GSM3074440: 300_120L_20W_C3_H3K27ac; Homo sapiens; ChIP-Seq
Sample containing 10^6 cells was used as the starting material and centrifuged at 1,600 g for 5 min and washed twice with 1 ml cold PBS. Cells were crosslinked by adding 1 ml freshly made 1% formaldehyde and incubating at room temperature for 5 min on a shaker. Crosslinking was promptly terminated by adding 50 µl of 2.5 M glycine and shaking for 5 min. The pellet was resuspended in 130 μl Covaris buffer (10 mM Tris-HCl, pH 8.1, 1 mM EDTA, 0.1% SDS and 1× protease inhibitor cocktail) and sonicated using the following conditions. Using Covaris M 220 (Covaris), the sample was sheared at 4 oC with 75 W peak incident power, 5% duty factor, and 200 cycles per burst for 12 min. The sheared sample was centrifuged in a 4 oC centrifuge at 14,000g for 10 min, and the supernatant was transferred to a new tube. Sheared chromatin was diluted with IP buffer (20 mM Tris-HCl, pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% (w/v) sodium deoxycholate, 0.1% SDS, 1% (v/v) Triton X-100, with 1% freshly added PMSF and PIC) and aliquoted into 25 μl samples containing 250, 500, 1500, and 5,000 cell samples and stored at -80 oC. Library preparation was performed using the Swift Bio S2 library preparation kit (Swift Biosciences) using 40 μl of purified DNA. 2.5 μl of the low EDTA TE buffer in the 50 μl amplification cycle reaction mix was replaced with 2.5 μl of 20x EvaGreen, and amplification was terminated after samples saw an >3000 RFU increase (BioRad CFX Connect). Amplified DNA was then eluted into 7 μl low EDTA TE buffer where 2 μl could be used for qPCR analysis for preliminary quality control analysis, Kappa DNA quantification, and Tapestation fragment size analysis and the other 5 μl for library pooling. Libraries were pooled at 10 nM for sequencing by Illumina HiSeq 4000 with single-end 50 nt read. LIFE-ChIP-seq (Low-Input Fluidized-bed Enabled Chromatin Immunoprecipitation combined with sequencing), procedures are detailed in our paper
GEO Accession
GSM3074440