GSM2064462: ChIP input batch 5; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Uterus
Cell type
HeLa
Primary Tissue
Cervix
Tissue Diagnosis
Adenocarcinoma
Attributes by original data submitter
Sample
source_name
h3_k4me3_in_wt
cell line
HeLa Kyoto cells
chip antibody
none (input)
experimental batch
5
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
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