Illumina HiSeq 2500 sequencing; TTC7A deficiency Impacts the H3K27ac and H3K27me3 histone marks
TTC7A deficiency Impacts the H3K27ac and H3K27me3 histone marks
Original B-lymphobast cells come from peripheral blood mononuclear cells (PBMC). These cells are obtained from blood samples after ficoll separation. PBMC from controls and patients were transduced with the Epstein Barr Virus (EBV) to generate the B-lymphobast cells lines. Cells were expanded and frozen. The B-lymphobast are cultured in medium composed of RPMI, Glutamine, Fetal bovin serum and penicillin-streptomycin. B-lymphoblastoid cell lines transformed with EBV of 3 controls (C1 to C3) and 3 TTC7A deficient patients (P3_E71K, P4_E71K, P*/L478P) were cultured in RPMI medium 1640+Glutamax (Gibco, ref 61870-010) supplemented with 10% (vol/vol) Fetal Bovine Serum (Gibco), sodium pyruvate (1 mM, LifeTechnologies) and penicillin and streptomycin (100U/ml each, LifeTechnologies). Cells with blocked in early G1/S phase with thymidine double blockage (2mM, 18 h first treatment, 9 h release and 15 h second treatment). The antibodies used for ChIP were: H3K27ac (catalogue number: 39685, active motif), H3K27me3 (catalogue number: 39155, active motif) Immunoprecipitated DNA (ChIP) and whole cell extracts DNA (input) were treated with RNaseA and proteinase K then purified by using Qiaquick PCR purification kit (Qiagen, 28106). TruSeq ChIP kit from Illumina was used to perform library preparation according to the manufacturer's recommendations. Briefly, IP and input DNA were first size selected using SPRIselect beads (Agencourt) in order to have fragments between 100 and 500bp after BioAnalyzer profiling. 1 to 10ng of DNA were used in a first step of end-repair followed by the adenylation of 3'-end allowing further ligation of specific Illumina adaptors required for sequencing. These adaptors contain specific indexes (barcodes) different for each sample so that library from different samples can be mixed together before sequencing. After PCR amplification (12-15 cycles), quality assessment of DNA libraries was achieved using BioAnalyzer profiling and Qubit quantification. After equimolar pooling of libraries, the final dilution was accurately quantified using Kapa library quantification kit.
Experimental Factor: genotype
wild type genotype
Experimental Factor: immunoprecipitate
anti-H3K27ac (#39685, active motif)