title: GSM2064461: ChIP H3K4me3 batch 5; Homo sapiens; ChIP-Seq
library_construction_protocol: Immunoprecipitations were carried out with S1 mononucleosomes derived from HeLaK cells stably expressing GFP-tagged  H2A.Z.1, H2A.Z.2 or PWWP2A from 4 x 107 cells in 1.5 ml low-binding tubes. 25 µl slurry GFP-trap magnetic beads (ChromoTek) per IP were equilibrated in EX100 buffer (10 mM Hepes pH 7.6, 100 mM NaCl, 1.5 mM MgCl2, 0.5 mM EGTA, 10% glycerol (v/v), 10 mM β-glycerolphosphate, 1mM DTT, 1x protease inhibitors), added to mononucleosomes and incubated for 2.5 h at 4°C while rotating. Beads were quickly spun down, separated in a magnetic rack and the supernatant kept as non-bound fraction. For GFP-PWWP2A IPs, mononucleosomes prepared from stable HeLa Kyoto GFP-PWWP2A cell line were precleared with G-protein magnetic beads (Millipore) for 1 h at 4°C and subsequently incubated over night (ON) with 2 µl of GFP antibody (kind gift of Prof. Andreas Ladurner, LMU Munich) at 4°C while rotating. Next, 20 µl of magnetic G-protein beads equilibrated in EX100 buffer were incubated with the reaction for additional 4 h at 4°C while rotating. An IP with an unspecific rabbit IgG antibody served as negative control.  For native H3K4me3 IPs, mononucleosomes from wild type HeLaK cells were prepared. The MNase digestion protocol was optimized for the use of 4 x 106 cells per IP. The precleared mononucleosomes were incubated with 1 µl of a rabbit H3K4me3 antibody (Diagenode). Washed beads were resuspended in 100 µl TE, 3 µl 10% SDS and 5 µl of 20 mg/ml proteinase K were added and incubated for 1 h at 65 °C. Suspensions were vortexed briefly, magnetically separated and the supernatant transferred to a fresh tube. Beads were washed once with 100 µl TE containing 0.5 M NaCl, magnetically separated and the supernatant mixed with the first supernatant. Input fractions were processed in parallel. After Phenol/chlorophorm/isoamylalcohol extracting and ethanol precipitating the DNA, the IP DNA pellet was resuspended in 12 µl and the input DNA pellet in 32 µl 10 mM Tris-HCl (pH 7.5). DNA concentrations were determined with the Qubit dsDNA hs Kit (Invitrogen) and DNA size monitored on a 1000 DNA BioAnalyzer chip (Agilent). Illumina Sequencing libraries were established with the MicroPlex Library Preparation Kit (Diagenode) following the manufacturer’s instructions with following variations. The number of step 5 amplification cycles was scaled according to the amount of input material. When purifying libraries after amplification, samples were incubated 15 min with AMpure beads instead of 5 min, ethanol washed beads were dried for 3 min at RT instead at 37°C and DNA was eluted in 22 µl 10 mM Tris-HCL pH 7.5 instead of TE
attributes.tag: GEO Accession
attributes.value: GSM2064461