title: GSM5106466: DMSO1 ChIRP; Homo sapiens; OTHER
library_construction_protocol: ChIRP-seq experiments were performed according to Chu et al. (Chu et al., 2011) with modification as follows. 107 cells were washed once by PBS and resuspended in 10ml PBS. Cells were harvested by trypsin digestion and crosslinked with 2mM DSG (Thermo Fisher) in PBS at RT for 30 min with gentle end to end rotation. Formaldehyde was added to a final concentration of 3.7% to crosslink for 10 min further, then quenched with 250mM glycine at RT for 10 min. The cells were then washed twice by ice cold PBS, pelleted and snap frozen by liquid nitrogen and stored at -80℃. Cell pellets were resuspended with nuclear lysis buffer and incubated on ice for 10min. Chromatin was sonicated into 200-500bp, centrifuged at high speed for 10min and the supernatant was moved to a new tube, with 10ul reserved as DNA Input. For hybridization, samples were incubated with 100 pmol probes per 1ml lysate, supplemented with 250ul of 5x hybridization buffer. The hybridization was conducted at 39 oC rotating for 3h. Prewashed 50 uL streptavidin M280 beads were then added and the incubation continued for an additional 3h. The beads were washed 5 times total with 0.1 X SSC wash buffer (0.1 X SSC, 1% SDS) at 42 oC for 5min. For the Rnase treatment control, after 3 times of washing, the beads were treated once with 10 ug/ml Rnase A at 37 oC in Rnase digestion buffer, before washing two more times. Beads were resuspended with 1ml 0.1X SSC wash buffer. 900ul of the sample was separated for DNA elution, 100ul for RNA elution. Following ChIP de-crosslink method and PK, Rnase A and Rnase H were added in the second step. DNA was purified using the MinElute PCR Purification Kit (QIAGEN), followed by constructing the sequence library by NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®. The ChIRP-seq libraries were constructed by NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®. Briefly, the fragment DNA are ligated to adapters by TA ligation. Then PCR for 10 cycles with specific index for sequencing.