GSM3616007: Input lin35 26C rep1; Caenorhabditis elegans; ChIP-Seq
Crosslinked samples were resuspended in FA buffer, and sonicated in a Diagenode Bioruptor (60 pulses of 30 seconds at full power with 1 minute rest in between).  Extracts were clarified and protein concentrations determined using a Bradford reagents.  Histone modifications were individually IPed using 1 μg appropriate antibodies on prepared lysates. ChIPs were performed with 0.5 mg of extract, and 2% of the extract was set aside for an input reference control.  Two methods were used for ChIP, see Sample section above to match with sample.  Method 1: ChIPs were incubated overnight at 4°C with 1% sarkosyl. Mouse IgG Dynabeads equilibrated in 100 μL FA buffer were added and incubated for 2 hours at 4°C. ChIPs were washed with the following buffers: twice with FA buffer, once with FA buffer containing 1 M NaCl, once with FA buffer containing 0.5 M NaCl, once with TEL buffer (10 mM Tris-HCl pH 8.0, 0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA), and twice with TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA). 2 elutions of 150 μL elution buffer containing TE plus 1% SDS and 250 mM NaCl were incubated at 65°C. Eluted ChIP and input samples were incubated with RNAse One for 30 min at 37°C and then proteinase K for 1 hour at 55°C. Crosslinks were reversed overnight at 65°C. DNA was purified using a Qiagen PCR clean-up column and eluted in 25 μl of water.  Method 2: ChIPs were performed using an IP-Star Compact Automated System (Diagenode) according to the manufacturers' instructions with the following settings and reagents: the “indirect method” was used with 100 μL reaction, 80 μL Mouse IgG Dynabeads equilibrated in FA buffer; IP for 12 hr, bead incubation for 2 hr; 10 minute washes at middle speed with FA buffer containing 1 M NaCl, FA buffer containing 0.5M NaCl, TEL buffer, and TE buffer. Samples were eluted in 100 μL Elution Buffer. Eluted samples were incubated with RNAse One for 30 min at 37°C and then proteinase K for 1 hour at 55°C. Crosslinks were reversed overnight at 65°C. DNA was purified by phenol chloroform extraction, ETOH precipitated, and DNA resuspended in 20 μl of water. Two methods were used for library construction, see Characteristics section.  Method 1: Both ChIP and input DNA samples had the ends blunted and phosphorylated with KlenowDNAP, T4 DNAP, and T4 PNK. 3'-dA overhangs are then added with Klenow Fragment (3' to 5'exo minus), and Illumina adapters were subsequently ligated to the ends. Next, PCR amplification was performed using Illumina primers, for a total of 16 cycles. Amplified DNA libraries were size-selected on a 2% agarose gel so that fragments sized between 250-350bp were obtained; gel extraction was carried out at room temperature. Method 2: The NEBNext Ultra DNA library Prep Kit (NEB) was used following the manufacturers' instructions.  1 ng of starting DNA was used, adapters were diluted 1:40, and AMPure beads were used for size selection before amplification to enrich for fragments corresponding to a 200 bp insert size.
GEO Accession
GSM3616007