title: GSM4333354: NS263_WT_ChIP_H2AZ; Trypanosoma brucei brucei; ChIP-Seq
library_construction_protocol: In brief, 2 x 10e8 cells were harvested, crosslinked in 1% formaldehyde, lysed using 200 µM digitonin (final concentration) and chromatin was fragmented using 1 U/µl MNase (Sigma-Aldrich). see Wedel et al. 2017 #1 (DOI: 10.15252/embj.201695323) and Wedel et al. 2017 #2 (10.1016/j.exppara.2017.03.003 ) Immunoprecipitation was performed using Dynabeads M-280 sheep anti-rabbit IgG (Invitrogen) coupled to 10 µg polyclonal affinity-purified H2A.Z rabbit antibody. Bound material was washed and eluted, and cross-links were reversed at 65 °C for ~11 h in the presence of 300 mM NaCl (final concentration). ChIP-seq libraries were constructed using 35 ng of immunoprecipitated or input DNA for sequencing on an Illumina platform. Following blunt-ending and poly(A)-tailing, Y-shaped adapters (Ford et al., 2014) containing the Illumina Truseq-Adapter-sequences are ligated to each end of a DNA molecule. The library concentrations were determined in duplicates using Qubit dsDNA HS Assay Kit (Invitrogen, cat. no. Q32854) for Qubit 2.0 Fluorometer (Invitrogen, cat. no. Q32866). ChIP-seq libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems, cat. no. KK4824) according to the manufacturer's instruction and sequenced in paired-end mode with 2 x 76 cycles using an Illumina NextSeq 500 platform.
identifiers.namespace: GEO
identifiers.id: GSM4333354