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| Status |
Public on Dec 12, 2013 |
| Title |
Whole Genome Shotgun Bisulfite Sequencing of Gastric Cells from Human STL002; methylC-seq_STL002GA-01b |
| Sample type |
SRA |
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| Source name |
Gastric tissue; methylC-seq_STL002GA-01b
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| Organism |
Homo sapiens |
| Characteristics |
sra sample accession: SRS366516
disease: iron deficiency, bipolar
biomaterial_provider: Shin Lin, Stanford University
biomaterial_type: Primary Tissue
tissue_type: Gastric
tissue_depot: N/A
collection_method: Autopsy
donor_id: STL002
donor_age: 30
donor_health_status: iron deficiency, bipolar disease (NO diabetes, hypertension, coronary artery disease, cancer)
donor_sex: Female
donor_ethnicity: Caucasian
experiment_type: DNA Methylation
extraction_protocol: Qiagen DNeasy mini kit, performed as per manufacturer's instructions
extraction_protocol_type_of_sonicator: Covaris S2
extraction_protocol_sonication_cycles: 200 bp, 3 cycles
dna_preparation_initial_dna_qnty: 1 ug
dna_preparation_fragment_size_range: 200
dna_preparation_adaptor_sequence: A: 5' P-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG, B: 5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
dna_preparation_adaptor_ligation_protocol: 16C for 16 hours with T4 DNA ligase (New England Biolabs)
dna_preparation_post-ligation_fragment_size_selection: Two rounds of purification with AMPure XP beads (Agencourt)
bisulfite_conversion_protocol: Invitrogen MethylCode
bisulfite_conversion_percent: 99.5% of cytosines converted based on shotgun sequencing of unmethylated lambda phage control spiked into original genomic DNA sample
library_generation_pcr_template_conc: >The adapter-ligated, bisulfite converted DNA was used in a 50 ul PCR reaction
library_generation_pcr_polymerase_type: KAPA HiFi HotStart Uracil+ ReadyMix
library_generation_pcr_thermocycling_program: 95C 2 min; 98C 30 sec, 4 cycles of 98C 15 sec, 60C 30 sec, 72C 4 min; 72C 10 min
library_generation_pcr_number_cycles: 4
library_generation_pcr_f_primer_sequence: 5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
library_generation_pcr_r_primer_sequence: 5' CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT
library_generation_pcr_primer_conc: 25 uM
library_generation_pcr_product_isolation_protocol: Two rounds of purification with AMPure XP beads (Agencourt)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Library construction protocol: One ug of genomic DNA was extracted from frozen ground tissue using the DNeasy Mini Kit (Qiagen, Valencia, CA) and spiked with 25 ng unmethylated cl857 Sam7 Lambda DNA (Promega, Madison, WI). The DNA was fragmented with a Covaris S2 (Covaris, Woburn, MA) to 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 16C 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 4 cycles of PCR with the following reaction composition: 25 ul 2x KAPA HiFi HotStart Uracil+ReadyMix, 2.5 ul Primer 1, 2.5 ul Primer 2 (50 ul final). The thermocycling parameters were: 95C 2 min, 98C 30 sec, then 4-8 cycles of 98C 15 sec, 60C 30 sec and 72C 4 min, ending with one 72C 10 min step. The reaction products were purified using AMPure XP beads (two rounds). Up to three 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 2000 |
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| Description |
sample_term_id: UBERON_0000945
assay_term_id: OBI_0001863
nucleic_acid_term_id: SO_0000352
Design description: Whole Genome Shotgun Bisulfite Sequencing of Gastric Cells from Human STL002
Library name: methylC-seq_STL002GA-01b
EDACC Genboree Experiment Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FEXPERIMENT%2FEDACC.18868
EDACC Genboree Sample Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSD%2FSAMPLE%2FEDACC.14004
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For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
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| Data processing |
Various levels of processed data files will be made available as this project proceeds.
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| Submission date |
Dec 06, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
UCSD AND SALK |
| Organization name |
University of California, San Diego
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| Street address |
Health Sciences Drive
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92092 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE16256 |
UCSD Human Reference Epigenome Mapping Project |
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| Relations |
| SRA |
SRX388742 |
| BioSample |
SAMN01737668 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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