GSM1705239: Primed_SMC1_ChIAPET_biologicalReplicate2_run2; Homo sapiens; OTHER
400 million naive or primed hES cells were cross-linked by 1% formaldehyde at RT 10 mintues and were sonicated to solubilize the chromatin. The chromatin complexes contain DNA fragments with an average size of 300-800 bp were immunoprecipitated by using SMC1a antibody. The ChIA-PET libraries were generated in three stages. In the first stage, ChIP was performed using 25 μg anti-SMC1 antibody (Bethyl Labs, A300-055A) and 250 μl protein G Dynabeads (Life technology, 10004D). This stage was the same as the experimental procedure described in the ChIP-seq library generation. The second stage was the ligation of the DNA fragments based on proximity. The ChIP-DNA with beads were washed once with TE buffer, then incubated with the end blunting mix (containing 7.2 μl T4 DNA polymerase (New England Biolabs, M0203S) and 7 μl 10 mM dNTP (Life Technologies, 18427013) in 700 μl total volume) at 37 °C for 40 min. The beads were then washed three times with ChIA-PET wash buffer (10 mM Tris-HCl pH7.5, 1 mM EDTA, 500 mM NaCl). The beads were incubated with klenow (3’-5’ exo-) master mix (containing 7 μl klenow fragment (3’-5’ exo-) (New England Biolabs, M0212S) and 7 μl 10 mM dATP (New England Biolabs, N0440S) in 700 μl total volume) at 37 °C for 50 min. The beads were then washed three times with ChIA-PET wash buffer. The beads were then incubated with proximity ligation mix (containing 42 μl T4 DNA ligase (Life Technologies, 46300018) and 4 μl bridge linker (200 ng/ μl including Forward: /5Phos/CGCGATATC/iBiodT/TATCTGACT; Reverse: /5Phos/GTCAGATAAGATATCGCGT) in 14 ml total volume) at 16 °C for 22 hr. The beads were then washed three times with ChIA-PET wash buffer. The beads were then incubated with an exonuclease reaction mix (containing 6 μl lambda exonuclease (New England Biolabs, M0262S), and 6 μl exonuclease I  (New England Biolabs, M0293S) in 700 μl total volume) at 37 °C for 1 hr. The DNA was eluted and reverse crosslinked with proteinase K (Life Technologies, AM2546) at 55 °C overnight. The DNA was purified by phenol-chloroform extraction and ethanol precipitation. The third stage was the tagmentation of ligated products, purification of the tagmented DNA fragments, amplification of the DNA by PCR, size selection and paired-end sequencing. The ChIA-PET proximity ligation products were tagmented with Tn5 Transposase (5 μl Tn5 transposase (Illumina, FC-121-1030) for 50 ng DNA) at 55 °C for 5 min, then at 10 °C for 10 min. The tagmented DNA was purified using a Zymo column (VWR, 100554-654) following the manufacturer’s instructions. The tagmented DNA was affinity purified with M280 streptavidin beads (50 μl for each library, Life Technologies, 11205D), followed by washing five times with 2×SSC/0.5% SDS and then two times with 1× B&W buffer (5 mM Tris-HCl pH7.5, 0.5 mM EDTA, 1 M NaCl). The buffer was discarded and the immobilized DNA library on beads was gently resuspended in 30 μl EB buffer (QIAGEN). The PCR amplifications of the DNA libraries were performed by using the Nextera DNA Sample Preparation Kit (Illumina, FC-121-1031) for 10-12 cycles. The DNA was selected for the size range of 300-500 bp and was purified by gel extraction. The ChIA-PET library was sequenced by paired-end sequencing (100×100) on the Illumina HiSeq 2000. Linker Forward: /5Phos/CGCGATATC/iBiodT/TATCTGACT; Reverse: /5Phos/GTCAGATAAGATATCGCGT
GEO Accession
GSM1705239