Truseq nano dna ht sample preparation kit

Formaldehyde-fixed cell pellets were snap-frozen in liquid nitrogen and stored at −80 °C before the ChIP assay. Frozen cells were lysed in 120 µl of lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 1 mM phenyl methane sulfonylfluoride, 20 mg/ml sodium butyrate, proteinase inhibitor cocktail; Sigma), and chromatin was sheared by sonication, using a BioRuptor (Diagenode), to generate 100- to 500-bp DNA fragments. Chromatin was diluted in 1 ml of radioimmunoprecipitation assay (RIPA) buffer and immunoprecipitation was done overnight at 4 °C by incubating 1 µl of H3K27ac antibody (rabbit polyclonal IgG, lot# Gr167929-1, Abcam) pre-coated on 10 µl protein A–coated magnetic beads (Invitrogen). Immunocomplexes were captured and washed, and ChIP DNA was eluted as described previously^{9 (link)}. 1 ng of ChIP DNA was PCR amplified for 18–22 cycles using whole-genome amplification (WGA) primers (WGA-SEQX, Sigma) following the manufacturer’s instructions. Approximately 100 ng amplified DNA was used for preparing a standard Illumina sequencing library (TruSeq Nano DNA HT Sample Preparation Kit, Illumina). Libraries were sequenced on the HiSeq2500 Illumina platform to obtain 50-bp single-end reads (TruSeq Rapid Kit, Illumina). Samples that failed quality control steps, as previously described^{9 (link)}, were eliminated from further downstream steps and analysis.