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| Status |
Public on Jul 02, 2014 |
| Title |
HESO-7 |
| Sample type |
SRA |
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| Source name |
Human Embryonic Stem Cell
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| Organism |
Homo sapiens |
| Characteristics |
tissue: pluripotent
age: fetal
genotype: pluripotent
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
One µg of genomic DNA was spiked with 5 ng unmethylated cl857 Sam7 Lambda DNA (Promega, Madison, WI). The DNA was fragmented with a Covaris S2 (Covaris, Woburn, MA) to 150-200 bp, followed by end repair and addition of a 3’ A base. Cytosine-methylated adapters provided by Illumina (Illumina, San Diego, CA) were ligated to the sonicated DNA at 16˚C for 16 hours with T4 DNA ligase (New England Biolabs). Adapter-ligated DNA was isolated by two rounds of purification with AMPure XP beads (Beckman Coulter Genomics, Danvers, MA). Adapter-ligated DNA (≤450 ng) was subjected to sodium bisulfite conversion using the MethylCode kit (Life Technologies, Carlsbad, CA) as per manufacturer’s instructions. The bisulfite-converted, adapter-ligated DNA molecules were enriched by 8 cycles of PCR with the following reaction composition: 25 µL of Kapa HiFi Hotstart Uracil+ Readymix (Kapa Biosystems, Woburn, MA) and 5 µl TruSeq PCR Primer Mix (Illumina) (50 µl final). The thermocycling parameters were: 95˚C 2 min, 98˚C 30 sec, then 4 cycles of 98˚C 15 sec, 60˚C 30 sec and 72˚C 4 min, ending with one 72˚C 10 min step. The reaction products were purified using AMPure XP beads. Up to two separate PCR reactions were performed on subsets of the adapter-ligated, bisulfite-converted DNA, yielding up to two One µg of genomic DNA was spiked with 5 ng unmethylated cl857 Sam7 Lambda DNA (Promega, Madison, WI). The DNA was fragmented with a Covaris S2 (Covaris, Woburn, MA) to 150-200 bp, followed by end repair and addition of a 3’ A base. Cytosine-methylated adapters provided by Illumina (Illumina, San Diego, CA) were ligated to the sonicated DNA at 16˚C for 16 hours with T4 DNA ligase (New England Biolabs). Adapter-ligated DNA was isolated by two rounds of purification with AMPure XP beads (Beckman Coulter Genomics, Danvers, MA). Adapter-ligated DNA (≤450 ng) was subjected to sodium bisulfite conversion using the MethylCode kit (Life Technologies, Carlsbad, CA) as per manufacturer’s instructions. The bisulfite-converted, adapter-ligated DNA molecules were enriched by 8 cycles of PCR with the following reaction composition: 25 µL of Kapa HiFi Hotstart Uracil+ Readymix (Kapa Biosystems, Woburn, MA) and 5 µl TruSeq PCR Primer Mix (Illumina) (50 µl final). The thermocycling parameters were: 95˚C 2 min, 98˚C 30 sec, then 4 cycles of 98˚C 15 sec, 60˚C 30 sec and 72˚C 4 min, ending with one 72˚C 10 min step. The reaction products were purified using AMPure XP beads. Up to two separate PCR reactions were performed on subsets of the adapter-ligated, bisulfite-converted DNA, yielding up to two independent libraries from the same biological sample.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
allc_HESO-7
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| Data processing |
Sequencing reads were first trimmed for adapter sequence using Cutadapt.
All cytosines in the trimmed reads were then computationally converted to thymines and mapped twice, to a converted forward strand reference and to a converted reverse strand reference both based on the hg19 reference genome.
A converted reference is created by replacing all cytosines with thymines (forward strand) or all guanines with adenines (reverse strand) in the reference FASTA file.
For mapping we used Bowtie3 with the following options: "-S","-k 1","-m 1","--chunkmbs 3072","--best","--strata","-o 4","-e 80","-l 20", and "-n 0".
Any read that mapped to multiple locations was removed and one read from each starting location on each strand from each library was kept (i.e., clonal reads were removed).
To call methylated sites, we summed the number of reads that supported methylation at a site and the number of reads that did not. We used these counts to perform a binomial test with a probability of success equal to the non-conversion rate, which was determined by computing the fraction of methylated reads in the lambda genome (spiked in during library construction). The false discovery rate (FDR) for a given p-value cutoff was computed using Benjamini-Hochberg approach. Because the p-value distributions for each methylation context are different, this procedure was applied to each three nucleotide context independently (e.g., a p-value cutoff was calculated for CAT cytosines).
Genome_build: hg19
Supplementary_files_format_and_content: The processed data for the allc_<sample>_<chr>.tsv files contain a row of headers indicating the chromosome, position, strand, methylation class, methylation count, total coverage, and methylation call. This is a tab delimited file.
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| Submission date |
May 13, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Joseph R Ecker |
| E-mail(s) |
ecker@salk.edu
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| Phone |
8584534100
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| Organization name |
HHMI-Salk-Institute
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| Department |
Genomic Analysis Laboratory
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| Lab |
Ecker lab
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| Street address |
10010 North Torrey Pines Road
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE57179 |
Epigenetic and transcriptional aberrations in human pluripotent stem cells reflect differences in reprogramming mechanisms |
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| Relations |
| BioSample |
SAMN02777316 |
| SRA |
SRX542417 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1385975_allc_HESO-7_1.tsv.gz |
347.9 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_10.tsv.gz |
204.6 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_11.tsv.gz |
206.6 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_12.tsv.gz |
203.8 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_13.tsv.gz |
142.3 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_14.tsv.gz |
137.6 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_15.tsv.gz |
126.3 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_16.tsv.gz |
128.7 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_17.tsv.gz |
130.8 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_18.tsv.gz |
114.0 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_19.tsv.gz |
98.6 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_2.tsv.gz |
359.8 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_20.tsv.gz |
100.0 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_21.tsv.gz |
54.4 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_22.tsv.gz |
60.5 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_3.tsv.gz |
294.3 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_4.tsv.gz |
271.4 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_5.tsv.gz |
262.8 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_6.tsv.gz |
251.1 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_7.tsv.gz |
232.7 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_8.tsv.gz |
214.7 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_9.tsv.gz |
174.0 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_L.tsv.gz |
108.6 Kb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_X.tsv.gz |
209.8 Mb |
(ftp)(http) |
TSV |
| GSM1385975_allc_HESO-7_Y.tsv.gz |
1.5 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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