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Sample GSM1233909

Query DataSets for GSM1233909

Status

Public on Oct 18, 2013

Title

GM12878_RZ_1

Sample type

SRA

Source name

Lymphoblastoid Cell Lines

Organism

Homo sapiens

Characteristics

cell line: 12878
antibody: RNA-seq
replicate: 1

Treatment protocol

For ChIP-Seq, cross linking was performed in 1% formaldehyde for 10 minutes at room temperature.

Growth protocol

Biological replicates were grown in separate batches and at separate times. Cells were grown to a density of 0.6-0.8 x 10^6/mL in 15% fetal bovine serum. RNA was obtained from lymphoblastoid cell lines (LCLs) of the same growth as used for the ChIP-seq experiments.

Extracted molecule

total RNA

Extraction protocol

For ChIP-Seq, nuclear lysates were sonicated using a Branson 250 Sonifier (power setting 2, 100% duty cycle for 7 x 30-s intervals). Clarified lysates corresponding to 20 million cells were treated with 1-5ug of antibody (Table S4) coupled to Protein G Dynabeads (Invitrogen #10003D, New York). The protein-DNA complexes were washed with RIPA buffer and eluted in 1% SDS TE at 65°C. An aliquot of clarified lysate was reserved as “input DNA” and handled in parallel with the immunoprecipitated samples following elution from Dynabeads. For RNA-Seq, Total RNA was extracted using Trizol (Lifetechnologies, Grand Island, NY) reagent according to the manufacturer’s instructions, then purified using the Qiagen RNeasy kit (Qiagen, Valencia, CA) and treated with RNAse-free DNase (Qiagen). RNA integrity was checked on a Bioanalyzer (Agilent, Santa Clara CA) and only RNA with an RNA integrity number (RIN) of > 9.5was used for subsequent library construction. Purified total RNA was depleted of ribosomal RNA using the Ribo-Zero Magnetic Gold Kit (Human/Mouse/Rat) (Epicentre Biotechnologies, Madison, WI).
ChIP DNA sequencing libraries were generated according to Illumina DNA Tru-Seq DNA Sample Preparation Kit Instructions (Illumina Part # FC-121-2001, San Diego, CA). For RNA-Seq, stranded libraries were prepared following a dUTP protocol. Briefly, ~100 ng of rRNA depleted RNA were fragmented with 10 x fragmentation buffer (Lifetechnologies, #AM8740) for 2 min at 70 °C. First-strand cDNA synthesis was primed with random hexamers. Actinomycin D was added to reduce anti-sense artifacts. For second-strand synthesis dTTP was replaced with dUTP. The cDNA was endrepaired, A-tailed and Illumina TruSeq adapters were added. After size-selection on an agarose gel the second strand was eliminated by digestion with AmpErase Uracil Nglycosylase (UNG) (Lifetechnologies, N8080096) and after PCR amplification samples were sequenced on the Illumina Hi-Seq 2000.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Personal genomes were created by adding the SNPs of each individual into hg19. SNPs were obtained from the 1000 Genomes Project and from Chen et al 2012
ChIP-seq reads were aligned to personal genomes with BWA 0.6.1 (options -q 20 and the rest set to defaults)
Peaks were called using MACS2. Genome-wide signal tracks (bigWig) were generated using wiggler.
RNA-seq reads were mapped to personal transcriptomes using TOPHAT 2.0.4.
RNA-seq reads overlapping exons (Gencode v10) were obtained using HT-Seq 0.5.4
Genome_build: hg19
Supplementary_files_format_and_content: Peaks are in the narrowPeak format. bigWig files were generated using wiggler. Abundance measurements from the RNA-seq data are presented in a matrix (geneCount_mat.txt), where each row is a gene and each column is a cell line. The values in the matrix are the total counts (from HT-Seq) across both RNA-seq replicates for that cell line.
Samples represent the individual replicates. The combined replicate data are on the Series record.

Submission date

Sep 18, 2013

Last update date

May 15, 2019

Contact name

Maya Kasowski

E-mail(s)

[email protected]

Phone

610-283-5618

Organization name

Stanford University