title: GSM2813775: sh-LSD1_LSD1; Homo sapiens; ChIP-Seq
library_construction_protocol: For ChIP, lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. For total RNA extraction, cells in culture were directly lysed in TRIzol (Life Technologies, cat#15596018) after medium removal. RNA extraction was performed according to manufacture's instructions Cell nuclei were obtained by lysing whole cells in hypotonic buffer (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% v/v glycerol, 1 mM DTT, and 0.1% v/v Triton X-100) supplemented with protease inhibitor. After washing with PBS, nuclei were fixed in 1% formaldehyde for 10 min at room temperature, followed by quenching in 125 mM glycine. Nuclei were then washed twice with ice-cold PBS, lysed in ChIP sonication buffer (50 mM HEPES pH7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.2% SDS) supplemented with protease inhibitor, and were subjected to sonication to obtain DNA fragments of 300-800 bp. Subsequent procedures were carried out by following the Epigenesys protocol (https://www.epigenesys.eu/en/). The following antibodies were used to ChIP: anti-LSD1 (Abcam, cat# ab17721) and anti-H3K4me2 (EMDMillipore, cat#07-030). ChIP-Seq libraries were prepared using NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (New England Biolabs, cat#E7370L) and NEBNext® Multiplex Oligos for Illumina® (New England Biolabs, cat# E7335L) according to manufacture's instructions. Library concentrations were quantified by Qubit (Invitrogen) and mixed equally for sequencing at HiSeq 2500 (Illumina) to generate 50 bp reads from single-end. ||Purified total RNA was quantified by Qubit (Invitrogen) and analyzed by Agilent Bioanalyzer to assess RNA integrity. 1 μg RNA (RIN>9) was used to generate rRNA-depleted RNA with NEBNext® rRNA Depletion Kit (New England Biolabs, cat# E6310S) according to manufacturer's instructions. The rRNA-depleted RNA was subjected to Agilent Bioanalyzer to ensure the complete removal of rRNA, and then used to generate directional RNA library with NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, cat# E7760L) and NEBNext® Multiplex Oligos for Illumina® (New England Biolabs, cat# E7335L) according to manufacturer's instructions. Library concentrations were quantified by Qubit (Invitrogen) and mixed equally for sequencing at HiSeq 2500 (Illumina) to generate 50 bp reads from paired-ends.
attributes.tag: GEO Accession
attributes.value: GSM2813775