title: NextSeq 500 sequencing; FOXM1 dependent regulation of retinal pigment epithelium cell fate
design: FOXM1 dependent regulation of retinal pigment epithelium cell fate
library_construction_protocol: RPE were generated from the hESC cell line SHEF1.3 or iPSC reprogrammed from erythroblasts using the spontaneous differentiation method described previously (Vugler et al., 2008). Pluripotent cells were cultured as feeder-free colonies on hESC-qualified Matrigel (BD) in mTesR1 (StemCell Technologies) media. RPE foci were excised with a scalpel and dissociated into a single cell suspension using Accutase (Gibco) and plated onto CellStart (Invitrogen) coated surfaces. ChIP was performed essentially as described before (Sanders et al., 2013). Briefly, 1 million cells were crosslinked with 1% Formaldehyde for 10 minutes and the reaction was quenched with 125mM glycine. Sonication was performed on isolated nuclei using the Bioruptor Plus with cooling system for 35 cycles of each 30 seconds on and 30 seconds off at the maximum setting leading to a fragment size between 200-300bp. For each IP, 50 ul of Protein A Dynabeads were incubated with 5 ug of the appropriate antibody (anti-FOXM1 [Santa Cruz Biotechnology, SC-502] or Rabbit IgG REF) overnight at 4oC. Chromatin immunoprecipitation was performed by adding the 100 ul of preblocked antibody-bead complexes per sample and incubating overnight at 4oC on a rotator. Elution and crosslink reversal of both sample and input was performed by incubating the samples for 6 hours at 65oC in a waterbath. The DNA was purified using the Zymo ChIP DNA Clean & Concentrator kit (Zymo Research) according to the manufacturer's instructions. The sequencing library was generated using the MicroPlex Library Preparation kit (Diagenode) and size selected using Agencourt AMPure XP. The libraries were quantified using the qPCR based Illumina library quantification kit (Kappa Biosystems) .