GSM1607179: N440 RNA-seq_rep2; Homo sapiens; RNA-Seq
For DNase I digestion, 0.2 to 1 unit of Dnase I was added to the cells and incubated at 37°C for 5 minutes. The reaction was stopped by adding 80 ul of Stop Buffer (10mM Tris-HCl, pH 7.5, 10mM NaCl, 0.15% SDS, 10mM EDTA), 1 ul of 20mg/ml Proteinase K and 5 ul of 6ng/ul circular carrier DNA. The mixture was incubated at 55°C for 1 hour and DNA purified by Phenol-chloroform extraction, followed by precipitation with ethanol in the presence of glycogen. The library was prepared using Illumina kits as described G. Hu et al. (Nature immunology 2013). The libraries were amplified using a two-step method to preferentially amplify the small DNA fragments derived from the DNA hypersensitive sites and to reduce non-specific amplification of the carrier DNA. The first amplification was done with index primers with the PCR condition: 98°C, 10"; 67°C, 30"; 72°C, 30" for 6 cycles. The second amplification was done with the P5 and P7 primers with the condition: 98°C, 10"; 68°C, 30"; 72°C, 30" for 22 cycles. The fragments between 160bp to 300bp were isolated on E-gel and sequenced on Illumina HiSeq2000.
GEO Accession
GSM1607179