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| Status |
Public on Jan 31, 2017 |
| Title |
EWS_T103 |
| Sample type |
SRA |
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| Source name |
EWS_tumor
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| Organism |
Homo sapiens |
| Characteristics |
tissue: tumor
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was isolated from 10 to 25 mg of snap-frozen tumors, cell lines, and MSCs by standard proteinase K digestion and phenol/chloroform extraction. DNA was quantified using a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Q32866) and the Qubit dsDNA BR Assay Kit (ThermoFisher Scientific, Q32850).
For RRBS, 100 ng of genomic DNA was digested for 12 hours at 37°C with 20 units of MspI (New England Biolabs, R0106L) in 30 μl of 1x NEB buffer 2. To retain even the smallest fragments and to minimize the loss of material, end preparation and adaptor ligation were performed in a single-tube setup. End fill-in and A-tailing were performed by addition of Klenow Fragment 3’ > 5’ exo- (New England Biolabs, M0212L) and dNTP mix (10 mM dATP, 1 mM dCTP, 1 mM dGTP). After ligation to methylated Illumina TruSeq LT v2 adaptors using Quick Ligase (New England Biolabs, M2200L), the libraries were size selected by performing a 0.75x clean-up with AMPure XP beads (Beckman Coulter, A63881). The libraries were pooled in combinations of six based on qPCR data and subjected to bisulfite conversion using the EZ DNA Methylation Direct Kit (Zymo Research, D5020) with the following changes to the manufacturer’s protocol: conversion reagent was used at 0.9x concentration, incubation performed for 20 cycles of 1 min at 95°C, 10 min at 60°C and the desulphonation time was extended to 30 min. These changes increase the number of CpG dinucleotides covered, by reducing double-strand break formation in larger library fragments. Bisulfite-converted libraries were enriched using PfuTurbo Cx Hotstart DNA Polymerase (Agilent, 600412). The minimum number of enrichment cycles was estimated based on a qPCR experiment. After a 2x AMPure XP clean-up, quality control was performed by a Qubit dsDNA HS (Life Technologies, Q32854) and Experion DNA 1k assay (BioRad, 700-7107). The RRBS libraries were sequenced on the Illumina HiSeq 2000 platform in 50 bp single read mode.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
adapter trimming with trimmomatic (ILLUMINACLIP ":2:40:7 SLIDINGWINDOW:4:15 MAXINFO:20:0.50 MINLEN:18")
alignment with bsmap (RRBS mode; equal_best_hits=100;mismatch rate=.08; report_repeat=1; seed_size=12; random_number=0; filter=5; quality_threshold=0)
methylation calling with biseqmethcalling (minFragmentLength=20; maxFragmentLength=1000;pfStatus=All;maxMismatches=0.1;baseQualityScoreC=20;baseQualityScoreNextToC=10;genomeFraction=50;smartWindows=250000)
Genome_build: hg38
Supplementary_files_format_and_content: bed file format containing for each CpG the number of methylated reads and the total number of reads
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| Submission date |
Jan 29, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Christoph Bock |
| E-mail(s) |
cbock@cemm.oeaw.ac.at
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| Organization name |
CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences
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| Street address |
Lazarettgasse 14
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| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE88826 |
DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma |
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| Relations |
| BioSample |
SAMN06281627 |
| SRA |
SRX2531944 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2471604_EWS_T103.txt.gz |
29.5 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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