GSM8521145: MEFs_WT_INPUT_Rep1; Mus musculus; ChIP-Seq
MEFs Chip seq: We used three confluent 150cm2 dishes of primary MEFs cells. First, we cross-linked proteins using 1% of formaldehyde, quenched with glycine and scraped dishes to get cell pellet. Then, nuclear extracts were obtained and chromatin was prepared by sonication to an average fragment size of 300-500 bp using Covaris M220 focused-ultrasonicator. Then, shearing chromatin was diluted in dilution buffer and incubated with primary antibody (H4K20me1, ab9051) overnight at 4°C on a rotator. After that, beads were added to all samples and IP 6 hours with rotation at 4°C. After fives washes with RIPA buffer and two washes with TE buffer, DNA elution and reversal of DNA cross-links were performed.                                                  Hela ChIPseq: Chromatin was prepared using the truChIP® Chromatin Shearing kit (Covaris) with modifications. 12 million Hela cells were harvested and washed once in PBS before fixation in 1% formaldehyde for 10 min. The crosslinking reaction was stopped and fixed cells were washed twice with ice-cold PBS. Nuclei were purified and sonicated following the manufacturer instructions. One volume of 2X dilution buffer supplemented with 0.2% SDS and protease inhibitors was added before clarifying the lysate (10 000 g, 5 min, 4oC). Chromatin immunoprecipitation was performed overnight at 4oC with 5µg of an anti-H4K20me1 antibody (ab9051, Abcam). Then, 20 µL Pierce ChIP-grade Protein A/G Magnetic Beads (ThermoFisher) were added during 3 hours. After immunoprecipitation, samples were serially washed once with Low salt wash buffer (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), High salt wash buffer (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), LiCl immune complex wash buffer (10 mM Tris-HCl pH 8, 250 mM LiCl, 1% NP40, 1% sodium deoxycholate, 1mM EDTA) supplemented with protease inhibitors, and twice with modified TE buffer (0.1mM EDTA, 10 mM Tris-HCl pH 8). Crosslink was reversed in elution buffer (NaHCO3 0,1M+ 1% SDS) with 10 µg RNase A (ThermoFisher) for 30 min at 37oC followed by an additional 6 h incubation at 65oC with 50 µg Proteinase K (Apollo Scientific). DNA was subsequently cleaned up using NucleoSpin Gel and PCR Clean-up columns (Macherey-Nagel) before library construction. MEFs purified DNA was quantified with Qubit dsDNA kit (Invitrogen, Q32854) and 10ug was used for library preparation. Sequencing was carried out on an Illumina HiSeq-2500 sequencing system with a 50-bp singled-end read length in the genomic unit of CRG (Centre for Genomic Regulation, Barcelona).   HeLa purified DNA was quantified with Qubit dsDNA kit (Invitrogen, Q32854) and 10ug was used for library preparation. Sequencing was carried out in BGI using DNBSeq PE100 sequencingsystem.