title: GSM3677396: DNase-seq_EILP_rep3_GB7347; Mus musculus; OTHER
library_construction_protocol: For DNase I digestion, 300 cells from each cell type were collected by FACS sorting. 0.3 unit of DNase I (Roche, 04716728001) was added to each cell type and incubated at 37 °C for 5 minutes. Reaction were stop by adding 80 μl of stop buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.15% SDS, 10 mM EDTA) containing 1 μl of 20 mg/ml proteinase K. Samples were incubated at 55 °C for overnight and DNA purified by phenol–chloroform extraction, followed by precipitation with ethanol in the presence of 20 μg 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 HiSeq3000.
attributes.tag: GEO Accession
attributes.value: GSM3677396