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| Status |
Public on Feb 07, 2020 |
| Title |
CBP11 |
| Sample type |
SRA |
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| Source name |
human CD3+ T-cell
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| Organism |
Homo sapiens |
| Characteristics |
offspring gender: female
gestational age (weeks): 38.3
birth weight (g): 2820
maternal age (years): 22
bmi at term: 24.4
weight at term (lbs): 150
tissue: CD3+ T-cell
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
High molecular weight genomic DNA was extracted from CD3+ T-cell from human cord blood.
Custom adapters were created for HELP-tagging, referred to as AE and AS Index. As well as an Illumina adapter sequence, adapter AE contains an EcoP15I recognition site and a T7 promoter sequence, and adapter AS contains index sequences for multiplexing . Adapter AS contains an Illumina sequencing primer sequence. Five hundred nanograms of genomic DNA were digested with HpaII in separate 50 μl reactions and purified by phenol/chloroform extraction followed by ethanol precipitation. The digested genomic DNA was ligated to adapter AE using an NEB Quick Ligation Kit (25 μl of 2x Quick ligase buffer, HpaII-digested DNA, 0.5 μl of Adapter AE (0.1 μM), 1 μl of Quick Ligase in a final volume of 10 μl). After AE ligation, the products were purified using AMpure XP (Agencort), then digested with EcoP15I (NEB). The restriction fragments were end-repaired to inhibit to dimerization of adapters, and tailed with a single dA, at the 3’ end. After the dA tailing reaction, adapter AS was ligated to the dA-tailed fragments using an NEB Quick Ligation Kit (12.5 μl of 2x Quick ligase buffer, 1.25 μl of Adapter AS (1 μM), 1.25 μl of Quick Ligase in a final volume 25 μl). After ligation, products were purified using the MinElute PCR purification kit (Qiagen) and in vitro-transcribed using MEGAshortscript (Ambion). Following in vitro transcription, products were purified with an RNeasy clean-up kit (Qiagen) before reverse transcription was performed using the SuperScript III kit (Invitrogen). The first strand cDNA produced was used as a template for PCR using the following conditions: 96°C for 2 minutes, then 18 cycles of 96°C for 15 seconds and 72°C for 15 seconds followed by 5 minutes at 72°C for the final extension. After PCR, the library was purified using a QIAQuick PCR clean-up kit (Qiagen).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
HpaII test for HELP-tagging
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| Data processing |
Images generated by the Illumina sequencer were analyzed by Illumina pipeline software (ELAND - 1.7.0). Initial data processing was performed using the default read length of 37 or 42 bp, after which we isolated the sequences in which we found adapter-tags on the 3’-end, replaced the tag sequence with a poly(N) sequence of the same length), and re-ran the Illumina ELAND pipeline again on these sequences with the sequence length set at 27 bp (the 2-28 bp subsequence). The data within the ELAND_extended.txt files were used for counting the number of aligned sequences adjacent to each CCGG (HpaII/MspI) site annotated in the hg19 freeze of the human genome at the UCSC genome browser. We permitted up to two mismatches in each sequence, and allowed a sequence to align to up to a maximum of 10 locations within the genome. For non-unique alignments, a sequence was assigned a partial count for each aligment location amounting to 1/n, where n represents the total number of aligned positions. To normalize the data between experiments, the number of sequences associated with each HpaII site was divided by the total number of sequences (including partial counts) aligning to all HpaII sites in the same sample.
Illumina Casava was used for basecalling.
After the sequence data was obtained from Illumina HiSeq 2500, we isolated the sequences in which we found adapter-tags on the 3’-end, replaced the tag sequence with a poly(N) sequence of the same length), and re-ran the Illumina ELAND pipeline again (ELAND - 1.7.0) on these sequences with the sequence length set at 27 bp (the 2-28 bp subsequence).
The data within the ELAND_extended.txt files were used for counting the number of aligned sequences adjacent to each CCGG (HpaII/MspI) site annotated in the hg19 freeze of the human genome at the UCSC genome browser. We permitted up to two mismatches in each sequence, and allowed a sequence to align to up to a maximum of 10 locations within the genome. For non-unique alignments, a sequence was assigned a partial count for each aligment location amounting to 1/n, where n represents the total number of aligned positions.
To normalize the data between experiments, the number of sequences associated with each HpaII site was divided by the total number of sequences (including partial counts) aligning to all HpaII sites in the same sample.
Genome_build: hg19
Supplementary_files_format_and_content: Format: bedGraph; content: Chromosome, start, end, processed angle value
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| Submission date |
Feb 09, 2017 |
| Last update date |
Feb 07, 2020 |
| Contact name |
Maureen J Charron |
| Organization name |
Albert Einstein College of Medicine
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| Department |
Biochemistry
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| Street address |
1300 Morris Park Ave
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| City |
Bronx |
| State/province |
NY |
| ZIP/Postal code |
10461 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE94729 |
Sexually dimorphic methylation of CD3+ T-lymphocyte DNA in offspring of overweight and obese mothers in a high risk, minority population in the Bronx |
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| Relations |
| BioSample |
SAMN06317997 |
| SRA |
SRX2548074 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2481456_CBP11.bedGraph.gz |
14.9 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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