title: GSM9459567: shControl_Control_Input_Rep4; Homo sapiens; ChIP-Seq
library_construction_protocol: H3K27Ac: Samples were sent to Active Motif (Carlsbad, CA) for ChIP-Seq. Active Motif prepared chromatin, performed ChIP reactions, generated libraries, sequenced the libraries and performed basic data analysis. In brief, cells were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. Chromatin was isolated by adding lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500 bp with Active Motif's EpiShear probe sonicator (cat# 53051). Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by SPRI beads clean up (Beckman Coulter) and quantitation by Qubit. Extrapolation to the original chromatin volume allowed determination of the total chromatin yield. An aliquot of chromatin (30 ug) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4ug of antibody against H3K27Ac (Active Motif, catalog number 39133, lot number 06921014). Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation.   FLAG and H3K36Me: FLAG-KDM2A samples were double-fixed using 2mM ethylene glycol bis(scuccinimidyl succinate) (EGS) (Thermo Scientific cat. 21565) for 1h followed by 1% formaldehyde for ten minutes.  H3K36 methylation samples were fixed with 1% formaldehyde alone.  Reactions were quenched with glycine to a final concentration of 0.125M. Pellets were washed with PBS and lysed in complete sonication buffer (20mM Tris pH 8.0, 2mM EDTA, 0.5mM EGTA, 1x protease inhibitors (Roche), 0.5% SDS, and 0.5mM PMSF) prior to sonication with a QSonica Q800. Fragment size (200-500bp) from extracted DNA was confirmed by agarose gel and quantified by Qubit. Input was aliquoted from 20% of total material, and chromatin samples were subsequently pre-cleared for 1 hour at 4ºC with immunoprecipitation (IP) buffer (20mM Tris pH 8.0, 2mM EDTA, 0.5% Triton X-100, 150mM NaCl, and 10% Glycerol) and Protein A/G beads. IP for proteins of interest was conducted using protein A/G beads and 10µg of antibody. For H3K36 methylation IPs Drosophila spike-in chromatin (100ng)  (Active Motif cat. 53083) and spike-in antibody (5μg) (Active Motif cat. 61686)were added. IPs were washed then eluted in a buffer of 100mM NaHC03, 1% SDS. Both IP and input samples were proteinase K treated, reverse crosslinked, and purified by phenol-chloroform extraction with ethanol precipitation. H3K27Ac: Illumina sequencing libraries (a custom type, using the same paired read adapter oligonucleotides described by Bentley et al., 2008)) were prepared from the ChIP and Input DNAs on an automated system (Apollo 342, Wafergen Biosystems/Takara). After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on Illumina's NextSeq 500  (75 nt reads, single end).  FLAG and H3K36Me: Samples were checked by Agilent Bioanalyzer, and libraries were prepared by the using ThruPlex DNA Seq (Takara Bio. cat. R400674) for H3K36 libraries, and DNA SMART ChiP-Seq (Takara Bio.cat. 634865) for FLAG libraries. Libraries were barcoded, pooled, and sequenced (PE) on Illumina NovaSeq X and NovaSeq 6000 machines. Please note, H3K36 methylation libraries were  initially sequenced on the NovaSeq 6000 then subsequently on the NovaSeq X  for additional coverage depth. Fastq files from these runs were concatinated head to tail prior to any anaylsis, and these pooled files are provided here for convenience. Files from each individual sequencing run are available upon request.