Ez magna chip a kit

ChIP assays were performed with the EZ-Magna Chip™ A kit (Millipore, Boston, MA, USA) following the manufacturer's instructions. A total of 1×10⁷ HUVECs co-cultured with A549 conditioned media or control conditioned media were cross-linked with 1% formaldehyde at room temperature for 10 minutes. Sonication was performed on ice to get 200 to1000 bp DNA fragments. The chromatin was then immunoprecipitated with anti-IgG antibody (Millipore, Boston, MA, USA) and anti-GATA3 antibody (Abcam, Cambridge, UK). After reverse cross-linking and DNA purification, DNA from input (1:10 diluted) or immunoprecipitated samples were assayed by semi-quantitative PCR. The primers amplifying the GATA binding motif on the vWF promoter are as follows: sense 5′-TGGGCGGCACCATTGT-3′, and antisense 5′- CATACCTTCCCCTGCAAATGA-3′. The PCR products were analyzed by agarose gel electrophoresis.

ChIP assays were performed using the EZ magna ChIP A kit (Millipore, Billerica, MD, USA) with a modified protocol. For small interfering (si)RNA treatment, 10⁷ AC1 cells in 10-cm dishes were transfected with mouse scrambled or ETV6 siRNAs for 48 h. Cells were cross-linked with 1% formaldehyde in culture medium at room temperature for 15 min and then quenched with the addition of 1 ml of 10× glycine. Cells were washed twice with cold phosphate-buffered saline (PBS) containing a protease inhibitor (Roche) and centrifuged at 10⁵ rpm. Cell pellets were resuspended in 0.5 ml of cell Lysis Buffer (BioRad, Hercules, CA, USA) and incubated on ice for 15 min. Nuclei were collected by centrifugation at 10⁵ rpm and 4 °C for 10 min and resuspended in nuclear lysis buffer. Genomic DNA was sheared by a microtip during sonication (Branson Sonifier 250, Germany) following 15 cycles of a 20-s burst then 1 min of cooling on ice. This procedure resulted in DNA fragments sized approximately 100~ 300 bp. Sheared chromatin was aliquoted to perform immunoprecipitation with a control immunoglobulin G (IgG) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or antibodies against ETV6 or Gapdh at 4 °C overnight. A qPCR was performed in triplicate with 2 μl of eluted chromatin. ChIP antibodies and PCR primers are listed in Additional file 1; Table S3.

Chromatin Immunoprecipitation (ChIP) assays were performed using EZ-Magna ChIP™ A kit (Millipore, Cat. no. 17–408) according to the manufacturer's instructions. Antibodies raised in rabbit against H3K4Me3, H3K9me3, H3K27me3, and H4K20me3 were purchased from Diagenode (Cat. no. pAb-003–050, pAb-056–050, pAb-069–050 and pAb-057–050, respectively). Rabbit IgG was provided with the ChIP kit. Cells were crosslinked in their culture vessel with the appropriate media containing 1% formaldehyde (Sigma, Cat. no. F8775) for 10 min at room temperature. Cells were sonicated at 4°C using a Diagenode Bioruptor. Cells (10⁶) were used for each IP and the recovered material was analyzed by real-time PCR using custom designed Taqman assays for LCT13, LCT14, and APRT (Table S3). GAPDH primers were provided in the kit.

3 × 10⁸ exponentially growing cells were fixed with 1% formaldehyde for 25 min at 30 °C. After quenching by 250 mM glycine, cells were harvested and washed with Buffer 1 (1 M Tris-HCl (pH 8.0), 167 mM NaCl, 1.2 mM EDTA, 1% TritonX-100, 0.1% Na-deoxycholate). Cells were resuspended in Buffer 1 supplemented with protease inhibitors cocktail (05892970001, Roche Applied Science) and homogenized with a bead-beater (FastPrep-24, MP, California, USA) by glass beads. The cell extract was sonicated for 15 min with a sonicator (Sonics & Materials, Connecticut, USA) and centrifuged. Supernatant was incubated with anti-HA (M20003L, Abmart, Shanghai, China), anti-H3K9me2 (07-441, Millipore, Massachusetts, USA), anti-Ago1 (ab18190, Abcam, Cambs, UK, Abcam), anti-RNA polymerase II 8WG16 (MMS-126R, Covance, New Jersey, USA), anti-FLAG (F1804-200UG, Sigma-Aldrich, St Louis, MO, USA) or anti-Chp1 (ab18181, Abcam) antibody for 4 hour. Samples were subjected to purification by using an EZ-Magna ChIP A Kit (17-408, Millipore). Eluted DNA was subjected to qPCR as described above. Primers used are listed in Supplementary Table S2.

ChIP assays were performed using the EZ-Magna ChIP™ A kit (Millipore) according to the manufacturer's protocol. Aliquots of chromatin immunoprecipitation lysates were diluted and incubated in the indicated antibody and protein A magnetic beads at 4°C for 2 h. The beads were separated using a magnetic separator and sequentially washed with low-salt buffer, high-salt buffer, LiCl Immune Complex Wash Buffer and TE Buffer. After de-cross-linking and proteinase K digestion, the DNA was cleaned using a spin filter. PCR was performed in the exponential linear zone of amplification using the following primers:
ZNF32 gene promoters, ⁻¹⁴⁰⁹ACA GCC GGT CTT GAC CTT AAC⁻¹³⁸⁸ (forward) and ⁻¹²¹³GGG GTG TTG CAC AGC TAA GTC⁻¹¹⁹³ (reverse); ⁻¹⁸⁵GCT GTG AGT CTC CGG ATT ACC AG⁻¹⁶³ (forward) and ⁺⁷⁵CGG CCT CAC TCA CCG CA⁺⁹¹ (reverse); C1QBP gene promoters, ⁻¹¹³³CAC AAG CAT GAG TTC CGA GCC⁻¹¹¹³ (forward) and ⁻⁹⁶⁶GTT GGG ATT GAA TTG GAG GAC AC⁻⁹⁴⁴ (reverse).