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Sample GSM1603856

Query DataSets for GSM1603856

Status

Public on Feb 01, 2016

Title

GM12878 Rep1

Sample type

SRA

Source name

GM12878 Lymphoblastoid Cells

Organism

Homo sapiens

Characteristics

cell type: Lymphoblastoid Cell Line (LCL)
passages: 8-9
donor: NA12878

Growth protocol

Lymphoblastoid cell lines (LCLs) were generated by culturing adherent cells in RPMI medium (Gibco) supplemented with 15% FBS and 1% P/S.

Extracted molecule

genomic DNA

Extraction protocol

In preparation for Hi-C assay, cells were pelleted and resuspsended in media supplemented with formaldehyde at a final concentration of 1% for chromatin crosslinking. Crosslinking was halted in ~200mM Glyceine, pelleted, washed with cold PBS, pelleted, snap frozen, and stored at -80 until experimentation.
Hi-C libraries were prepared using the published method (Lieberman-Aiden, Science, 2009). Briefly, cross-linked cell pellets were lysed and chromatin within intact nuclei were digested using HindIII. Digested ends were filled in using biotinylated dCTP and an E. coli DNA Pol I large fragment (Klenow polymerase). Blunt-ended DNA molecules were then proximally ligated using T4 DNA ligase. After chromatin cross-linking was reversed, ligated DNA was purified. Next, purified ligation products were treated with T4 DNA polymeras, dATPs and dGTPs to remove biotinylated dCTPs from unligated DNA molecules and column-purified. Then, ligation products were fragmented to ~400bp using a Covaris S2 instrument. Ends of fragmented molecules were then repaired using a combination of T4 DNA polymerase, Klenow polymerase and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (exo minus) and dATP to yield a protruding 3- 'A' base for eventual ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. Yet after dA-tailing, DNA molecules were size selected on agarose gel from 200-600bp. After gel purification, biotinylated DNA molecules were pulled down using C1 streptavidin beads (Life Tech) and Illumina sequencing adapters were ligated using T4 DNA ligase. The bead-bound library was then PCR amplified using 14-15 cycles and purified using AMPure XP beads prior to target enrichment. For target enrichment, 500ng of Hi-C library was mixed with 500ng of RNA probes and incubated overnight at 65 degrees. The following day, captured DNA:RNA duplexes were bound to T1 streptavidin-coated beads (Life Tech), washed rigorously, and PCR amplified using 10-11 cycles. Final PCR amplicons were purified using AMPure XP beads and captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the HiSeq2500 following the manufacturer's protocols for 100 cycle, paired-end sequencing.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

library strategy: HiC

Data processing

Basecalls performed using CASAVA version 1.4
Targeted HaploSeq reads were aligned as single ends to the hg19 genome assembly using BWA MEM with the following command - bwa mem -B 8 -M
Single ends from bwa were paired using inhouse scripts, followed by GATK pipeline to recaliberate based and realign Indels.
The final bam file from the above steps were used for haplotyping and genotyping. More specifically, genotype calls from parent-child trio data was used as input for HaploSeq based haplotyping.
Accuracy of haplotype calls from HaploSeq were compared with parent-child trio dataset to estimate accuracy.
Genome_build: hg19
Supplementary_files_format_and_content: VCF files were generated as targeted HaploSeq output

Submission date

Feb 06, 2015

Last update date

May 15, 2019

Contact name

Anthony D Schmitt

E-mail(s)

aschmitt1987@gmail.com

Phone

6178429022

Organization name

UC San Diego