P-GSE24241-3 - P-GSE24241-3
nucleic acid library construction protocol
Worm_chromatin_immunoprecipitation_vKI2. Antibody was incubated with protein A/G beads at 4 oC for at least 1 hr to prepare antibody-beads complex. For each reaction, 0.5-2 mg of protein extract was taken and sarkosyl was added to 1% final concentration. The extract was precleared by incubating with protein A/G beads for 30 min at 4 oC. Before immunoprecipitation, 10% of extract was taken and treated with RNase. The extract was mixed with the antibody-beads complex and incubated overnight at 4C. After washing beads, immune complexes were eluted from beads twice with 150uL elution buffer (1% SDS in TE with 250 mM NaCl) for 15minutes at 65C. Samples were treated with proteinase K at 55C for 1-2hrs then transfer to 65C overnight to reverse crosslinks. DNA was cleaned up Qiagen PCR purification kit. For a detailed protocol see
. || Solexa_Library_Prep_vKI1. DNA was incubated with End Repair Enzyme mix (Epicentre, Madison) to ensure blunt ends and then with Exo(-) Klenow fragment in the presence of dATP to add adenosine at the 3\` ends. The DNA fragments were ligated with single-end adaptor (Illumina) and then amplified by PCR with single end primers. The amplicon was loaded into an agarose gel, and size-selected DNA was recovered from the gel.