3.20.1 Cell culture and infection.
Approximately 2 x 107 AR42J and INS-1 cells were plated in 100 mm cell culture dishes in the appropriate medium and infected with 20 MOI of AdNgn3-EGFP and Ad- EGFP, respectively, for 4 hr and then the medium was changed with fresh one. The cells were maintained in culture for two days.
3.20.2 In Vivo Crosslinking and Lysis
For the ChiP-cloning experiments a modification of the EZ-ChiP kit has been used. To the cell plates prepared as above 540 μl of fresh 18.5% formaldehyde (final concentration is 1%) prepared dissolving 925 mg of paraformaldehyde into 4.8 ml of dH2O in presence of 35 μl of 1N KOH were added. The plates were gently swirled and incubated at room temperature for 10 min. After that 1 ml of 10X Glycine to each dish was added to quench unreacted formaldehyde and the plates incubated at room temperature for 5 min. The plates were placed on ice and the medium aspirated. The cells were washed twice with 10 ml of cold PBS. 1 ml cold PBS containing Protease Inhibitor Cocktail was added to the dish. The cells were then scraped into a microfuge tube and spun at 700 x g at 4°C for 2-5 min to pellet cells and the supernatant discarded. 1 ml of SDS Lysis Buffer containing Protease Inhibitor Cocktail was then added, the cell pellet resuspended and aliquoted between 300-400 μl per microcentrifuge tube. Cells in SDS Lysis Buffer at a cell concentration of 2 x 107 per ml were sheared with 4-5 sets of 10- second pulses on wet ice using a 60 SONIC Dismembrator (Fisher Scientific) equipped with a 2 mm tip and set to 30% of maximum power. The sheared cells were then spun down at a minimum of 10,000 x g at 4°C for 10 min to remove insoluble material and the supernatant collected in fresh microcentrifuge tubes in 100 μl aliquots. Each 100 μl aliquot contains ~2 x 106 cell equivalents of chromatin.
3.20.3 Immunoprecipitation (IP) of Crosslinked Protein/DNA
To at least 2 x 100 μl aliquots (experiment + negative control) 900 μl of Dilution Buffer containing Protease Inhibitor Cocktail were added. Sixty μl of Protein G magnetic beads for each IP were then added and the tubes incubated for 1 hour at 4°C with rotation. The cleared supernatants were separated from the Protein G magnetic beads using a magnetic separation rack. Ten μl (1%) of the supernatant were collected as Input
and to the remaining supernatant fractions 10 μg of rabbit anti-Ngn3 and 10 μg of normal rabbit IgG were added to the “experiment” and “negative control” tubes, respectively. The tubes were incubated overnight at 4°C with rotation. To recover the antibody/antigen/DNA complexes 60 μl of Protein G magnetic beads were added and the tubes incubated for 1 hour at 4°C with rotation. The magnetic beads were recovered using a magnetic separation rack as above and the beads were washed as follows by resuspending the beads in 1 ml each of the cold buffers in the order listed below and incubating for 5 min on a rotating platform followed by recovery of the supernatant using a magnetic separation rack:
a. Low Salt Immune Complex Wash Buffer, one wash. b. High Salt Immune Complex Wash Buffer, one wash. c. LiCl Immune Complex Wash Buffer, one wash. d. TE Buffer, two washes.
3.20.4 Elution of Protein/DNA Complexes
To each tubes (IPs and inputs) obtained as described above 100 μl of Elution Buffer were added mixed by flicking and incubated at room temperature for 15 min. The magnetic beads were pelleted using the magnetic separation rack and the supernatant collected. The steps were repeated with an extra 100 μl of Elution Buffer and the eluates combined.
3.20.5 Reverse Crosslinks of Protein/DNA Complexes to Free DNA
added and incubated for 30 min at 37°C. Four μl 0.5 M EDTA, 8 μl 1M Tris-HCl and 1 μl Proteinase K were added and incubated at 45°C for 2 h.
3.20.6 DNA Purification Using Spin Columns
One ml of Bind Reagent A was added to each 200 μl DNA sample tube (IPs and Inputs), and the sample/Bind Reagent A. mixture transferred to the Spin Filter in Collection Tube and centrifuged for 30 sec at a minimum of 10,000 x g. Five hundred μl of the Wash Reagent B were then added to the Spin Filter in Collection Tube and centrifuged for 30 seconds at a minimum of 10,000 x g. The Spin Filter was put into a clean Collection Tube, added with 50 μl of Elution Buffer C and centrifuged for 30 sec at a minimum of 10,000 x g.
3.20.7 Blunting reaction, ligation and bacteria transformation.
For blunting reaction in a sterile 200 μl tube were mixed:
Purified DNA 19 μl
10X Blunting Buffer 2.5 μl
1 mM dNTP Mix 2.5 μl
Blunting Enzyme Mix 1.0 μl
Sterile dH20 variable To 25 μl
The mixtures were incubated at room temperature for 30 min. Immediately after that the enzymes were inactivated by heating at 70°C for 10 min.
Blunted DNA 10 μl
10X T4 DNA ligase Buffer 2.0 μl
pCR-BLUNT vector 1.0 μl
T4 DNA ligase 1.0 μl
Sterile dH2O variable To 20 μl
The mixtures were incubated at 16°C for 16 h. The reaction was placed on ice and the transformation was performed as described above.
Single colonies from the transformed bacteria plates obtained as above were inoculated into 3 ml of LB medium containing 50 μg/ml of kanamycin, and grown with vigorous shaking for 12–16 h. The plasmid DNA was then extracted as described.
The purified plasmid DNA was quantified by use of a ND-1000 Spectrophotometer (NanoDrop Technologies).
3.20.8 Bioinformatics analysis.
About 400 ng of each mini-prep DNA samples were sequenced using the M13 forward (-20) (5´-GTAAAACGACGGCCAG-3´) and the M13 reverse (5´- CAGGAAACAGCTATGAC-3´) primers at the Brigham and Women’s DNA Core Sequencing Facility (Boston, MA, USA).
The sequences not belonging to the vector were used to perform BLAST analyses using the blastn that compares a nucleotide query sequence against a nucleotide
sequence database. The rat genome database (http://www.ncbi.nlm.nih.gov/genome/seq/BlastGen/BlastGen.cgi?taxid=10116) has been analyzed using the default filters setting. The output results were analyzed for sequences that span the 5’term in the proximity of transcription factors and that contain at least one DNA binding motif for the bHLH family of transcription factors (E-box, CANNTG).
3.20.9 Confirmation of the results
The sequences showing the presence of E-boxe(s) and localized at the 5’-term of transcription factors were confirmed by independent classical ChiP experiments. Approximately 2 x 107 AR42J cells were plated in three 100 mm cell culture dishes in the appropriate medium and infected with 20 MOI of AdNgn3-EGFP, 20 MOI of AdNeuroD and 20 MOI of AdEGFP, respectively, for 4 hr and then the medium was changed with fresh one. The cells were maintained in culture for two days. The DNAs and protein complexes were crosslinked and immunoprecipited using anti-Ngn3 and anti- NeuroD/BETA2 antibodies, respectively as described above. The purified DNA was analyzed by PCR using clone specific primer(s).
Following 35 cycles of amplification the PCR products were run on a 1% agarose gel and analyzed by ethidium bromide staining.