GSM1536379: WT_NC13-pSer5; Drosophila melanogaster; ChIP-Seq
150 embryos (NC12 and NC13) or 100 embryos (NC14 E, M, or L) were hand selected on the basis of nuclear morphology and density following 15’ cross linking in 1% formaldehyde/Heptane/PBS/0.5% Triton X-100. Samples were homogenized in 1% RIPA buffer plus protease and phosphatase inhibitors and 1mM DTT. Cleared nuclear pellets were sonicated for 7x 20 seconds to shear chromatin to ~300 bp average size followed by overnight immunoprecipitation and 1h complex capture on Protein G dynabeads. Following two low and high salt washes, purified complexes were eluted, RNase treated, and cross links were reversed in the presence of Proteinase K. DNA was purified via Qiagen spin column. NEBNext ChIP Seq library master mix kit for Illumina was used according to the manufacturer’s specifications, using 1 ng input chromatin or the entire ChIP sample for library preparation. Ampure beads were used for size-selection prior to PCR amplification with barcoding primers. Mean library fragment size was ~270 bp. Libraries were sequenced on an Illumina HiSeq 2500 with read length of 67 bp except for mei-41 samples which were sequenced at 141 bp.
GEO Accession
GSM1536379