macroH2A1/2 double knockout with dox-inducible macroH2A2-GFP transgene
treated with
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Nuclei isolation for nChIP was performed as previously described (Fontanals-Cirera et al., 2017). Nuclei were resuspended with 100 ul Buffer A (0.32 M sucrose, 50 mM Tris, pH 7.5, 4 mM MgCl2 and 1 mM CaCl2) per 10 million cells. For each ChIP, an aliquot of 5 million cells was used and diluted with 350 ul of Buffer A. CaCl2 was added to a final concentration of 3 mM. 8.5 U MNase (Affymetrix, 70196Y) was added and the reaction was incubated at 37ºC for 10 min. The reaction was stopped by adding EGTA to a final concentration of 10 mM. Nuclei were spun down at 10,000g for 7 min. The supernatant, which contains mostly mononucleosomes, was collected as S1. The pellet was gently resuspended with 400 ul of Buffer B (50 mM Tris, pH 7.5, 300 mM NaCl, 2 mM EDTA and 0.1% NP-40) and incubated at 4ºC for 2 hours with head-to-head rotation. Nuclei were spun down at 10,000g for 7 min and the supernatant was collected as S2, which contains longer chromatin fragments. S1 and S2 were pooled, OD260 was taken and 100 ug chromatin was usde for each ChIP. The material was further clarified at maximum speed for 5 min and the supernatant was collected and diluted with Buffer C (50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.05% NP-40) to 1 ml. 50 ul of the resulting material was taken as input. For GFP IP, 20 ul GFP-Trap_MA (ChromoTek) bead slurry was equilibrated and added to the material followed by a 2.5 hour incubation at 4ºC with head-to-head rotation. Beads were collected using a magnetic rack and washed once with Buffer G 150 (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% NP-40), twice with Buffer G 250 (50 mM Tris, pH 7.5, 250 mM NaCl, 0.5% NP-40), and once with TE buffer (10 mM Tris, pH 7.5 and 1 mM EDTA). Input and beads were incubated with 50 ug/ml RNase A for 1 hour at 37ºC in 200 ul TE. SDS was then added to 0.5% and Proteinase K 500 ug/ml. Samples were incubated overnight at 56ºC with constant vortexing at 800 r.p.m. in a thermomixer. The supernatant was collected and purified with QIAquick PCR purification columns (Qiagen). The input and ChIP DNA were subsequently analyzed and quantified using the Agilent 2100 Bioanalyzer High Sensitivity Kit. nChIP-seq libraries were prepared as previously described with minor changes (Fontanals-Cirera et. al., 2017). Briefly, 8-10 ng input or ChIP DNA was end-repaired with T4 DNA polymerase (NEB), DNA polymerase I large (Klenow) fragment (NEB) and T4 polynucleotide kinase (NEB) in the presence of dNTPs (Roche). Next, dA-tailing was performed using Klenow fragment (3'->5' exo-, NEB) with dATP (Roche) in NEB Buffer 2. Illumina Truseq adaptors with barcodes were ligated using Quick Ligase (NEB). Libraries were size-selected at the 300-400 bp range through agarose gel electrophoresis corresponding to mononucleosomal DNA. The libraries were then amplified using KAPA HiFi DNA Polymerase (KAPA biosystems). Optimal number of PCR cycles was determined by qPCR as described (Buenrostro et al., 2015). The size distribution and level of amplification were further controlled by analysis with the Agilent bioanalyzer and Qubit quantification. Resulting libraries were subject to 80-bp, single-end sequencing with an Illumina NextSeq 500 instrument.