title: GSM2474304: MBD_rep2; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: 500 ug of inflorescence tissues were collected for each genotype and cross linked with 1% formaldehyde in water for 10 min with vacuum. Cross linking was stopped by adding glycine to a final concentration of 0.125M. Liquid Nitrogen was used to grind the tissues  to a fine powder, which was mixed to 30ml of Extraction Buffer 1 (0.4M sucrose, 10mM Tris-HCl pH 8, 10mM MgCl, 5mM BME, 0.1mM PMSF, 1 tab Protease inhibitors (Roche Biochemicals) per 50mls), mixed gently and let on ice for 15 min. The solution was filtered twice with Miracloth into a fresh 50ml falcon tube, and spinned for 20 minutes at 4000 rpm in a centrifuge at 4°C. The pellet was resuspended in 1ml of Extraction Buffer 2 ( 0.25M sucrose, 10mM Tris-HCl pH 8, 10mM MgCl2, 1% Triton X-100, 5mM BME, 0.1mM PMSF, Protease inhibitors (Roche Biochemicals, 1 mini tab per 5mls) and transferred to 1.5ml eppendorf tube, followed by 10 min centrifugation (12,000 g at 4 degrees). The resulting pellet was resuspend in 300ul of Extraction Buffer 3 (1.7M sucrose, 10mM Tris-HCl pH 8, 0.15% Triton X-100, 2mM MgCl2, 5mM BME, 0.1mM PMSF, protease inhibitor (Roche Biochemicals, 1 mini tab per 5mls), layered on top of 300ul of Extraction Buffer 3 in a clean tube and spin for 1 hour at 16,000 at 4 degrees. The chromatin pellet was then resuspended in 300ul of Nuclei Lysis Buffer (50mM Tris-HCl ph 8, 10mM EDTA, 1% SDS, Protease inhibitors (Roche Biochemicals, 1 mini tab per 5mls) and sonicated with a Diagenode Bioruptor machine (10 cycles, 30 seconds/1 minute ON/OFF), spinned at full speed for 5 minutes at 4 degrees to pellet debris, the supernatant collected to a fresh tube, and the chromatin solution brought to 3ml with ChIP Dilution Buffer (1.1% Triton X-100, 1.2mM EDTA, 16.7mM Tris-HCl pH 8, 167mM NaCl). 1ml of chromatin was added to new low-binding tubes and pre-cleared with 50ul of agorose beads for 1h on a rotating wheel. 1ml of pre-cleared chromatin was mixed with 80µl of equilibrated agarose beads and 3µg of anti-GFP antibody, and incubated overnight at 4°C on a ratting wheel. Beads were washed with the following sequence: twice 5min with Low Salt Wash Buffer (150mM NaCl, 0.1% SDS, 1% TritonX-100, 2mM EDTA, 20mM Tris-HCl pH8.1), twice 5min with High Salt Wash Buffer (500mM NaCl, 0.1% SDS, 1% TritonX-100, 2mM EDTA, 20mM Tris-HCl (pH8.1)), twice 5min with LiCl Wash Buffer (0.25 LiCl, 1% NP40, 1% sodium deoxycholate, 1mM EDTA, 10mM Tris-HCl pH 8.1), and twice 5min with TE Buffer:  (10mM Tris-HCl pH 8, 1mM EDTA). Immune complexes were eluted twice with 250ul of Elution Buffer (1% SDS, 0.1 M NaHCO3) by incubating 65 degrees for 15 minutes with gentle agitation (mix tubes during incubation), and combining both two eluates. 20ul 5M NaCl was added to the combined eluates, which were reverse-cross linked at 65°C overnight, and treated with 2ul of 10mg/ml proteinase K for one hour at 45°C.  Note:  Don't forget to get your total DNA out of the freezer and reverse X-link with other samples (Add 500 ul of Elution buffer + 20ul 5M NaCl). The resulting DNA fragments were purified using Diagenode ChIP purification kit (catalogue #C03010011), and resuspended in 50ul of distilled water. Library constructed using Illumina's TruSeq ChIP Sample Prep Kit, and following manufacturer's protocol.
attributes.tag: GEO Accession
attributes.value: GSM2474304