Instrument: Illumina HiSeq 2500
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: SINGLE
Construction protocol: ChIP-seq: Briefly, the cells were crossed linked with 1% formaldehyde at ambient temperature for 10 min. The reaction was quenched by 125mM glycine for 5 min at room temperature. Cells were washed with PBS and treated with hypotonic buffer (20mM Hepes pH7.9, 10mM KCl, 1mM EDTA, 10% Glycerol and 1mM DTT with additional protease inhibitor (Roche)) to isolate nuclei. The nuclei were suspended with RIPA buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate with protease inhibitor) and sonicated using Covaris S220 Focused-ultrasonicator. Fragmented chromatin was pre-cleared with protein G conjugated sepharose beads (GE). Antibodies were used to pull down the respective proteins and their associated chromatin. Washes with different concentration of NaCl were performed. The enriched protein-DNA complexes were reverse crosslinked at 65°C over night with proteinase K (NEB). DNA was purified with Qiagen MinElute kit. STARR-seq: The plasmid pool was transfected into HEK293T or HepG2 cell lines using Fugene HD and continued culturing for 48 hours before harvest. Total RNA was extracted with RNeasy kit (Qiagen, 74104) and mRNA was enriched with poly(dT)25 Dynabeads (Invitrogen, 61002). First strand cDNA was synthesized using a specific primer (5'-CAAACTCATCAATGTATCTTATCATG) with high High-Capacity cDNA Reverse Transcription kit (ThermoFisher Scientific, 4368814). The nested PCR was used to amplify the SNP specific fragments from cDNA, first using two reporter-specific PCR primers (5'-GGGCCAGCTGTTGGGGTGTCCAC & 5'-CTTATCATGTCTGCTCGAAGC) and then generic primers used in HT-SELEX. DNA was purified with AMPure beads. Hi-C: Briefly, the human islets were washed with cold PBS and cut into small pieces. For HepG2 cells, the cells were trypsinized and washed with PBS. The chromatin was cross-linked with 1% formaldehyde (Sigma) at ambient temperature for 10 min and quenched with 125mM glycine for 5 min. PBS washed tissue was homogenized with loose fitting douncer for 30 strokes before centrifugation to isolate the nuclei. Nuclei were isolated and directly applied for digestion using 4 cutter restriction enzyme MboI (NEB) at 37 °C o/n. The single strand overhang was filled with biotinylated-14-ATP (Life Tech.) using Klenow DNA polymerase (NEB). Different from tradition Hi-C, with in situ protocol the ligation was performed when the nuclear membrane was still intact. DNA was ligated for 4h at 16 °C using T4 ligase (NEB). Protein was degraded by proteinase K (NEB) treatment at 55 °C for 30 min. The crosslinking was reversed with 500 mM of NaCl and heated at 68 °C o/n. DNA was purified and sonicated to 300-700 bp small fragments. Biotinylated DNA was selected with Dynabeads My One T1 Streptavidin beads (Life Tech.). Sequencing library was prepared on beads and intensive wash was performed between different reactions. Whole genome sequencing: The genomic DNA was extracted using Qiagen kit (cat. no. 69506). The DNA was then fragmented with Covaris S220 ultrasonicator to 300-500 bp long. RNA-seq: The total RNA was isolated using Qiagen RNeasy mini kit. The sequencing library was prepared using Truseq illumine RNA Library Prep Kit v2 (cat. #RS-122-2001). SELEX: Briefly, the E. coli expressed 6xHis-tagged TF proteins were immobilized to Ni sepharose beads (GE, 17-5318-01) in Promega binding buffer (10mM Tris pH7.5, 50mM NaCl, 1mM MgCl2, 4% glycerol, 0.5mM EDTA, 5µg/ml poly-dIdC). Oligos from input or previous HT-SELEX cycles were added into the protein beads mixture and incubated at ambient temperature for 30 min. After binding, the beads were consecutively washed for 12 times with the Promega binding buffer. After final wash, TE (10mM Tris pH 8.0, 1mM EDTA) was used to re-suspend the beads and for PCR amplification. The PCR products