title: ASY1_ChIP_Rep2
organism: Arabidopsis thaliana
description: Protocols: Unopened floral buds at meiotic stage or leaf tissue at rosette stage were collected from 6-week old plants. Plant tissue was immediately snap frozen in liquid nitrogen and stored at -80oC. Not applicable Plant were grown in growth chambers under the following conditions: 60% humidity, long day conditions (16 hours light/ 8 hours dark) at 20oC. No treatment was applied. Not applicable 8g of tissue were ground in liquid nitrogen and resuspended in 4 tubes of 25 ml nuclear isolation buffer each (60mM Hepes pH 8.0, 1M sucrose, 5mM KCl, 5mM MgCl2, 5mM EDTA, 0.6% Triton X100, 0.4mM PMSF, protease inhibitors). Chromatin was crosslinked by adding 680ul of 37% formaldehyde and incubated for 25 minutes under rotation. Crosslinking was stopped with 1.7ml of 2M glycine for 25 minutes under rotation. The tubes were centrifuged for 25 minutes at 3,000g and the supernatant was resuspended in 1ml of EB2 buffer (0.25M sucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl2, 1% Triton X100, 1mM EDTA, 5mM BME, 0.1mM PMSF and protease inhibitors). The tubes were centrifuged and the supernatants were resuspended in 600ul of Nuclei Lysis Buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS, 0.1mM PMSF and protease inhibitors) and the solution was transferred to 2 tubes of 300ul solution each. Chromatin was sheared using a Bioruptor instrument (Diagenode) for 15 minutes at high power (alternating 30 seconds on and 30 seconds off). 200ul of ChIP Dilution Buffer (1.1% Triton X100, 1.1 mM EDTA, 20mM Tris-HCl pH 8.0, 167 mM NaCl and protease inhibitors) was added followed by another round of sonication 25 minutes at high power (alternating 30 seconds on and 1 minute off). The tubes were centrifuged at 4oC, 5,000rpm for 10 minutes and the supernatant was recovered. Chromatin immunoprecipitation was performed using a-ASY1 antibody or the pre-immune serum at a dilution of 1/160 overnight at 4oC and under rotation. Magnetic beads bound with the antibody and DNA were washed twice with a solution of Low Salt Wash buffer (150mM NaCl, 0.1% SDS, 1% Triton X100, 2mM EDTA, 20mM Tris-HCl pH 8.0), once with a solution of High Salt Wash buffer (500mM NaCl, 0.1% SDS, 1% Triton X100, 2mM EDTA, 20mM Tris-HCl pH 8.0) and once with a solution of LiCl Wash Buffer (250mM LiCl, 1% NP-40, 1% Deoxycholate, 1mM EDTA, 10mM Tris-HCl pH 8.0) with an incubation time of 5 minutes under rotation at 4oC for each step. The magnetic beads were then washed with TE buffer (10mM Tris-HCl pH 8.0, 1mM EDTA) and the DNA was eluted with Elution Buffer (100mM NaHCO3, 1% SDS). 400ul of DNA solution is recovered and 20ul of 5M NaCL was added and the solution was incubated at 65oC overnight for reverse-crosslinking. Phenol chloroform extraction, AMPure XP (Beckman Coulter) for DNA extraction and purification. Approximately 10 ng of purified DNA was used to generate a library using the TruSeq prep kit v2 (Illumina).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-05-18T04:06:27Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-01-20T15:29:10Z
attributes.tag: External Id
attributes.value: SAMEA6492183
attributes.tag: INSDC center name
attributes.value: University of Cambridge, Department of Plant Sciences
attributes.tag: INSDC first public
attributes.value: 2020-05-18T04:06:27Z
attributes.tag: INSDC last update
attributes.value: 2020-01-20T15:29:10Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8705:ASY1_ChIP_Rep2
attributes.tag: age
attributes.value: 6
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: thale cress
attributes.tag: developmental stage
attributes.value: meiotic-stage
attributes.tag: ecotype
attributes.value: Columbia
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: organism part
attributes.value: flower bud
attributes.tag: sample name
attributes.value: E-MTAB-8705:ASY1_ChIP_Rep2
attributes.tag: scientific_name
attributes.value: Arabidopsis thaliana