GSM5501184: CHIP_LSD1i; Homo sapiens; ChIP-Seq
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). 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 ~200 bp in average size length by using a Branson Sonifier 250. Chromatin pre-clearing was obtained with protein A-sepharose beads (Amersham).  For histone modification 250ug of chromatin were incubated with 2ug of primary antibody; for LSD1, 2mg of chromatin were incubated with 10ug of primary antibody. Before IP 2.5% of input was stored prior to the decrosslinking procedure. After overnight IP,  protein G magnetic beads have been added to the samples and incubated for 4 hours.  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 with Qubit and processed for ChIP-Seq library preparation (as described for the Illumina protocol). For libraries preparation, 2ng of immunoprecipitated DNA was used.