Induced pluripotent stem cells from human fibroblasts from patients with FA disease
genotype correction status
Sequenced DNA Library
Immunocomplexes were incubated with 1:1 mix of protein A and G Dynabeads (Invitrogen, Carlsbad, CA) for 2 hours at 4°C, with rotation. Beads were immobilised on magnetic racks and the supernatants discarded, after which the beads were washed with low salt buffer, high salt buffer and LiCl buffer. All washes were carried out for 5 minutes at 4°C on a rotating wheel in the presence of protease inhibitors. Beads were then resuspended in 100 μl of elution buffer and DNA complexes were decrosslinked at 65 °C for 3 hour with shaking. DNA was then eluted in 50 μl of water using the PCR purification kit (QIAGEN). Libraries were prepared using the NEBNext® ChIP-Seq Library Prep Reagent Set for Illumina® kit (ref. E6200S) according to the manufacturer's protocol. Briefly, 10 ng of input and ChIP enriched DNA were subjected to end repair, addition of “A” bases to 3′ ends and ligation of PE adapters. All purification steps were performed using Qiagen PCR purification columns (refs. 50928106 and 50928006). Library size selection was done with 2% low-range agarose gels and DNA was extracted using QIAquick Gel extraction kit (ref. 50928706, Qiagen) and eluted in 36 µl EB. Library amplification was performed by PCR on the size selected fragments. Final libraries were analyzed using Agilent DNA 1000 chip to estimate the quantity and check size distribution, and were then quantified by qPCR using the KAPA Library Quantification Kit (ref. KK4835, KapaBiosystems) prior to amplification with Illumina’s cBot. Sequencing was done on the HiSeq2000, Single Reads, 50nts.