Illumina MiSeq paired end sequencing; Amplicon based single molecule footprinting on ES cells and derivatives
Amplicon based single molecule footprinting on ES cells and derivatives
Wild-type embryonic stem cells (ESC), DNA methylation-null ESC (DNMT TKO) and REST knockout ESC were grown on gelatin-coated 10cm dishes, using Dulbecco's Modified Eagle Medium (DMEM), supplemented with 15% FBS, LIF, 2-Mercaptoethanol, 1mM L-Glutamine and 1x non-essential amino acids (ThermoFisher 11140050) with regular splitting of cells. Cells are trypsinized, counted and washed in ice cold PBS. For 1 reaction, material equivalent to 250.000 cells were pelleted and resuspended in 1mL ice cold hypertonic buffer (10mM Tris (pH = 7.4), 10mM NaCl, 3mM Mgcl2, 0.1mM EDTA, 0.5% NP40) to release the nuclei, span down and washed in the same buffer without NP40, eliminating detergents. Nuclei were then span down.in 1x M.CviPI reaction buffer (50mM Tris (pH 8.5), 50mM NaCl, 10mM DTT). The nuclei were then incubated with 200U of M.CviPI (NEB-M0227L) at 37?C for 7.5 min (in presence of 0.6mM SAM, and 300mM Sucrose). The reaction was supplemented with 100U of M.CviPI and 128pmol of SAM before a second incubation round of 37C for 7.5 min. For ES samples, reactions were stopped at this point by adding a SDS containing buffer (20mM Tris, 600mM NaCl, 1%SDS 10mM EDTA).  For TKO samples, where dual enzyme footprinting was applied, 10mM MgCl2, 128pmol of SAM and 60U of M.SssI (NEB- M0226L) were added for a third incubation round of 37C for 7.5 min followed by stoppage of the reaction by adding the same SDS containing buffer. Samples were then incubated at 55degrees over night, followed by phenol / chloroform extraction of DNA. Single molecule footprinted DNA (approximately 1microg per reaction) was bisulfite converted (Qiagen Epitect Bisulfite Kit) adn eluted in 100ul total volume from the column. This material was used for PCR amplification, using bisulfite specific primers (625nanomolar in final reaction) and KAPA U+ Polymerase master mix, in a final volume of 16microL. To this end, premixed forward and reverse primers were ordered in 96 well format and the 96well format was maintained during PCR (total of  3 sets of primers were used in the study, denoted as P1,2,3). After PCR, samples were run on gel to confirm specific PCR band sizes, pooled in one tube per 96well plate and bead purified using 0.8x beads to DNA ratio. 1microG of purified DNA was used to generate a sequencing library using NEBNext Ultra II kit according to manufacturers guidelines, where a size selection compatible with designed amplicons (300-500bp) was utilized. Up to 12 samples were multiplexed per library using NEBNext Multiplex Oligos system, each of which were measured on Bioanalyzer to confirm size distribution and molarity, followed by equimolar pooling of barcoded libraries and sequencing on an Illumina MiSeq platform, generating reads of 500bp.