GSM4119577: NEG Acute DMSO FAIRE Replicate 2; Homo sapiens; FAIRE-seq
Cells were crosslinked and nuclei were prepared using the Active Motif Chromatin Preparation kit (53046) according to manufacturer's instructions except that 0.25 million formaldehyde crosslinked Drosophila S2 cells were spiked into the SUM-229 cells just prior to nuclei preparation. Nuclei were resuspended in 90 uL Lysis Buffer A (Simon et al., 2012) and 10 uL of MegaShear (Triangle Biotechnology, Inc.) was added to each tube prior to sonication. Sonication was performed in a Covaris E110 instrument for 4 minutes per sample using conditions described in (Chiarella et al., 2018). Following sonication, insoluble debris was removed by centrifugation and samples were digested with 200 ug RNase (Qiagen) at 37˚C for 30 minutes. Ten uL of sample was removed for input and digested with 40 mg Proteinase K (Worthington) for 1 hour at 55˚C, followed by 2 hours at 80˚C to reverse the crosslinks, then purified using a silica matrix column (Zymo Research, ChIP DNA Clean and Concentrate kit, D5201). FAIRE was performed on the remaining 90 uL of sonicated chromatin by purification on a silica matrix column as described in (Pattenden et al., 2016). DNA was quantitated using fluorometry (Qubit dsDNA High Sensitivity Assay Kit, Invitrogen). Average peak fragment size of input and FAIRE DNA was confirmed using gel electrophoresis. Approximately 50 ng of fragmented DNA was adapter ligated and purified using the KAPA stranded HyperPrep Kit (Roche cat. # 07962347001) and Illumina TruSeq Indexed Adapters according to the manufacturer's instructions. Dual size selection was performed after 18 cycles of PCR amplification of the cDNA library according to manufacturer recommendations. A multiplexed pool of libraries was sequenced using the 75-cycle NextSeq 500/550 High Output v2 sequencing kit (Illumina cat. # FC-404-2005) on the Illumina NextSeq500 to yield approximately 5.0 x 10e7 75 bp single-ended reads per sample.
GEO Accession
GSM4119577