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GenomeTiling

ABSTRACT: Samples were purchased from Clontech as mRNA. Hybridization material was generated through a random-priming amplification procedure (RP-AMP) using primers with a random sequence at the 3' end and a fixed motif at the 5' end. shDNP256 (first strand synthesis): 5'- TAG ATG CTG TTG NNN NNN NNN -3' shT7N9 (second strand synthesis): 5'- ACT ATA GGG AGA NNN NNN NNN -3' 1.5 g of mRNA was reverse-transcribed with Superscript II and the DP256 random primer for 20 minutes at 42 C (10 mM DTT, 50 mM Tris-HCl pH 8.3, 75 mM KCl, 8 mM MgCl2, 0.5 mM dNTPs, 5 U/ l Superscript II). The RNA was degraded with the addition of 20 l volume of 0.5N sodium hydroxide and 0.25 M EDTA for 20 minutes at 65 C. The single stranded cDNA was purified using a commercial kit (Qiagen Qiaquick). The resulting product of single stranded cDNA was placed in its entirety in a second strand reaction. Second strand synthesis reactions utilized shT7N9 random primer and the Klenow fragment of DNA polymerase utilizing standard reaction conditions (37 C for 60 minutes, 0.2 mM DTT, 2.1 mM Tris-HCl pH 7.9, 2.1 mM MgCl2, 10.7 mM NaCl, 1.07 mM dNTPs, 0.1U/ l Klenow), followed by another Qiaquick purification. Multiple polymerase chain reactions were run using 0.15 g of double stranded DNA and standard reaction conditions. Amplification was achieved using 10 cycles of PCR with the DP256 and T7 primers (20 mM Tris-HCl pH 8.4, 50 mM KCl, 0.01 mM dNTPs, 1.5 mM MgCl2, 0.01 U/ l Taq Polymerase) DP256: 5'- GTT CGA GAC CTC TAG ATG CTG TTG -3' T7: 5'- AA TTA ATA CGA CTC ACT ATA GGG AGA -3' followed by Qiaquick purification. Further amplification was achieved using in vitro transcription reactions with X0.5 g dsDNA and T7 RNA Polymerase (7.5 mM DTT, 40 mM Tris-HCl pH 7.5, 14.25 mM MgCl2, 10 mM NaCl, 2 mM Spermidine, 125 U/ml RNAguard, 2.5 mM dNTPs, 15 U/ml IPPase, 25kU/ml T7 Polymerase) for 16 hours at 42 C. The cRNA was purified (RNeasy) and reverse transcribed using Superscript and random 9-mers and amino-allyl dUTP (42 C for 20 minutes, 10 mM DTT, 50mM Tris-HCl pH 8.3, 75 mM KCl, 8 mM MgCl2, 0.5 mM dNTPs, 0.5 mM aa-dUTP, 5 U/ l Superscript II). The cDNA for each channel was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively, in 1M sodium bicarbonate, pH 9.0, followed by quenching with 4.0 M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were purified with a QIAquick PCR purification kit and the percentage dye incorporation and total cDNA yield was determined spectrophotometrically. Cy5 and Cy3 labelled cDNAs were combined and added to 2 mls 30% formamide with Herring Sperm DNA for hybridization to the microarray. Further details can be found in Castle et al., Genome Biol 2003;4(10):R66, http://genomebiology.com/2003/4/10/R66 , Roberts CJ et al., Science 2000, 287:873-880, and Schadt, Edwards, et al., unpublished. Normalization of single channel data is described in Ying, et al., "Identification of Chromosomal Regions Containing Transcribed Sequences Using Microarrays and Computational Methods" , 2003 Proceedings of the American Statistical Association, Biopharmaceutical Section [CD-ROM], Alexandria, VA: American Statistical Association, 2003. Briefly, an invariant set of probes was selected based on their intensity ranks. Then, a LOESS regression model was fit for Cy5 vs. Cy3 intensities for all probes in the invariant set. Finally, for all probes on the array the Cy5 intensities were normalized to the corresponding Cy3 intensities using prediction values from the regression model. Note that the normalized intensity values given here were used for all analyses reported in the accompanying paper. The ratio values reported here were not used for the analysis described in the manuscript and are not calculated from the normalized intensity values given below. Keywords: other

