title: GSM4478949: WT OE Rep3 ChIPseq; Myotis velifer; ChIP-Seq
library_construction_protocol: For each KRABINER expression construct, 2 × 107 cells were pelleted at 500xg for 5 min at room temperature and washed once with 1 mL of PBS + 1X Pierce Protease Inhibitors [Thermo Fisher]. Cells were then fixed with 1% formaldehyde in PBS + inhibitors for 10 minutes at room temperature, with rocking. The fixation reaction was quenched with 0.125M glycine solution for 5 minutes at room temperature, with rocking.  Pellets were washed twice with ice cold PBS + inhibitors and stored at -80C until use. Frozen fixed cell pellets were then resuspended in 2 mL lysis buffer (1 % SDS, 10 mM EDTA, 10 mM Tris-Cl pH 8.0, and 1X Pierce Protease Inhibitors [Thermo Fisher]) and sonicated to average fragment size of ~500-800 bp using a Diagenode Bioruptor. Sonicated chromatin was diluted in 1X TBST+PI (25 mM Tris-Cl pH 8.0, 0.15 M NaCl, 0.05% Tween-20, and 1X Pierce Protease Inhibitors [Thermo Fisher]). For input libraries, 3.33% of total sonicated chromatin from each was pooled across genotypes of the same KRABINER variant for total input material of ~10% respective to each individual IP. For each IP, 100 µL Pierce c-Myc magnetic beads (Thermo Fisher) were washed twice in ice cold TBST+PI and then added to sonicated lysate and incubated for 16 h at 4 °C. Beads were washed three times with low salt wash buffer (0.1% SDS, 1% Triton-X100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-Cl pH 8.0), twice with high salt wash buffer (0.1% SDS, 1% Triton-X100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-Cl pH 8.0), once with lithium chloride wash buffer (0.1% SDS, 1% Triton-X100, 2 mM EDTA, 500 mM LiCl, 20 mM Tris pH 8.0), and three times with final wash buffer (2 mM EDTA, 10% glycerol, 20 mM Tris-Cl pH 8.0). Beads were the resuspended in TE buffer (10 mM Tris-Cl pH 8.0, 10 mM EDTA). Crosslinks were reversed at 65 °C for 16 h using 0.5% SDS and 0.4 µg/µl Proteinase K (Ambion) in a thermomixer. DNA was extracted twice with phenol:chloroform, once with chloroform, precipitated in ethanol Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) according to manufacturer's instructions with 4 cycles of PCR amplification
attributes.tag: GEO Accession
attributes.value: GSM4478949