title: GSM2044775: Lmo2Del_rep2_hiseq; Homo sapiens; OTHER
library_construction_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. 
attributes.tag: GEO Accession
attributes.value: GSM2044775