ORGANISM(S): Homo sapiens

PROVIDER: GSE1097 | GEO | 2004/03/04

SECONDARY ACCESSION(S): PRJNA87205

REPOSITORIES: GEO

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Project description:Samples were purchased from Clontech as mRNA. Hybridization material was generated through a random-priming amplification procedure (RP-AMP) using primers with a random sequence at the 3' end and a fixed motif at the 5' end. shDNP256 (first strand synthesis): 5'- TAG ATG CTG TTG NNN NNN NNN -3' shT7N9 (second strand synthesis): 5'- ACT ATA GGG AGA NNN NNN NNN -3' 1.5 g of mRNA was reverse-transcribed with Superscript II and the DP256 random primer for 20 minutes at 42 C (10 mM DTT, 50 mM Tris-HCl pH 8.3, 75 mM KCl, 8 mM MgCl2, 0.5 mM dNTPs, 5 U/ l Superscript II). The RNA was degraded with the addition of 20 l volume of 0.5N sodium hydroxide and 0.25 M EDTA for 20 minutes at 65 C. The single stranded cDNA was purified using a commercial kit (Qiagen Qiaquick). The resulting product of single stranded cDNA was placed in its entirety in a second strand reaction. Second strand synthesis reactions utilized shT7N9 random primer and the Klenow fragment of DNA polymerase utilizing standard reaction conditions (37 C for 60 minutes, 0.2 mM DTT, 2.1 mM Tris-HCl pH 7.9, 2.1 mM MgCl2, 10.7 mM NaCl, 1.07 mM dNTPs, 0.1U/ l Klenow), followed by another Qiaquick purification. Multiple polymerase chain reactions were run using 0.15 g of double stranded DNA and standard reaction conditions. Amplification was achieved using 10 cycles of PCR with the DP256 and T7 primers (20 mM Tris-HCl pH 8.4, 50 mM KCl, 0.01 mM dNTPs, 1.5 mM MgCl2, 0.01 U/ l Taq Polymerase) DP256: 5'- GTT CGA GAC CTC TAG ATG CTG TTG -3' T7: 5'- AA TTA ATA CGA CTC ACT ATA GGG AGA -3' followed by Qiaquick purification. Further amplification was achieved using in vitro transcription reactions with X0.5 g dsDNA and T7 RNA Polymerase (7.5 mM DTT, 40 mM Tris-HCl pH 7.5, 14.25 mM MgCl2, 10 mM NaCl, 2 mM Spermidine, 125 U/ml RNAguard, 2.5 mM dNTPs, 15 U/ml IPPase, 25kU/ml T7 Polymerase) for 16 hours at 42 C. The cRNA was purified (RNeasy) and reverse transcribed using Superscript and random 9-mers and amino-allyl dUTP (42 C for 20 minutes, 10 mM DTT, 50mM Tris-HCl pH 8.3, 75 mM KCl, 8 mM MgCl2, 0.5 mM dNTPs, 0.5 mM aa-dUTP, 5 U/ l Superscript II). The cDNA for each channel was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively, in 1M sodium bicarbonate, pH 9.0, followed by quenching with 4.0 M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were purified with a QIAquick PCR purification kit and the percentage dye incorporation and total cDNA yield was determined spectrophotometrically. Cy5 and Cy3 labelled cDNAs were combined and added to 2 mls 30% formamide with Herring Sperm DNA for hybridization to the microarray. Further details can be found in Castle et al., Genome Biol 2003;4(10):R66, http://genomebiology.com/2003/4/10/R66 , Roberts CJ et al., Science 2000, 287:873-880, and Schadt, Edwards, et al., unpublished. Normalization of single channel data is described in Ying, et al., "Identification of Chromosomal Regions Containing Transcribed Sequences Using Microarrays and Computational Methods" , 2003 Proceedings of the American Statistical Association, Biopharmaceutical Section [CD-ROM], Alexandria, VA: American Statistical Association, 2003. Briefly, an invariant set of probes was selected based on their intensity ranks. Then, a LOESS regression model was fit for Cy5 vs. Cy3 intensities for all probes in the invariant set. Finally, for all probes on the array the Cy5 intensities were normalized to the corresponding Cy3 intensities using prediction values from the regression model. Note that the normalized intensity values given here were used for all analyses reported in the accompanying paper. The ratio values reported here were not used for the analysis described in the manuscript and are not calculated from the normalized intensity values given below.

