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| Status |
Public on Nov 20, 2014 |
| Title |
NBM_3_hmeSeal |
| Sample type |
SRA |
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| Source name |
Normal CD34+ bone marrow cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Normal CD34+ bone marrow cells
experiment type: hMe-seal
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted using Qiagen Puregene kit protocol;
hMe-seal 5hmC labelling: Genomic DNA (gDNA) was sonicated into 200-400bp long fragments (set up: high, 30 times, 30s interval pulse followed by 30 s pause). Subsequently 2 g units are wrong of gDNA were labeled with chemically modified uridine diphosphoglucose glucose (UDP-6-N3-Glu) by viral β-glucosyltransferase (β-GT) as described previously(Song et al., 2011, Nature Biotech.). Afterwards the labeled DNA was purified by QIAquick PCR Purification Kit (Qiagen Inc.,Valencia CA) and eluted in water. The click chemistry reaction was performed by addition of 150 μM dibenzocyclooctyne modified biotin into the eluted DNA, the reaction mixture was incubated for 2 h at 37 °C. The DNA samples were then purified by MinElute Reaction Cleanup Kit (Qiagen Inc., Valencia CA) and the amount of eluted DNA was determined by Nanodrop UV spectroscope (Thermo, Wilmington, DE)
ERRBS: Library construction protocol: Genomic DNA was purified from cell lines and primary human samples, digested with MspI restriction enzyme (200U MspI per reaction), subjected to end repair and adenylation followed by ligation to cytosine methylated paired-end Illumina adaptors (2000U T4 DNA ligase per reaction). Fragments of 150-250 bp (reduced representation bisulfite sequencing) and 150-400 bp (for enhanced reduced representation bisulfite sequencing) were isolated after agarose gel electrophoresis and subjected to bisulfite conversion (single conversion round using Zymo Research EZ DNA Methylation Kit) and PCR amplification using paired-end Illumina primers (18 cycles). Libraries were sequenced using an Illumina Genome Analyzer II or HiSeq2000 per manufacturer’s recommended protocol for 50bp single end read runs.
Bisulfite-seq (RRBS) and OTHER (hMe-Seal: chemical labeling,biotnylation and pulldown of regions with 5hmC )
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
hMe-seal
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| Data processing |
For ERRBS:Adaptors are trimmed with cutadapt,then we aligned trimmed reads to genome (hg18) with Bismark aligner (v 0.6.3). We called for methylation percentage for each CpG with at least 10X coverage and each base on the reads that cover the C must have at least phred Quality score of 20.
For hMe-Seal: reads are aligned to genome using eland (from CASAVA 1.8 pipeline) and peaks are called using Chipseeqer (arguments: -t 15 -fold_t 2 - uniquereads 1 )
Genome_build: hg18
Supplementary_files_format_and_content: For ERRBS, text files for location of CpGs and their methylation information: column 1: base id column 2: chromosome column 3: base position column 4: strand column 5: coverage column 6: number of methylated Cs on that position column 7: number of unmethylated Cs on that position For hMeSeal, text file containing peak locations and statistics Chromosome : column 1 : the first (genomic) coordinate of the peak column 2 : the second (genomic) coordinate of the peak column 3 : average log p-value of the nucleotides in the normalized peak region column 4 : score estimated as the average ChIP reads/length of peak (minus the average INPUT reads/length of peak - if INPUT is available) column 5 : the position of the maximum height of the peak column 6 : the maximum peak height (in reads) column 7 : the relative position of the maximum height of the peak, e.g., 50% means the highest point is at the middle of the peak column 8 : the size of the peak (in bp) column 9 : the middle position of the peak column 10 : the distance of the maximum height from the middle of the peak
Supplementary_files_format_and_content: peak locations and statistics from Chipseeqer
Supplementary_files_format_and_content: CpG methylation information: location, number of methylated Cs(freqCs),number of unmethylated Cs(freqTs) and coverage
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| Submission date |
Nov 03, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Ari Melnick |
| Organization name |
Weill Cornell Medical College
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| Street address |
1305 York Ave.
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| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE52945 |
Epigenomic profiling of Acute myeloid leukemia subtypes from primary tumor samples |
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| Relations |
| BioSample |
SAMN03159559 |
| SRA |
SRX750051 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1536365_NBM_3_hmeSeal.peak.txt.gz |
5.2 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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