title: GSM3061403: ko_6: LSD1 KO rep2; Mus musculus; RNA-Seq
library_construction_protocol: For MCF-7 cells, 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. For GFP-labeled B16 cells isolated from tumor-bearing immunocompetent mice, intact cells were used for the following steps. After washing with PBS, nuclei/cells were fixed in 1% formaldehyde for 10 min at room temperature, followed by quenching in 125 mM glycine. Nuclei/cells 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), anti-H3K4me1 (Abcam, cat#ab8895), anti-H3K4me2 (EMDMillipore, cat#07-030), and mouse IgG (Santa Cruz Biotechnology, cat# sc-2025). || For RNA extraction, cells weredirectly lyzed in TRIzol and total RNA was extracted according to manufacture's instructions. 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 the 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 from MCF-7 cells or GFP-labeled B16 cells isolated from tumor-bearing immunocompetent mice 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) or poly(A)+ RNA with Magnetic mRNA Isolation Kit (New England Biolabs, cat# S1550S) according to the manufacturer's instructions. The rRNA-depleted or poly(A)+ 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 the manufacturer's instructions. Library concentrations were quantified by Qubit (Invitrogen) and mixed equally for sequencing at HiSeq 2500 or Nextseq 500 (Illumina) to generate 50 bp reads (MCF-7) or 75 bp (B16) from paired-ends.
attributes.tag: GEO Accession
attributes.value: GSM3061403