Project description:Glioblastoma stem cells (TS-543) were harvested and cross-linked with 1% formaldehyde for 10 mins at room temperature. The cells were lysed using SDS Lysis buffer for ChIP (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then sonicated for 25 cycles at 30% amplitude (15 s ON and 45 s OFF). The sonicated samples were then diluted in ChIP dilution buffer (0.01% SDS, 1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) and used for the immunoprecipitation with H3K27ac (Abcam, Ab4729), H2AZ (Active motive, cat#39113) and H3K27ac (Abcam cat#ab8580). After an over-night incubation with antibody, the bound DNA was washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1%deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) to remove non-specific sequences and eluted in the elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). Then the samples were reverse cross-linked using NaCl at 65°C overnight. The eluted DNA was purified and used for library preparation. Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.

Project description:Human Umbilical Vein Endothelial Cells pooled from several donors, were purchased from Lonza and cultured in FBS reduced Endopan 3 media. HUVEC cells of passages 5-10 were used for experiments. MPO treatment was performed with protein purified from human blood (Planta) and at 1 μg/ml final concentration. Immunoprecipitation was performed from isolated nuclei (after 8 hours MPO treatment), non-treated cells were used as negative control. For immunoprecipitation antibody against MPO (Calbiochem, 475915) was used. Cell nuclei were isolated by incubating cells for 15 min on ice in NIB buffer (15 mM Tris-HCL pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 250 mM sucrose) containing 0.3% NP-40. Nuclei were pelleted for 5 min 800 × g at 4 °C, washed twice in the same buffer, lysed for 10 min on ice in IP buffer (150 mM LiCl, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.5% Empigen) freshly supplemented with 2 mM sodium vanadate, 1× protease inhibitor cocktail (Roche), PMSF (10 µl), 0,5 mM DTT, and 50 units Benzonase per ml of IP buffer, before preclearing cell debris by centrifugation at >15,000 × g at 4 °C. Finally, 1 mg of the lysate was incubated with anti-MPO antisera overnight at 4 °C. Magnetic beads (Active Motif) were then washed once with 1× PBS-Tween and combined with the antibody-lysate mixture. Following a 2-h incubation at 4 °C, beads were separated on a magnetic rack and washed 5×, 5 min each in wash buffer (150 mM KCl, 5 mM MgCl2, 50 mM Tris-HCl pH 7.5, 0.5% NP-40) and another two times in wash buffer without NP-40. Captured proteins were predigested and eluted from the beads using digestion buffer (2 M Urea, 50 mM Tris-HCl pH 7.5, 1 mM DTT) supplemented with trypsin and eluted from the beads with elution buffer (2 M Urea, 50 mM Tris-HCl pH 7.5, 5 mM chloroacetamide) supplemented with trypsin and LysC, before subjected to mass-spectrometry on a Q-Exactive Plus Orbitrap platform coupled to an EASY nLC (Thermo Scientific). Peptides were loaded in solvent A (0.1% formic acid in water) onto an in-house packed analytical column (50 cm length, 75 µm I.D., filled with 2.7 µm Poroshell EC120 C1; Agilent)

