GSM1217497: seq-SDQ0809_COH1_fem2_AD_Input_Rep1; Caenorhabditis elegans; ChIP-Seq
Worms are frozen, ground, crosslinked for 20 minutes in EGS, and for an additional 10 minutes in EGS plus formaldehyde. Later, washed pellets are resuspended in FA buffer and subjected to sonication in Bioruptor (1 hour of 30 seconds with 1 minute rests in between). Extracts are then spun down and soluble fraction is stored for quality tests and future ChIP. Worm extract from adult worms and 5 ?g of affinity purified antibody was used for ChIP. Dynal Protein A or G beads were used to recover the ChIPed DNA.  Samples were then treated with RNase A and Proteinase K, and reverse crosslinked at 65°C. ChIPed DNA was purified and then quantified using the Pico Green HS fluorescent assay. DNA for Library Prep is incubated with an End Repair Enzyme mix (NEB Klenow, T4 DNA polymerase and T4 PNK) to ensure blunt ends. It is then purified and incubated with Exo(-) Klenow fragment in the presence of dATP to add adenosine at the 3? ends (a single A-overhang for more efficient and directed ligation of the adaptors). After a second purification, the DNA fragments is ligated with single-end ?Homebrew? adaptors which contain an index sequence within (Corbin Jones? lab). After ligation, the samples are purified TWICE using SPRI beads, allowing for a size selection step getting rid of excess adaptors. Samples are then amplified by PCR with single end primers. Depending on the original fragmentation of the DNA, samples can either be purified by SPRI or by separation and purification from an agarose gel. Prepared sample are sequenced using Illumina GAII or HiSeq2000 at the High Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.
GEO Accession
GSM1217497