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Sample GSM301562

Query DataSets for GSM301562

Status

Public on Jul 03, 2008

Title

HEK Input DNA rep1

Sample type

SRA

Source name

Input DNA

Organism

Homo sapiens

Characteristics

n/a

Growth protocol

HEK 293T cells were cultured in parallel in 2*150 cm3 flasks with DMEM (penicillin-streptomycin) (Gibco) supplemented with 10% Fetal Calf Serum (FCS) (Biochrom) and Glutamax (1x) (Gibco). In short, 5-10 ? 107 293T/17 cells were cross-linked with 1% formaldehyde for 10 min and the reaction stopped by adding 1/20 volume of 2.5 M glycine. The cross-linked material was washed with PBS, lysed, and sonicated to an average size of 300 bp.

Extracted molecule

genomic DNA

Extraction protocol

The sheared chromatin was incubated with 10 µg specific antibody to RNA polymerase II (ab5408, recognizing the hypophosphorylated form of RNA pol II) coupled to protein G magnetic beads (Invitrogen) for 16 h at 4°C. An aliquot of the input DNA was saved prior to immunoprecipitation as reference sample. The DNA was finally recovered, after washing (wash buffer: 50 mM Hepes-KOH, pH 7.6), 500 mM LiCl;1mM EDTA; 1% NP-40; 0.7% Na-Deoxycholate) and elution, by reversing the cross-linking overnight at 65° C in the elution buffer (50 mM Tris-HCl, pH 8.0; 10mM EDTA; 1% SDS). 80 ng of input DNA and 10 ng of ChIP DNA (see above) were taken for library preparation. The amplification was performed prior to gel size fractionation. 18 cycles of PCR with Illumina PCR primers were performed using an elongation time of 45 seconds. PCR-products were size fractionated and 150-300bp fragments were excised.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina Genome Analyzer

Description

raw sequence 070710_4

Data processing

After mapping of the reads to the genome, the replicates were pooled and resulted in 3,198,755 unique reads for two lanes of the RNA Pol II experiment and in 8,017,459 unique reads for three lanes of the control input DNA. The reads were sorted by their position on the chromosome and the start positions were used for the analysis. The RNA-Seq reads were analyzed for clusters of 10 reads in an initial window of 100 base pairs. 198 of the Pol-II clusters were overlapping with one of the input control clusters and thus were discarded.

Submission date

Jun 30, 2008

Last update date

May 15, 2019

Contact name

Marc Sultan

E-mail(s)

sultan@molgen.mpg.de

Organization name

Max Planck Institute for molecular genetics

Street address

Ihnestr. 73

City

Berlin

ZIP/Postal code

14195

Country

Germany

Platform ID

GPL9052

Series (1)

GSE11892

A Global View of Gene Activity and Alternative Splicing by Deep Sequencing of the Human Transcriptome

Relations

SRA

SRX003879

BioSample

SAMN02195283

Data table header descriptions
SEQUENCE	dots in sequences denote ambiguous base calls
COUNT	number of times sequenced

Data table
SEQUENCE	COUNT
..CGTACATCCAGTCTGTGATATTGTTCTTAATATT	0
..GAAATGTTGATTATTTTACTGGCACATCTTTGGC	0
..TAGCATTCAGGTTTGCGGGATGGGGCCTCTCACT	0
..TCACGATTACCCCGCTCAACGCCCAAACCCAAAC	0
..TTCTTGGTGTGGGGCTCATGCACTTATTTATTCC	0
.A.GTAAGGAGCTGTCTTGTGGGGTGAGACATTGAT	0
.A.TAAAAAAGTTTTGTCACTTAACAAGATAAACCC	0
.A.TAATTTTCATTTACTAATAGAACAAATATCTAA	0
.AA.AAGTTTCTGGGGTTTACCTCTCCGTGCTACTC	0
.AA.ATTTCATTGGTAGTGTTTTAGGACAATATCTT	0
.AA.CACAACGCCATTGAGGATTAAAGGCTGACACT	0
.AA.CACATAAGCATTTCCTTTAGGCTCTGGCTTCT	0
.AA.CCTATTCCCATGTCGTTGCGATACCCCCCCAT	0
.AA.GAGTGGAATTGGATAGTTTTATAACACAAAGA	0
.AA.GAGTGTAGTGGCGTGATCTCTGCCCAACGCCA	0
.AA.GCCAGGATAACCCGGTGGTCAAACTTTCTGCA	0
.AA.GGCACAACTGAAGCACACGCCCCTCCCCCCCC	0
.AA.GGGCAGCATAAGTAAACCTGATCTGCCAGGAT	0
.AA.TTTTTAACTAAGGGTGATGGACCTCTTGGGCT	0
.AAAAAAAAAATTGCAATTTGGACTCTTGTGGAGAA	0

Total number of rows: 12814371

Table truncated, full table size 449566 Kbytes.

Supplementary data files not provided

SRA Run Selector

Raw data are available in SRA

Processed data included within Sample table