alias: GSM3678999
title: GSM3678999: ChIP-Seq_H3K4me_DMSO; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were cross-linked in culture medium with 1% formaldehyde in PBS and the reaction was stopped after 10 min at RT by adding 0.125 M glycine for 5 min at 4°C. The cells were washed twice with PBS and collected by centrifugation. Pellets were stored at -80° in SDS buffer (50 mM Tris•HCl pH 8.1, 0.33% SDS, 150mM NaCl, 5 mM EDTA, and protease inhibitor cocktail) or directly processed. Fixed cells were resuspended in IP buffer (100mM tris ph 8.6 0.3% SDS 1.7% TRITON x-100 and 5mM EDTA). Chromatin was then fragmented to obtain ~300 bp in average size length by using a Branson Sonifier 250. Chromatin pre-clearing was obtained with protein A-sepharose beads (Amersham). Then, the supernatant was immunoprecipitated overnight in the presence of 30-50 µl of protein G magnetic beads. For histone modification 1 ml corresponding to 3x106 cells per each IP and 4ug/ml primary antibody were used; for LSD1, GFI1 and PML Chip-Seq 40x106 cells per each IP; 10ug/ml. Before IP 2.5% of input was stored prior to the decrosslinking procedure. Decrosslinking was performed for all the IP samples and corresponding inputs, overnight in 0.1%SDS and 0.1% NaCOH3. The day after, the enriched DNA was treated with proteinase K at 56°C for 40 min and purified with a DNA purification kit (Qiagen). The obtained DNA was then quantified by picogreen and processed for ChIP-Seq library preparation (as described for the Illumina protocol). For libraries preparation, 2ng of immunoprecipitated DNA was used.
identifiers.namespace: GEO
identifiers.id: GSM3678999