alias: GSM4104414
title: GSM4104414: Nrd1 fusion FLAG ChIP-Seq rep2; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: Cells were lysed in FA buffer 0.5% SDS using glass beads. Cross-linked extracted were sonicated using a Misonix 300 with cup horns, with 10s on/off pulses for a total sonication time of 20 min. Soluble chromatin was recovered by centrifugation and protein concentration quantified by Bradford assay. A total of 700 ug of chromatin was used per immunoprecipitation. Where S.pombe “spike-in” was used, 10 % S.pombe chromatin prepared similarly was added to the immunoprecipitation. The indicated antibody and 10 ul of Protein-G sepharose beads were added, followed by overnight incubation at 4 C. Beads were washed once with FA buffer, 0.1% SDS, 275 mM NaCl, once with FA buffer, 0.1% SDS, 500 mM NaCl, once with 10 mM Tris-HCl, pH 8.0, 0.25 M LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% Na Deoxycholate, and once with TE (10 mM Tris-HCl, pH 8.0, 1mM EDTA). Immunoprecipitated material was eluted with 50 mM Tris-HCl 7.5, 10 mM EDTA, 1% SDS by incubating at 65 C for 20 min. Beads were washed once with TE, and the supernatant added to eluted material. Recovered immunoprecipitate was de-crosslinked with 40 ug of Pronase 1 hour at 42 C and overnight at 65 C. Samples were treated with 5 ug RNAse A and phenol-chloroform/chloroform extracted followed by precipitation with LiCl, 40 ug Glycogen, and 100 % EtOH. Precipitated DNA was resuspended in appropriate volume of water and quantified using Qubit assay. 1 ng of immunoprecipitated DNA was used for ChIP-Seq library production. Immunoprecipitated DNA was end repaired using T4 DNA polymerase, T4 PNK and DNA Polymerase I, Large (Klenow) fragment. A single adenosine “tail” was added to the 3' end of fragments using Klenow (3' to 5' exo minus) and adapters containing inline multiplexing barcodes were added using T4 Quick DNA ligase. Adapter ligated DNA fragments between 200 and 500 bp were gel purified and PCR amplified with 16 cycles. Library quality was assessed using Agilent 2100 Bioanalyzer. Equal molarity of each library was mixed.
identifiers.namespace: GEO
identifiers.id: GSM4104414
attributes.tag: GEO Accession
attributes.value: GSM4104414