Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Fresh-frozen cancer and normal tissues were dissected using a razor blade in liquid nitrogen to obtain ~5mg sized pieces (~ 5 ml by apparent volume). Tissue pieces were fixed in 1% formaldehyde/TBSE buffer for 10 minutes (min) at room temperature. Fixation was stopped by addition of glycine to a final concentration of 125 mM. Tissue pieces were washed 3 times with TBSE buffer, and transferred into Lysonator cartridges (SG Microlab Devices, Singapore). Tissues were dissociated following the manufacturer’s guidelines (4K Hz for 3 min), and taken directly to the lysis step in the Nano ChIP assay. Dissociated tissues were lysed in 200 ml of lysis buffer and divided into two 1.5 ml tubes for sonication (6 min) using a Bioruptor (Diagenode). 5 CHiPs were performed on individual chromatin preparations using the following antibodies: H3K4me3 (07-473, Millipore); H3K4me1 (ab8895, Abcam); H3K27ac (ab4729, Abcam); H3K36me3 (ab9050, Abcam); H3K27me3 (07-449, Millipore). Amplified DNA was digested with BpmI (New EngliandBiolabs), ligated to a 2ndBpmI adaptor and digested again to trim the WGA primer regions and semi-random priming ends. 15 ng of amplified DNA was used for each Illumina sequencing library using the ChIPseq kit (Illumina). Each library was sequenced on one lane of HiSeq2000 to obtain either 36- or 101-base single reads.