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| Status |
Public on Apr 12, 2010 |
| Title |
Human ES MspI rep2 |
| Sample type |
SRA |
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| Source name |
human embryonic stem cell
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| Organism |
Homo sapiens |
| Characteristics |
cell type: H1 (WA01) human embryonic stem (ES) cell line
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| Growth protocol |
H1 human embryonic stem cells (hESCs, NIH code WA01 from Wicell Research Institute, Madison WI) were cultured on matrigel (BD Biosciences, San Diego), at 37 °C, 5% O2 and 5% CO2. Amplified hESC pluripotency was assessed by flow cytometry with SSEA4, CD24 and Oct4 markers. To extract DNA, the cells were suspended in 10 ml of a solution of 10 mM Tris-HCl (pH 8.0), 0.1 M EDTA and 1 ml of 10% SDS to which 10 μl of RNase A (20 mg/ml) was added. After incubation for 1 hour at 37 oC, 50 μl of proteinase K (20 mg/ml) was added and the solution was gently mixed and incubated in a 50 oC water bath overnight. To purify the lysate, it was extracted three times using saturated phenol, then twice with chloroform, and dialyzed for 16 hours at 4 oC against three changes of 0.2x SSC. Following dialysis, the DNA was concentrated by coating the dialysis bags in polyethylene glycol (molecular weight 20,000). The purity and final concentration of the purified DNA was checked by spectrometry (Nanodrop, Wilmington, DE).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Two custom adapters were created for HELP-tagging, referred to as AE and AS. As well as an Illumina adapter sequence, adapter AE contains an EcoP15I recognition site and a T7 promoter sequence. Adapter AS contains an Illumina sequencing primer sequence. Five micrograms of genomic DNA were digested with HpaII and MspI in separate 200 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, 3 μg of HpaII-digested DNA or 1 μg of MspI-digested DNA, 0.1 μl of Adapter AE (1 μM), 3 μl of Quick Ligase in a final volume of 50 μl). After AE ligation, the products were purified using AMpure (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 (25 μl of 2x Quick ligase buffer, 2.5 μl of Adapter AS (10 μM), 2.5 μl of Quick Ligase in a final volume 50 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 Genome Analyzer II |
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| Description |
MspI reference for HELP-tagging
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| Data processing |
Images generated by the Illumina sequencer were analyzed by Illumina pipeline software (versions 1.3-1.4). Initial data processing was performed using the default read length of 36 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 hg18 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.
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| Submission date |
Jan 19, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
John Greally |
| E-mail(s) |
john.greally@einsteinmed.edu
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| Phone |
7186781234
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| Organization name |
Albert Einstein College of Medicine
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| Department |
Genetics
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| Street address |
1301 Morris Park Avenue
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| City |
Bronx |
| State/province |
NY |
| ZIP/Postal code |
10461 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (1) |
| GSE19937 |
Human embryonic stem cells HELP-tagging cytosine methylation data (Albert Einstein College of Medicine) |
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| Relations |
| SRA |
SRX018699 |
| BioSample |
SAMN00010844 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM498219_hg18.Human_ES_MspI.30FRTAAXX.lane_8.eland_multi.bed.gz |
41.9 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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