Illumina HiSeq 2500 paired end sequencing; TTC7A Localizes to Specific Regions of Chromatin
TTC7A Localizes to Specific Regions of Chromatin
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. 2 control B-lymphoblastoid cell lines were established to stably express TTC7A_WT-3*Flag-HA-ires-EGFP. ChIP was performed using two distinct Flag antibodies: IP-1, sigma F1804 with 100*10e6 cells and IP-2, sigma F3165 with 30*10e6 cells. Cells 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).  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
TTC7A_WT-3*Flag-HA-ires-EGFP
Experimental Factor: immunoprecipitate
anti-Flag (#F1804 Sigma)