 mM sodium ascorbate) at 37 °C for 1 hr. After ethanol precipitation, DNA was incubated with Dynabeads Streptavidin C1 beads on a rotor for 30 min at RT, and washed according to the manufacturer’s instructions. Beads were incubated with End Repair Enzyme Mix on a rotor for 30 min at RT. After washing, beads were incubated with Klenow Fragment (3'→5' exo-) reaction mix on a rotor for 30 min at RT. After washing, beads were incubated with T4 DNA ligase reaction mix and methylated adaptors on a rotor for 2 hr at RT. After washing, beads were incubated in 150 mM NaOH for 3 min at RT. Supernatant was collected as the fraction containing the parental strand DNA (bio-). Beads were washed 3 times with NaOH, re-suspended in 95% formamide and 10 mM EDTA pH 8.2, and incubated at 90 °C for 3 min. Supernatant was collected as the fraction containing the daughter strand DNA (bio+). Both fractions were ethanol precipitated, mixed with 1 pg of lambda DNA, and bisulfite converted with the EZ DNA Methylation-Lightning Kit. PCR amplification was done with HiFi Uracil+ polymerase. For each library, the minimal number of PCR cycles were used to yield at least 20 ng of DNA measured after purification with AMpure XP beads.
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Library strategy |
Bisulfite-Seq |
Library source |
genomic |
Library selection |
RANDOM |
Instrument model |
Illumina HiSeq 2500 |
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Description |
H9_pulse_par_Wat_C(CpG).bdg.gz H9_pulse_par_Cri_C(CpG).bdg.gz H9_pulse_intraCpG.bed.gz
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Data processing |
50 bp paired-end reads were first trimmed with Trimmomatic to remove any remnant adaptor sequence and bases with Phred quality score <20. Only pairs with both reads ≥20 bp were retained for subsequent analysis. Reads were aligned to the human genome (hg38) using bismark with bowtie2. Alignment score was controlled by --score-min L,0,-0.4. Mapping uniqueness was controlled by selecting alignments with mapping quality Q>10. Duplicated alignments were removed by bismark. For each library, reads were specifically mapped to either the Watson or Crick strand, and methylation of single cytosines was called from strand-specific alignments. For each strand, methylation calls from all biological replicates were pooled together. Methylation of CpGs were extracted from certain pairs of Watson/Crick strand methylation calls using bedtools intersect. Genome_build: hg38 Supplementary_files_format_and_content: bdg files were generated by bismark and represent methylation frequency of cytosines in the context of CpG on each strand. bw files were normalized to counts per one million PE reads and represent occupancy frequency.
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Submission date |
Apr 04, 2017 |
Last update date |
Mar 13, 2018 |
Contact name |
Chenhuan Xu |
E-mail(s) |
chxu02@gmail.com
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Organization name |
Beijing Institute of Genomics
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Street address |
1 Beichen West Road
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City |
Beijing |
ZIP/Postal code |
100101 |
Country |
China |
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Platform ID |
GPL16791 |
Series (1) |
GSE97394 |
Mapping DNA methylation and CTCF/cohesin occupancy on nascent chromatin and DNMT-targeted nascent chromatin |
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Relations |
BioSample |
SAMN06685691 |
SRA |
SRX2708709 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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