cell type: Diffuse large B cell lymphoma cell line chip antibody: RNA PolII cell line: SUDHL16
Treatment protocol
None
Growth protocol
cells were grown in RPMI1640 medium with 10%FBS, 1mM L-Glutamine and Penicillin and Streptomycin
Extracted molecule
genomic DNA
Extraction protocol
For analysis of Histone modifications, native ChIP without formaldehyde crosslinking was performed followed by micrococcal nuclease digestion of the chromatin to obtain mononucleosomes. Briefly, 20x106 cells were lysed with the buffer (Tris-HCl pH7.6-50mM, CaCl2-1mM, TritonX-100-0.2%, Sodium Butyrate-5mM, PMSF-0.5mM, Protease Inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA)-20ul) on ice and digested with micrococcal nuclease 1Unit (Sigma-Aldrich, St. Louis, MO, USA) in the same buffer at 37°C for 6min. The reaction was stopped by addition of Tris-HCl pH7.6 and EDTA to a final concentration of 10mM and 5mM respectively. The cell lysate was then sonicated (Intensity -3, Pulse on-20”, Pulse off- 2’, Number of pulses-6) using Fisher F550 Sonic dismembrator. The chromatin was then dialyzed into RIPA buffer (10mM Tris pH7.6, 1mMEDTA, 0.1%SDS, 0.1% Sodium deoxycholate, 1% TritonX-100) for 2hours at 4°C and proceeded to the immuno precipitation after saving 10% of the dialysate as input. For analysis of PolII binding, 100x106 cells were cross-linked with formaldehyde (1% final concentration) for 10min and then quenched with Glycine (0.125M final concentration). The cells were washed with ice cold PBS and lysed using 5ml of the lysate buffer (1%SDS, EDTA-10mM, Tris HCl pH8.1-50mM, PMSF-1mM) on ice for 10 min. Chromatin was solubilized by performing sonication (Intensity-5, Pulse on-30”, Pulse off-1’, Number of pulses- 15) to break down the DNA to a size ranging from 200-400bp. The chromatin was then dialyzed overnight into RIPA buffer at 4°C. Chromatin equivalent to 20x106 cells was used for one ChIP experiment and 10% of it was saved as input. Chromatin immunoprecipitation was performed overnight at 4°C using specific antibody (4ug) bound protein A Dynabeads (Dynal Biotech, Oslo, Norway). The antibody- chromatin complexes were washed with RIPA buffer, High salt RIPA buffer (10mM Tris pH7.6, 1mMEDTA, 0.1%SDS, 0.1% Sodium deoxycholate, 1% TritonX-100, 300mM NaCl), Lithium chloride buffer (0.25M LiCl, 0.5% NP40, 0.5% Sodium Deoxycholate), TE+0.2% TritonX-100 and TE buffer. The DNA was eluted by incubation of the beads at 65°C in the presence of SDS and simultaneous proteinase K (Roche diagnostics) digestion. The ChIP DNA was validated for enrichment by real time quantitative-PCR amplification (primer sequences shown in the supplementary data) and the enriched input and ChIP DNA samples were later amplified.The ChIP DNA was amplified by using the LMPCR technique as described previously (Oberley et al., Methods Enzymol 376:316, 2004) with a few modifications. The ChIP DNA obtained was repaired using the DNA End Repair Kit (Epicentre Biotechnologies). dATP was added to the 3’ ends of ChIP DNA by incubating with dATP and Taq DNA polymerase (New England Biolabs) at 70°C for 30 min. The ChIP DNA was then ligated to unidirectional adaptors. The sequences of the oligonucleotides used as adaptors are as follows: Adaptor 3a-forward 5’- TCGGTAACGACTCACTTGGGTTCCT-3’ and Reverse 5’- GGAACCCAAGTG-3’, Adaptor 3b-forward 5’-TGGCAATTCCCTCACTAAAGGGCT-3’ and Reverse-5’-GCCCTTTAGTGA. The adaptors were annealed and ligated to the ChIP DNA by incubating ChIP DNA with the adaptors in excess at 16°C overnight using T4 DNA ligase (New England Biolabs). The ChIP DNA was then amplified by PCR using the following protocol: 1 cycle of 55°C 2 min, 72°C 5 min, 95°C 2 min; 30-35 rounds of amplification at 95°C 0.5 min, 55°C 0.5 min and 72°C 1 min; and final extension at 72°C for 4 min. The resulting LMPCR-amplified ChIP DNA was then purified using PCR purification kit (Qiagen) and validated for enrichment by real-time PCR to verify that the enrichment ratios are retained. The ChIP DNA was then hybridized to a custom oligonucleotide chip (NimbleGen Systems, Madison, WI).
