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Sample GSM2044774 Query DataSets for GSM2044774
Status Public on Jan 23, 2016
Title Lmo2Del_rep1_hiseq
Sample type SRA
Source name Lmo2Del
Organism Homo sapiens
Characteristics cell line: Human embryonic kidney cell line HEK293T
genotype/variation: Lmo2 deletion
Growth protocol For each 5C library, 50 million cells were resuspended in 40 mL DMEM (high glucose, pyruvate; Invitrogen, 11995-073) followed by the addition of 4 mL 11% formaldehyde solution (11% formaldehyde, 50 mM HEPES pH 7.3, 100 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0). Cells were cross-linked for 10 minutes with gentle agitation every 2 minutes. Cross-linking was quenched by the addition of 2 ml 2.5M glycine followed by two washes with ice cold PBS. Crosslinked cells were snap-frozen in liquid nitrogen and stored at -80oC until further processing.
Extracted molecule genomic DNA
Extraction protocol 3C was performed with HindIII restriction enzyme as previously described with some minor modifications. Ligation was performed in a final volume of 1,200 µL with 10 U of T4 ligase (Invitrogen) and 1% of triton X-100. For 50 million cells, 10 ligation reactions were performed, and pooled after DNA purification. The 3C libraries were then interrogated by 5C. The multiplex annealing reaction was performed overnight at 50°C. Pairs of annealed 5C primers were ligated at the same temperature using Taq DNA ligase for 1 h. 7 independent ligation reactions were performed for each 5C library, each containing an amount of 3C template that represents 600,000 genome equivalents and 0.3 fmol of each primer. Ligated 5C primer pairs, which represent a specific ligation junction in the 3C library and thus a long-range interaction between the two corresponding loci, were then amplified using 20 cycles of PCR with T7 and T3R universal tail primers that recognize the common tails of the 5C forward and reverse primers. Four separate amplification reactions were carried out for each of 7 annealing reactions described above and all the PCR products were pooled together. This pool constitutes the 5C library. The libraries were concentrated using Amicon Ultra Centrifugal filters - 0.5ml 30K (Millipore) and purified with Qiaquick PCR purification kit. Index adaptors (Illumina) were ligated to the 5C library using the Illumina protocol (TruSeq Nano DNA sample Prep kit). The linkered 5C libraries were then amplified by PCR (6 cycles), using the Illumina PCR mix. The 5C libraries were gel purified and sequenced on the Illumina HiSeq 2000 platform, generating 50-bp paired-end reads. The indexed adapter was AD005 for WT, AD006 for TAL1 deletion and AD015 for LMO2 deletion. 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 2000
Description Lmo2Del_rep1
Merged processed data files:
Data processing library strategy: 5C
Sequencing data was obtained from both an Illumina MiSeq and an Illumina HiSeq 2000 machine and was processed by a custom pipeline to map and assemble 5C interactions to hg19. We used an updated version of the Novoalign mapping algorithm (V3.02.00). Data from the two biological replicates were pooled, producing a single interaction map for the wild type (wt), TAL1-deletion (TAL-ΔCTCF), and LMO2-deletion (LMO2-ΔCTCF) samples. The summary statistics and the read depth of each 5C libraries can be found in Table S7.
We merged primer interactions from the same fragments and binned the data using a binsize=20 kb, binstep=2.5kb, binmode=median. We calculated the difference of the z-score matrices to quantify the amount of the differential interactions observed between the WT and deletion datasets.
Genome_build: hg19
Supplementary_files_format_and_content: *cis.matrix.gz [binned_interaction_matrix], *subtract.matrix.gz [log2_distadj_comparison], all other *.matrix.gz [raw_interaction_matrix]
Submission date Jan 22, 2016
Last update date May 15, 2019
Contact name Richard A Young
Phone 617-258-5219
Organization name Whitehead Institute for Biomedical Research
Lab Young Lab
Street address 9 Cambridge Center
City Cambridge
State/province MA
ZIP/Postal code 02142
Country USA
Platform ID GPL11154
Series (2)
GSE68978 Activation of proto-oncogenes by disruption of chromosome neighborhoods
GSE77142 Activation of proto-oncogenes by disruption of chromosome neighborhoods [5C-Seq]
BioSample SAMN04435520
SRA SRX1542063

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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