GSM2843461: wt_CENP-ACnp1_2; Schizosaccharomyces pombe; ChIP-Seq
Cells were fixed with 1% formaldehyde (Sigma) for 15 min at room temperature and lysed using a bead beater (Biospec Products). Cell lysates were sonicated in a Bioruptor (Diagenode) (30/40 min, 30 s On and 30 s Off at 'High' (200W) position). For CENP-ACnp1 ChIPs, anti-CENP-ACnp1 serum was used with Protein G sephrose beads (Roche). For all other ChIPs, Protein G Dynabeads (Life Technologies) were used along with anti-Myc 9B11 (Cell Signaling) or anti-Flag M2 antibody (Sigma), as appropriate. Immunoprecipitated DNA was recovered using Qiagen Min-Elute PCR purification kit. ChIP-seq libraries were generated using an Illumina-based protocol with custom reagents using NEXTFlex-96 DNA Barcodes (Bioo Scientific). Briefly, immunoprecipitated DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt phosphorylated ends were treated with Klenow fragment (3' to 5' exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina adapters, which have a single 'T' base overhang at the 3' end. After adapter ligation, DNA fragments were size-selected using AmpureXP beads to remove excess adapters and adapter-modified DNA fragments were enriched by PCR amplification with Illumina primers for 12 cycles. The purified ChIP-seq libraries thus generated were validated and subsequently captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq2500/ HiSeq4000 system following the manufacturer's protocols.
GEO Accession
GSM2843461