ChIP DNA Sample Preparation Customers provide us with ChIP enriched, qPCR verified DNA, approximately 10ng in 30ul of water. Zymo Research (www.zymoresearch.com) can supply a ChIP DNA clean & concentrator kit.

ChIP DNA Sample Preparation 2. Perform End Repair 5’-AGTCTTGGATCGACTCT-3’ 3’-ACCTAGCTG-5’ Convert the overhangs from into phosphorylated blunt ends, using T4 polymerase & E. coli DNA Polymerase I large fragment (Klenow polymerase), & T4 polynucleotide kinase (PNK). P5’-AGTCTTGGATCGAC-3’ 3’-TCAGAACCTAGCTG-5’P The 3’ to 5’ exonuclease activity of these enzymes removes 3’ overhangs & the polymerase activity fills in the 5’ Overhangs. 3. Add ‘A’ Bases to the 3’ End of the DNA Fragments P5’-AGTCTTGGATCGAC-3’ 3’-TCAGAACCTAGCTG-5’P An ‘A’ base is added to the 3’ end of the blunt phosphorylated DNA fragments, using the polymerase activity of Klenow fragment (3’ to 5’ exo minus). This prepares the DNA fragments for ligation to the adapters, which have a single ‘T’ base overhang at their 3’ end P5’-AGTCTTGGATCGACA-3’ 3’-ATCAGAACCTAGCTG-5’P

ChIP DNA Sample Preparation 4. Ligate Adapters to the DNA Fragments P5’-AGTCTTGGATCGACA-3’ 3’-ATCAGAACCTAGCTG-5’P Adapters are ligated to the ends of the DNA fragments, preparing them to be hybridized to the flow cell P5’-AGTCTTGGATCGACA-3’ 3’-ATCAGAACCTAGCTG-5’P 5. Purify Ligation Products The products from the ligation are purified on a 2% agarose gel, to remove all unligated adapters, Remove any adapters that may have ligated to one Another, & select a size range of templates to go on The cluster generation platform Excise a band in the gel from the 200 +/- 25bp range. Also excise a gel slice from an empty well in the same size range, to be used as a negative control.

ChIP DNA Sample Preparation 6. Enrich the Adapter-Modified DNA Fragments by PCR PCR is used to selectively enrich those DNA fragments that have adapter molecules on both ends, & to amplify the amount of DNA in the library. The PCR is performed with two primers that anneal to the ends of the adapters P5’-AGTCTTGGATCGACA-3’ 3’-ATCAGAACCTAGCTG-5’P Denaturation P5’-AGTCTTGGATCGACA-3’ Amplify using the following PCR protocol: *30 seconds at 980C *18 cycles of: 10 seconds at 980C 30 seconds at 650C 30 seconds at 720C *5 minutes at 720C *Hold at 40C Primer Annealing 3’-ATCAGAACCTAGCTG-5’P Extension P5’-AGTCTTGGATCGACA-3’ 3’-NNNNNTCAGAACCTAGCTGTNN-5’ As a negative control, perform PCR amplification on the sample extracted from the empty well. 5’-NNTAGTCTTGGATCGACNNNNN-3’ 3’-ATCAGAACCTAGCTG-5’P

ChIP DNA Sample Preparation 7. Validate the Library The amount of starting material is very low (10ng), and after 18 cycles of PCR the yield could still be too low to see on a regular gel, even though it is enough for cluster generation. The more sensitive method of quality control analysis is to run the library on an Agilent 2100 Bioanalyzer, to check the size, purity and concentration. Can not use an OD260/280 ratio for concentration measurements, since this will not distinguish dsDNA from primers, and therefore cannot be used to validate the library. High MW Marker Fluorescent Units (FU) Sample Low MW Marker Fragment Size (bp) Ladder Sample