GSM1442895: e1a_WT_RB1_ChIP-seq; Homo sapiens; ChIP-Seq
24 h post infection (p.i.), formaldehyde was added for 10 min at 37°C. After PBS washing, cross-linked cells were scraped from the plates and washed with 1 ml of PBS containing protease inhibitors (Roche). 2x10^7 Cells were resuspended in 450 ul of lysis buffer and incubated for 10 min on ice and immediately sonicated using Misonix cup-horn sonicator. 100 ul of the lysate (corresponding to 5x10^6 cells) were used for each immunoprecipitation with a given antibody (listed below); 10 ul of the lysate was used as input. After overnight reversal of crosslinking at 65°C, samples were treated with RNase A for 30 min at 37°C, proteinase K for 2 h at 56°C. DNA was subsequently purified using phenol/chloroform extraction and precipitation. DNA concentration was measured using Qubit (Invitrogen). At least 10 ng of dsDNA for both input and IP was used for library preparation according to the manufacturer's instructions (NuGen). Sequencing libraries were constructed from 1 ng of immunoprecipitated and input DNA using the NuGen Ovation Ultralow DR Multiplex System 1-8 kit according to protocol.
GEO Accession
GSM1442895