title: GSM8000381: cKO15 hypothalamus, knockout, week 5; Mus musculus; ChIP-Seq
library_construction_protocol: each cross-linked hypothalamic sample (1% formaldehyde) was sonicated in cell lysis buffer, resulting in chromatin fragments ranging from 200 to 1000 bp. These fragments incubated in ChIP dilution buffer with an αSMC3 antibody (5 μg/sample; Bethyl, Montgomery, Texas, USA, #A300-060A). Simultaneously, 5% input (without immunoprecipitation step) was utilized to normalize the amount of chromatin in each sample. Subsequently, DNA was isolated from each immunoprecipitated/input sample and subjected to genomic library preparation 20ul of DNA were used for libraries preperation using the NEBNext Ultra II DNA kit (NEB, #E7645). Quantification of the library was performed using dsDNA HS Assay Kit and Qubit 2.0 (Molecular Probes, Life Technologies) and qualification was done using the Agilent D1000 Tapestation Kit and Tapestation 4200.  25nM of each library was pooled together and was diluted to 4nM according to NextSeq manufacturer's instructions. 1.5pM was loaded onto the Flow Cell with 1% PhiX library control. Libraries were sequenced on an Illumina NextSeq 500 instrument, 75 cycles single read sequencing.