GSM2046085: Mud1 rep1 ChIP-nexus in T4V1-26 mutant; Saccharomyces cerevisiae BY4741; OTHER
For NET-seq frozen cells were cryogenically pulverized for lysis and nascent RNA was isolated via an immunoprecipitation at 4°C of Rpb3-3xFLAG and purified using the miRNeasy kit (Qiagen, 217004). For ChIP-nexus cells were crosslinked in 1% formaldehyde, lysed by bead beating, and sonicated. Chomatin was isolated via immunoprecipitation using phospoh-CTD antibodies or TAP tagged subunits. For NET-seq nascent immunoprecipitated RNA was fragmented by alkaline hydrolysis followed by size selection after electrophoresis on a polyacrylamide gel.RNA was treated with polynucleotide kinase (NEB) followed by ligation to a preadenylated RNA linker. The remainder of the library construction was done as described inte NET-seq library construction. All samples were sequenced using an Illumina NextSeq 500 in 75 base pair reads. For ChIP-nexus the library constrution was preformed as desribed in He, Q., Johnston, J., Zeitlinger, J. (2015) "ChIP-nexus enables improved detection of in vivo transcription factor binding footprints." Nature Biotechnology. Samples were sequenced on an Illumina MiSeq in 60 bp single end reads.
GEO Accession
GSM2046085