GSM1541816: 38-Per_1_K27me3; Homo sapiens; ChIP-Seq
For ChIP-seq, Input and IP DNA were extracted with Phenol:Chloroform. Libraries were prepared from 10-20 ng of IP ChIP DNA and 100-150 ng of input DNA according to Illumina’s instructions along with the ChIP-seq DNA Sample Prep Kit (IP-102-1001). Briefly, samples were checked for quality and concentration from 150-250 bp on a bioanalyzer. DNA was end-repaired using Klenow polymerase in 58 μL of reaction buffer. For IP DNA, Klenow was diluted 1:5. Samples were incubated at 20°C for 30 minutes and subsequently purified on QIAquick PCR purification columns. A-tails were then added to the DNA with Klenow and dATP in NEB buffer 2 at 37°C for 30 minutes and cleaned with Qiagen MiniElute PCR purification columns. Sequencing adapters were then ligated onto the DNA for 15 minutes at room temperature followed by cleaning with MiniElute columns. Samples were then run on 2% agarose gels and DNA from 216-366 bp (DNA plus adapters) were cut from the gel and purified with a Qiagen QIAquickGel Extraction kit. Concentrations were then checked on a bioanalyzer and 8 ng were PCR amplified with Phusion polymerase (Fisher) for 15 cycles (10 sec 98°C, 30 sec 65°C, 30 sec 72°C) followed by 5 minutes at 72°C. Samples were then cleaned with Ampure kits (Illumina) and washed with 80% ethanol. DNA samples were resuspended at the end of the cleanup into 17.5 μL buffer EB (Qiagen) and subjected to next generation sequencing on Illumina HiSeq platform according to manufacturers instructions.