Library construction was performed using the Illumina ChIP-Seq library preparation kit according to the standard protocol. ChIP-Seq fragments were separated on an agarose gel, and fragments of approximately 200 bp were ligated to Illumina-specific adapters to generate libraries. Six cycles of PCR amplification were used to select for fragments containing both Illumina-specific adapters before bead-based elimination of unligated adapters. ChIP enrichment of HA-EKLF-bound chromatin obtained from fetal liver progenitors and erythroblasts was performed as previously described.9,27,34 Chromatin was processed using the Magna ChIP A kit (#17-610; Millipore) according to the manufacturer’s instructions. Chromatin was immunoprecipitated with monoclonal antibodies against HA (F-7, sc-7329X, Santa Cruz Biotechnology). As a background genomic control, sheared chromatin from E13.5 HA-EKLF fetal liver cells was processed in parallel, minus incubation with the antibodies. Fetal livers were dissociated to single- cell suspension and stained with anti-CD71–FITC and anti-Ter119–PE antibodies (BD Biosciences PharMingen). Cell populations were isolated using a FACSAria flow cytometer running FACSDiva 6.1.3 software (BD Biosciences). Cells were collected as erythroid progenitors (Ter119-CD71- and Ter119-CD71+) or erythroblasts (Ter119+CD71+). At least 3 indepen- dent cell sorts for each population were performed.
GEO Accession
GSM1165498