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Sample GSM1385975 Query DataSets for GSM1385975
Status Public on Jul 02, 2014
Title HESO-7
Sample type SRA
 
Source name Human Embryonic Stem Cell
Organism Homo sapiens
Characteristics tissue: pluripotent
age: fetal
genotype: pluripotent
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.
 
Library strategy Bisulfite-Seq
Library source genomic
Library selection RANDOM
Instrument model Illumina HiSeq 2500
 
Description allc_HESO-7
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.
 
Submission date May 13, 2014
Last update date May 15, 2019
Contact name Joseph R Ecker
E-mail(s) ecker@salk.edu
Phone 8584534100
Organization name HHMI-Salk-Institute
Department Genomic Analysis Laboratory
Lab Ecker lab
Street address 10010 North Torrey Pines Road
City La Jolla
State/province CA
ZIP/Postal code 92037
Country USA
 
Platform ID GPL16791
Series (1)
GSE57179 Epigenetic and transcriptional aberrations in human pluripotent stem cells reflect differences in reprogramming mechanisms
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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