GSM1665612: input-L2; Caenorhabditis elegans; ChIP-Seq
Larvae were cross-linked at RT for 30 min in 2% formaldehyde (Fisher). Crosslinking was quenched with 0.25 M glycine for another 30 min at RT. For each immunoprecipitation, 10 mg 4H8 antibody was coupled to 50 ml Dynabeads (Invitrogen) coated with protein A and G (25 ml protein A plus 25 ml protein G). After sonication and centrifugation, 1.5 ml supernatant (around 30 mg protein) was pre-cleared by rotating for 2 hrs with 30 ml empty Dynabeads (15 ml protein A plus 15 ml protein G) before incubating with 4H8-coupled Dynabeads at 4°C overnight. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, 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 (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
GEO Accession
GSM1665612