title: GSM1701080: ChIP-Seq analysis of TRB1 associated sequences_(Wt(-)_anti-TRB1); Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: The ChIP assay was performed as described Bowler et al. (2004) with modifications. Chromatin extracts were prepared from seedlings treated by 1% formaldehyde. The chromatin from isolated nuclei was sheared to an average length of 250-500 bp by sonication (Bioruptor, Diagenode) and spun. Matrix GFP-TRAP_A (Chromtec) was blocked against non-specific interaction with 200 mM ethanolamine, 1% BSA and TR10-24-G and TR10-24-C (i.e. DNA sequences, that are not recognised by TRB proteins (Schrumpfova et al., 2004). The pretreated matrix was incubated with chromatin diluted with ChIP dilution buffer (16.7 mM Tris-HCl, pH=8,0, 1.2 mM EDTA, 167 mM NaCl, 0.1% TritonX-100, PMSF and protease inhibitors) for 4°C / 4h and consequently washed with low salt; high salt; LiCl; TE buffers (in contrast to Bowler et al. (2004) levels of detergents (TritonX-100, NP-40 and sodium deoxycholate) were lowered to 0.1%). The cross-linking was reversed by 0.2 M NaCl O/N and followed by proteinase K (Serva) treatment, phenol/chlorophorm extraction and RNase A (Serva) treatment according to Bowler et al. (2004). Fifty microliters of immunoprecipitated DNA (ranging from 0.2-6 ng DNA (measured by Qubit dsDNA HS Assay Kit (Invitrogen)) was used for library preparation using NEBNext ChIP Seq Liabrary Prep Master Mix (NEB), with Agencourt XP beads (Beckman) in the ratios described in the protocol only with one exception: the Adapter was diluted 1:17 in water. DNA fragments were selected by 2% e-gel (Invitrogen) in the 270-300bp size range. Subsequently, a PCR reaction (18 cycles) with indexed Primers 1-11 from NEBNext Multiplex Oligo set (NEB) was performed. Libraries were sequenced (50 bp single-end reads) on an Illumina HiSeq 2500 using TruSeq SBS Kit v3-HS (Illumina) sequencing reagents.  The quality of the final libraries was checked on the Bioanalyzer (Agilent) and the quantity was checked with Qubit dsDNA HS Assay Kit (Invitrogen). The libraries were pooled equimolar and diluted to 10 pM (denaturation in NaOH) in Hyb Buffer 1 (HT1) from TruSeq SR Cluster Kit v3- cBot HS (Illumina).
identifiers.namespace: GEO
identifiers.id: GSM1701080