title: GSM7162785: GATA2_DMSO; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP-Seq libraries were prepared using the following protocol. End repair of immunoprecipitated DNA was performed using the End-It End-Repair kit (Epicentre, ER81050) and incubating the samples at 25°C for 45 min. End-repaired DNA was purified using AMPure XP Beads (1.8X of the reaction volume) (Agencourt AMPure XP – PCR purification Beads, BeckmanCoulter, A63881) and separating beads using DynaMag-96 Side Skirted Magnet (Life Technologies, 12027). A-tails were added to the end-repaired DNA using NEB Klenow Fragment Enzyme (3'-5' exo, M0212L), 1X NEB buffer 2 and 0.2mM dATP (Invitrogen, 18252-015) and by incubating the reaction mix at 37μC for 30 min. A-tailed DNA was cleaned up using AMPure beads (1.8X of reaction volume). Subsequently, cleaned up A-tailed DNA went through Adaptor ligation reaction using Quick Ligation Kit (NEB, M2200L) following manufacturer's protocol. Adaptor-ligated DNA was first cleaned up using AMPure beads (1.8X of reaction volume), eluted in 100μl and then size-selected using AMPure beads (0.9X of the final supernatant volume, 90μl). Adaptor-ligated DNA fragments of proper size were enriched with PCR reaction using Fusion High-Fidelity PCR Master Mix kit (NEB, M0531S) and specific index primers supplied in NEBNext Multiplex Oligo Kit for Illumina (Index Primer Set 1, NEB, E7335L). Conditions for PCR used are as follows: 98°C, 30 sec; [98°C, 10 sec; 65°C, 30 sec; 72°C, 30 sec] X 15 to 18 cycles; 72°C, 5 min; hold at 4°C. PCR enriched fragments were cleaned up using AMPure beads (1X of reaction volume). Libraries were eluted in 20μl elution buffer. All the libraries went through quality control analysis using an Agilent Bioanalyzer and subjected to next-generation sequencing using Illumina Hiseq 2500 platform.