from each HT-SELEX cycle were purified (Qiagen, 28004). ChIP-seq: Briefly, the cells were crossed linked with 1% formaldehyde at ambient temperature for 10 min. The reaction was quenched by 125mM glycine for 5 min at room temperature. Cells were washed with PBS and treated with hypotonic buffer (20mM Hepes pH7.9, 10mM KCl, 1mM EDTA, 10% Glycerol and 1mM DTT with additional protease inhibitor (Roche)) to isolate nuclei. The nuclei were suspended with RIPA buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate with protease inhibitor) and sonicated using Covaris S220 Focused-ultrasonicator. Fragmented chromatin was pre-cleared with protein G conjugated sepharose beads (GE). Antibodies were used to pull down the respective proteins and their associated chromatin. Washes with different concentration of NaCl were performed. The enriched protein-DNA complexes were reverse crosslinked at 65°C over night with proteinase K (NEB). DNA was purified with Qiagen MinElute kit. STARR-seq: The plasmid pool was transfected into HEK293T or HepG2 cell lines using Fugene HD and continued culturing for 48 hours before harvest. Total RNA was extracted with RNeasy kit (Qiagen, 74104) and mRNA was enriched with poly(dT)25 Dynabeads (Invitrogen, 61002). First strand cDNA was synthesized using a specific primer (5'-CAAACTCATCAATGTATCTTATCATG) with high High-Capacity cDNA Reverse Transcription kit (ThermoFisher Scientific, 4368814). The nested PCR was used to amplify the SNP specific fragments from cDNA, first using two reporter-specific PCR primers (5'-GGGCCAGCTGTTGGGGTGTCCAC & 5'-CTTATCATGTCTGCTCGAAGC) and then generic primers used in HT-SELEX. DNA was purified with AMPure beads. Hi-C: Briefly, the human islets were washed with cold PBS and cut into small pieces. For HepG2 cells, the cells were trypsinized and washed with PBS. The chromatin was cross-linked with 1% formaldehyde (Sigma) at ambient temperature for 10 min and quenched with 125mM glycine for 5 min. PBS washed tissue was homogenized with loose fitting douncer for 30 strokes before centrifugation to isolate the nuclei. Nuclei were isolated and directly applied for digestion using 4 cutter restriction enzyme MboI (NEB) at 37 °C o/n. The single strand overhang was filled with biotinylated-14-ATP (Life Tech.) using Klenow DNA polymerase (NEB). Different from tradition Hi-C, with in situ protocol the ligation was performed when the nuclear membrane was still intact. DNA was ligated for 4h at 16 °C using T4 ligase (NEB). Protein was degraded by proteinase K (NEB) treatment at 55 °C for 30 min. The crosslinking was reversed with 500 mM of NaCl and heated at 68 °C o/n. DNA was purified and sonicated to 300-700 bp small fragments. Biotinylated DNA was selected with Dynabeads My One T1 Streptavidin beads (Life Tech.). Sequencing library was prepared on beads and intensive wash was performed between different reactions. Whole genome sequencing: The genomic DNA was extracted using Qiagen kit (cat. no. 69506). The DNA was then fragmented with Covaris S220 ultrasonicator to 300-500 bp long. RNA-seq: The total RNA was isolated using Qiagen RNeasy mini kit. The sequencing library was prepared using Truseq illumine RNA Library Prep Kit v2 (cat. #RS-122-2001). SELEX: Briefly, the E. coli expressed 6xHis-tagged TF proteins were immobilized to Ni sepharose beads (GE, 17-5318-01) in Promega binding buffer (10mM Tris pH7.5, 50mM NaCl, 1mM MgCl2, 4% glycerol, 0.5mM EDTA, 5µg/ml poly-dIdC). Oligos from input or previous HT-SELEX cycles were added into the protein beads mixture and incubated at ambient temperature for 30 min. After binding, the beads were consecutively washed for 12 times with the Promega binding buffer. After final wash, TE (10mM Tris pH 8.0, 1mM EDTA) was used to re-suspend the beads and for PCR amplification. The PCR products from each HT-SELEX cycle were purified (Qiagen, 28004).