title: GSM8861684: Serie5, BlaER1 WT Lymphocytes, Input, REP3; Homo sapiens; ChIP-Seq
library_name: GSM8861684
library_construction_protocol: Cells were collected and fixed in 1% formaldehyde for 10 minutes at room temperature. The reaction was quenched using glycine at a final concentration of 125mM for 5 minutes. Cells were washed twice in ice cold PBS. For fragmentation by sonication, cells were lysed in nuclei extraction buffer (250mM sucrose, 10mM Tris-HCl, 10mM MgCl2, 1% Triton X-100, 5mM 2-mercaptoethanol, and cOmplete protease inhibitor cocktail (Roche)) for 10 minutes on ice. The extracted nuclei were resuspended in nuclei lysis buffer (50mM Tris-HCl, 10mM EDTA, 0,2% SDS, and cOmplete protease inhibitor cocktail (Roche)), and sonicated with a Bioruptor Pico (Diagenode) using 6 cycles (30 sec on, 30 sec off), until chromatin fragments reached a size of around 300 base pairs (bp). For fragmentation by Micrococcal Nuclease (Mnase) digestion, cells were lysed with SimpleChIP® Enzymatic Cell Lysis Buffers A & B (Cell signaling) following manufacturer's instructions. Nuclei were incubated with Mnase (Cell signaling) for 20 minutes at 37°C, until the chromatin fragments were digested to the size of 150 to 450 bp. ChIP-seq libraries were prepared using Diagenode MicroPlex kit following manufacturer's instructions