GSM4257230: IgG_1; Mus musculus; ChIP-Seq
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. For library preparation, purified input and IP DNA was end repaired using T4 DNA polymerase, Klenow polymerase and T4 Polynucleotide kinase. DNA was extracted using Agencourt Ampure XP beads and 'A' bases were added to the 3' end using Klenow exonuclease (3' to 5' exo minus) from NEB. DNA was purified using Agencourt Ampure XP beads and Illumina adapter sequences were ligated to the DNA fragments using the Quick Ligase (NEB). The library was size-selected using Agencourt Ampure XP beads. Using the multiplexing barcoded primers, the library was PCR amplified and purified and analyzed using a Fragment Analyzer (Advanced Analytical, performed by Molecular Biology Core Labs at UMMS). Libraries were pooled with equal molar ratios, denatured, diluted, and sequenced with NextSeq 500/550 High Output Kit v2.5 (Illumina, 75bp paired-end runs,) on a Nextseq500 sequencer (Illumina).