title: GSM5581271: MN_WT_ChIP_Ring1B; Mus musculus; ChIP-Seq
library_construction_protocol: Motor neurons differentiated for 6 days were dissociated with 0.05% trypsin, neutralized, fixed with 1% paraformaldehyde for 15 min and quenched with 0.125 M glycine for 5 min at RT, followed by twice wash with PBS. Cell pellets from above steps were lysed in LB1 buffer (50 mM HEPES, pH 7.5 at 4°C, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP40, 0.25% Triton X; 10 min at 4°C), LB2 buffer (10 mM Tris, pH 8 at 4°C, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA; 10 min at 4°C), and then resuspended in LB3 buffer  (10 mM Tris, pH 7.5 at 4°C, 1 mM EDTA, 0.5 mM EGTA, and 0.5% N-Lauroylsarcosine sodium salt). Chromatin was sheared using a Diagenode Bioruptor to an average size of ~250 bp. After pre-clearing with BSA-blocked protein G Dynabeads, chromatin was incubated with indicated antibodies at 4°C overnight. The chromatin immunocomplexes were recovered with BSA-blocked protein G Dynabeads for 2 hr at 4°C. Immunoprecipitated DNA was eluted from the beads, reversed crosslinked at 65°C overnight, digested with protease K and RNaseA and then purified for library construction. Immunoprecipitated DNA (∼1-30 ng) was end-repaired using End-It Repair Kit, tailed with deoxyadenine using Klenow exo-, and ligated to custom adapters with T4 Rapid DNA Ligase (Enzymatics). Fragments of 200-600 bp were size-selected using Agencourt AMPure XP beads (0.5X and 0.3X), and subjected to PCR amplification using Q5 DNA polymerase. Libraries were size-selected using Agencourt AMPure XP beads (0.75X), quantified by Qubit™ dsDNA HS Assay Kit and quality checked by High Sensitivity D1000 ScreenTape. Libraries were sequenced as 50 bp single-end reads on the Illumina HiSeq 4000 platform.