Label
Cy5
Label protocol
1µg each of control Input and experimental ChIP DNA were labelled by Roche-Nimblegen using Cy3-random heptamer and Cy5-random heptamer, respectively, according to manufacturer's protocols. (http://www.nimblegen.com/products/lit/lit.html).
chip antibody: None, Input DNA cell type: Diffuse large B cell lymphoma cell line cell line: SUDHL16
Treatment protocol
None
Growth protocol
cells were grown in RPMI1640 medium with 10%FBS, 1mM L-Glutamine and Penicillin and Streptomycin
Extracted molecule
genomic DNA
Extraction protocol
For analysis of Histone modifications, native ChIP without formaldehyde crosslinking was performed followed by micrococcal nuclease digestion of the chromatin to obtain mononucleosomes. Briefly, 20x106 cells were lysed with the buffer (Tris-HCl pH7.6-50mM, CaCl2-1mM, TritonX-100-0.2%, Sodium Butyrate-5mM, PMSF-0.5mM, Protease Inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA)-20ul) on ice and digested with micrococcal nuclease 1Unit (Sigma-Aldrich, St. Louis, MO, USA) in the same buffer at 37°C for 6min. The reaction was stopped by addition of Tris-HCl pH7.6 and EDTA to a final concentration of 10mM and 5mM respectively. The cell lysate was then sonicated (Intensity -3, Pulse on-20”, Pulse off- 2’, Number of pulses-6) using Fisher F550 Sonic dismembrator. The chromatin was then dialyzed into RIPA buffer (10mM Tris pH7.6, 1mMEDTA, 0.1%SDS, 0.1% Sodium deoxycholate, 1% TritonX-100) for 2hours at 4°C and proceeded to the immuno precipitation after saving 10% of the dialysate as input. For analysis of PolII binding, 100x106 cells were cross-linked with formaldehyde (1% final concentration) for 10min and then quenched with Glycine (0.125M final concentration). The cells were washed with ice cold PBS and lysed using 5ml of the lysate buffer (1%SDS, EDTA-10mM, Tris HCl pH8.1-50mM, PMSF-1mM) on ice for 10 min. Chromatin was solubilized by performing sonication (Intensity-5, Pulse on-30”, Pulse off-1’, Number of pulses- 15) to break down the DNA to a size ranging from 200-400bp. The chromatin was then dialyzed overnight into RIPA buffer at 4°C. Chromatin equivalent to 20x106 cells was used for one ChIP experiment and 10% of it was saved as input. Chromatin immunoprecipitation was performed overnight at 4°C using specific antibody (4ug) bound protein A Dynabeads (Dynal Biotech, Oslo, Norway). The antibody- chromatin complexes were washed with RIPA buffer, High salt RIPA buffer (10mM Tris pH7.6, 1mMEDTA, 0.1%SDS, 0.1% Sodium deoxycholate, 1% TritonX-100, 300mM NaCl), Lithium chloride buffer (0.25M LiCl, 0.5% NP40, 0.5% Sodium Deoxycholate), TE+0.2% TritonX-100 and TE buffer. The DNA was eluted by incubation of the beads at 65°C in the presence of SDS and simultaneous proteinase K (Roche diagnostics) digestion. The ChIP DNA was validated for enrichment by real time quantitative-PCR amplification (primer sequences shown in the supplementary data) and the enriched input and ChIP DNA samples were later amplified.The ChIP DNA was amplified by using the LMPCR technique as described previously (Oberley et al., Methods Enzymol 376:316, 2004) with a few modifications. The ChIP DNA obtained was repaired using the DNA End Repair Kit (Epicentre Biotechnologies). dATP was added to the 3’ ends of ChIP DNA by incubating with dATP and Taq DNA polymerase (New England Biolabs) at 70°C for 30 min. The ChIP DNA was then ligated to unidirectional adaptors. The sequences of the oligonucleotides used as adaptors are as follows: Adaptor 3a-forward 5’- TCGGTAACGACTCACTTGGGTTCCT-3’ and Reverse 5’- GGAACCCAAGTG-3’, Adaptor 3b-forward 5’-TGGCAATTCCCTCACTAAAGGGCT-3’ and Reverse-5’-GCCCTTTAGTGA. The adaptors were annealed and ligated to the ChIP DNA by incubating ChIP DNA with the adaptors in excess at 16°C overnight using T4 DNA ligase (New England Biolabs). The ChIP DNA was then amplified by PCR using the following protocol: 1 cycle of 55°C 2 min, 72°C 5 min, 95°C 2 min; 30-35 rounds of amplification at 95°C 0.5 min, 55°C 0.5 min and 72°C 1 min; and final extension at 72°C for 4 min. The resulting LMPCR-amplified ChIP DNA was then purified using PCR purification kit (Qiagen) and validated for enrichment by real-time PCR to verify that the enrichment ratios are retained. The ChIP DNA was then hybridized to a custom oligonucleotide chip (NimbleGen Systems, Madison, WI).
Label
Cy3
Label protocol
1µg each of control Input and experimental ChIP DNA were labelled by Roche-Nimblegen using Cy3-random heptamer and Cy5-random heptamer, respectively, according to manufacturer's protocols. (http://www.nimblegen.com/products/lit/lit.html).
Hybridization protocol
The labeled ChIP DNA was precipitated and hybridized to the custom arrays by Roche-Nimblegen according to the manufacturers instructions (http://www.nimblegen.com/products/lit/lit.html).