Project description:We determined genome-wide nucleosome occupancy in mouse embryonic stem cells and their neural progenitor and embryonic fibroblast counterparts to assess features associated with nucleosome positioning during lineage commitment. Cell type and protein specific binding preferences of transcription factors to sites with either low (e.g. Myc, Klf4, Zfx) or high (e.g. Nanog, Oct4 and Sox2) nucleosome occupancy as well as complex patterns for CTCF were identified. Nucleosome depleted regions around transcription start and termination sites were broad and more pronounced for active genes, with distinct patterns for promoters classified according to their CpG-content or histone methylation marks. Throughout the genome nucleosome occupancy was dependent on the presence of certain histone methylation or acetylation modifications. In addition, the average nucleosome-repeat length increased during differentiation by 5-7 base pairs, with local variations for specific genomic regions. Our results reveal regulatory mechanisms of cell differentiation acting through nucleosome repositioning. For chromatin immunoprecipitation, for each sample, 1 x 106 cells were cross-linked with 1% PFA and cell nuclei were prepared using a swelling buffer (25 mM Hepes pH 7.8, 1 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1 mM DTT). Chromatin was sheared to mononucleosomal fragments. After IgG preclearance the sheared chromatin was incubated with 4 Âµg of either a H3K9ac (Abcam, ab4441), a H3K27ac (Abcam, ab4729), or a H3K9me3 (Abcam ab8898) antibody over night. After washes with sonication (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% N-lauroylsarcosine, 0.1% Na-deoxycholate), high-salt- (50 mM Hepes pH 7.9, 500 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS), lithium- (20 mM Tris-HCl pH 8.0, 1mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate) and 10 mM Tris-HCl, chromatin was eluted from the protein G magnetic beads and the crosslink was reversed over night. After RNase A and proteinase K digestion, the DNA was purified and subsequently cloned into a multiplexed Illumina library according to standard protocols. Sequenced 50 bp reads were mapped with bowtie and subsequently clustered with MACS 55 implemented in the Genomatix software suite (Genomatix, Munich, Germany) using a p-value of 10-5.

Project description:Identification and characterization of HP1BP3 (a human histone H1 homologue) as a novel chromatin retention factor essential for the co-transcriptional processing of pri-miRNA. We generated BAC transgenic cells at 80% confluency (~1x107) were cross-linked with 1% formaldehyde for 10 minutes at 37Â°C, and quenched with 125 mM glycine at room temperature for 5 minutes. The fixed cells were washed twice with cold PBS, scraped, and transferred into 1 ml PBS containing protease inhibitors (Roche). After centrifugation at 700 g for 4 minutes at 4Â°C, the cell pellets were resuspended in 100 Î¼l ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1] with protease inhibitors) and sonicated at 4Â°C with a Bioruptor (Diagenode) (30 seconds ON and 30 seconds OFF at highest power for 15 minutes). The sheared chromatin with a fragment length of ~200 â 600 bp) was centrifuged at 20,000 g for 15 minutes at 4Â°C). 100 Î¼l of the supernatant was used for ChIP or as input. A 1:10 dilution of the solubilized chromatin in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl 16.7 mM Tris-HCl [pH 8.1]) was incubated at 4Â°C overnight with 6 Î¼g/ml of a goat anti-GFP (raised against His-tagged full-length eGFP and affinity-purified with GST-tagged full-length eGFP). Immunoprecipitation was carried out by incubating with 40 Î¼l pre-cleared Protein G Sepharose beads (Amersham Bioscience) for 1 hour at 4Â°C, followed by five washes for 10 minutes with 1ml of the following buffers: Buffer I: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl; Buffer II: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl; Buffer III: 0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]; twice with TE buffer [pH 8.0]. Elution from the beads was performed twice with 100 Î¼l ChIP elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature (RT) for 15 minutes. Protein-DNA complexes were de-crosslinked by heating at 65Â°C in 192 mM NaCl for 16 hours. DNA fragments were purified using QiaQuick PCR Purification kit (QIAGEN) and eluted into 30 Î¼l H2O according to the manufacturerâs protocol after treatment with RNase A and Proteinase K.

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GenomeTiling

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