NextSeq 500 sequencing; Genome-wide maps of nuclear lamina interactions in HeLa cells identified using MethylAdenine IDentification (MadID).
Genome-wide maps of nuclear lamina interactions in HeLa cells identified using MethylAdenine IDentification (MadID).
HeLa cells induced to express M.EcoGII-LaminB1 or M.EcoGII alone for 24hours by supplementing growing medium with 1 microM Shield1.  Genomic DNA was isolated using the Qiagen Blood & Cell Culture DNA Midi Kit (Genomic-tip 100/G) according to the manufacturer's recommendations together with RNAse treatment (200 µg/ml of RNAseA (Sigma) and RNase Cocktail™ Enzyme Mix (Ambion) (2.5U/ml RnaseA ; 100U/ml RnaseT1) at 37°C for 1 hour).  Double stranded genomic DNA (10 µg) was sheared into fragments of 200-400bp using Bioruptor Plus sonicator (Diagenode) – 300 µL of 33,3ng/µl in 1.5ml TPX tube; 40 cycles 30sec/60sec ON/OFF ; low power. The temperature was kept at 4°C (using the Diagenode Water cooler, Cat. No. BioAcc-Cool) for optimal shearing results. 1% of sample was taken as an input. Fragmented DNA was end-repaired and A-tailed according to manufacturer recommendations (NEBNext End-repair module (NEB #E6050), NEBNext A-tailing module (NEB #E6053)). Double Stranded TruSeq Illumina adapters were ligated (NEBNext Ligation Module (NEB #E6056). Then, samples were diluted in TE buffer up to 360 µl, denatured for 10 minutes at 95°C and snap cooled on ice for 10 minutes. Samples were supplemented with 10x m6AIP buffer (100mM Na-Phosphate buffer, pH 7.0; 3M NaCl; 0.5% Triton X-100), 2.5 µg m6A antibody (SYSY) and rotated overnight at 4ºC. Next 20 uL of protein A/G Dynabeads mix (Thermo Fisher Scientific), pre-blocked for 1 hour (PBS-0.5%BSA-0.1%Tween-20), was added and samples rotated at 4ºC for 3 hours. Beads were washed 4 times in 1 ml 1X m6AIP buffer. Beads and input samples were resuspended in 150 µl digestion buffer (50mM Tris, pH 8.0; 10mM EDTA; 0.5% SDS) containing 300 µg/ml proteinase K and incubated for 3 hours at 50°C with shaking. DNA was purified by phenol/chloroform/isoamyl alcohol extraction and resuspended in 21 µl milliQ water. IP efficiency was checked by qPCR using 1µL of IP and INPUT sample. DNA recovered from Immunoprecipitation was amplified (10 cycles) using KAPA Hifi DNA polymerase (Kapa Biosystems) using Illumina primers, and purified with AMPureXB beads (Beckmann Coulter).